Contribution of different IgGs to the total activity of nonfractionated Abs was calculated taking into account the relative content of these IgGs within polyclonal IgGmix and their RAs in the hydrolysis of MBP.

**Table 7.** Relative specific MBP-hydrolyzing activities (RAs) of IgGs of different types and their relative contributions to the total activity of polyclonal IgGmix [76].

hydrolyzed MBP within a wide range of pH values (5.3–9.5) and showed comparable pH dependencies, while the pH profiles for IgG1–IgG4 were unique (**Figure 17**).

These results clearly demonstrate that IgGs of all four subclasses are very heterogeneous and can consist of different sets of catalytic subfractions of polyclonal IgG having quite distinct pH dependencies. **Figure 17** shows the relative influence of PMSF and EDTA on the MBPhydrolyzing activity of different IgGs. The nonfractionated IgGs and lambda-IgGs demonstrated lower inhibition by PMSF than that for EDTA (**Figure 17**). The inhibition of serine-like and metal-dependent activities of kappa-IgGs were comparable. PMSF suppressed MBPhydrolyzing activity of IgG3, IgG2, and IgG1 by 13–17%, while the decrease of this activity by EDTA was significantly greater, 30–45%. There was no noticeable PMSF effect on the IgG4 activity, while EDTA decreased its activity by ~65% (**Figure 17**). Thus, IgG1–IgG4, kappa-IgGs, and lambda-IgGs are characterized by specific ratios of metal-dependent and serine-like proteolytic activities.

The cleavage site specificity of different IgG preparations in the case of four oligopeptides corresponding to four antigenic determinants of MBP was analyzed [76]. Overall, kappa-IgGs and lambda-IgGs, as well as IgG1–IgG4 demonstrated either different patterns of four oligopeptides cleavage, or at least stimulate the accumulation of the same products of the hydrolysis with different efficiency.

The dialysis of IgGs caused a more pronounced decrease in the activity of kappa-IgGs than of lambda-IgGs [76]. Addition to the reaction mixtures of Ca2+ + Mg2+ or Ca2+ + Co2+ led to

**Figure 17.** The pH dependence of RAs of MBP-hydrolyzing SLE kappa-and lambda-IgGs (A), as well IgG1, IgG2, IgG3, and IgG4 (B) [76]. Hydrolysis of MBP incubated without IgGs was used as control ("Con," A). The RAs of MBPhydrolyzing activity of SLE IgG1, IgG2, IgG3, IgG4, and total IgGmix (t-IgG) (C). The RAs were determined before (black columns) and after IgGs preincubation with PMSF (gray columns) and EDTA (white columns). The RAs of all IgGs in the absence of the inhibitors were taken as 100%.

 approximately comparable increase in the RAs of dialyzed lambda-IgG (1.6- to 1.7-fold), kappa-IgG (2.0- to 2.3-fold), and nonfractionated IgGs (1.7- to 1.8-fold). Ca2++Co2+ together cannot activate IgG1, while in the presence of Ca2+ + Mg2+ its activity increased by a factor of 1.6. Ca2+ + Co2+ increased the activity of IgG2 (~2.9-fold), IgG3 (~6.4-fold), and IgG4 (~6.0-fold). A significant increases in the RAs were revealed for Ca2+ + Mg2+ in the case of IgG3 (~3.5-fold), IgG4 (~4.4-fold), and IgG2 (~5.7-fold). While the Ca2+ + Mg2+ combination was the best for the activation of IgG2 and IgG1, IgG4, and IgG3 showed the highest activity in the presence of Ca2+ + Co2+. The ratios of RAs of all IgG preparations before and after their dialysis against EDTA, as well as in the presence of different metal ions, were individual for every preparation analyzed. These data indicate for an extreme Me2+-dependence diversity of different subclasses SLE IgGs hydrolyzing MBP.

The extraordinary diversity of polyclonal abzymes with DNase, RNase, and proteolytic activities was shown not only in the case of SLE, but also other diseases [13–22]. Very unexpected enzyme properties have been discovered in the case of monoclonal abzymes of patients with SLE.

#### **7.2. Monoclonal SLE abzymes hydrolyzing myelin basic protein**

For analysis of MBP-hydrolyzing activity of Abs, we have used the same phagemid library of kappa light chains [118–120] as for analysis of MLChs with DNase activity [111, 112]. The phage particles containing MLChs with different for MBP were separated by affinity chromatography on MBP-Sepharose (**Figure 18A**).

The pool of phage particles was distributed between 10 peaks eluted from the sorbent and all MLChs of fractions of 10 new small pools efficiently hydrolyzed MBP and four oligopeptides (OPs) corresponding to four immunodominant MBP sequences containing cleavage sites (**Figure 18B**). However, there were no any detectable particles peaks having considerable affinity for MBP after similar chromatography of phage particles with pCANTAB plasmid containing no cDNA of light chains (**Figure 18A**). Thus, the MLChs pools of all 10 phage particles fractions having different affinity to MBP contain both inactive and catalytically active light chains hydrolyzing MBP. Similar distribution all over the chromatography profiles was

 approximately comparable increase in the RAs of dialyzed lambda-IgG (1.6- to 1.7-fold), kappa-IgG (2.0- to 2.3-fold), and nonfractionated IgGs (1.7- to 1.8-fold). Ca2++Co2+ together cannot activate IgG1, while in the presence of Ca2+ + Mg2+ its activity increased by a factor of 1.6. Ca2+ + Co2+ increased the activity of IgG2 (~2.9-fold), IgG3 (~6.4-fold), and IgG4 (~6.0-fold). A significant increases in the RAs were revealed for Ca2+ + Mg2+ in the case of IgG3 (~3.5-fold), IgG4 (~4.4-fold), and IgG2 (~5.7-fold). While the Ca2+ + Mg2+ combination was the best for the activation of IgG2 and IgG1, IgG4, and IgG3 showed the highest activity in the presence of Ca2+ + Co2+. The ratios of RAs of all IgG preparations before and after their dialysis against EDTA, as well as in the presence of different metal ions, were individual for every preparation analyzed. These data indicate for an extreme Me2+-dependence diversity of different sub-

**Figure 17.** The pH dependence of RAs of MBP-hydrolyzing SLE kappa-and lambda-IgGs (A), as well IgG1, IgG2, IgG3, and IgG4 (B) [76]. Hydrolysis of MBP incubated without IgGs was used as control ("Con," A). The RAs of MBPhydrolyzing activity of SLE IgG1, IgG2, IgG3, IgG4, and total IgGmix (t-IgG) (C). The RAs were determined before (black columns) and after IgGs preincubation with PMSF (gray columns) and EDTA (white columns). The RAs of all IgGs in the

The extraordinary diversity of polyclonal abzymes with DNase, RNase, and proteolytic activities was shown not only in the case of SLE, but also other diseases [13–22]. Very unexpected enzyme properties have been discovered in the case of monoclonal abzymes of patients

classes SLE IgGs hydrolyzing MBP.

absence of the inhibitors were taken as 100%.

with SLE.

76 Lupus

**Figure 18.** Affinity chromatography on MBP-Sepharose of phage particles: (− −) and (—) absorbance at 280 nm of particles corresponding plasmid respectively without and with kappa light chains cDNA (A) [118]. Relative titres of phage particle and NaCl concentrations corresponding to various peaks are shown on panel A. The bars (B) indicate the RAs of 10 phage particles small pools of peaks 1–10 eluted from the MBP-Sepharose with different NaCl concentrations and acidic buffer (pH 2.6) (A); the reaction mixtures containing MBP (0.7 mg/ml) were incubated at 30°C for 12 h or different 1 mM OPs: OP17, OP19, OP21, and OP25 (see panel C) were incubated with 109 plaque-forming units/ml phage particles for 6 h and a complete hydrolysis of the substrates was taken as 100%. Complete MBP protein sequence and positions of four OPs sequences containing the protein cleavage sites (C).

observed for polyclonal IgGs from SLE and MS patients in the case of their chromatography on MBP-Sepharose [76, 77, 86, 87].

Phage particles eluted from MBP-Sepharose with 0.5 M NaCl (peak 7, **Figure 18A**) were used for preparation of individual colonies. Overall, 72 of 440 individual colonies choosing in a random way were used for study of MBP-hydrolyzing activity. MLChs of 22 of 72 single colonies (~30%) possess MBP-hydrolyzing activity. All 22 recombinant catalytically active MLChs containing a sequence of 6 histidine residues interacting with Ni2+ ions and 5 MLChs without activity were purified by chromatography on charged with Ni2+ ions HiTrap chelating Sepharose and by following FPLC gel filtration. Then a mixture of equal amounts of 22 catalytically active monoclonal MLChs (act-MLChmix) and second mixture of five preparations without activity (inact-MLChmix) were prepared. The electrophoretical homogeneity of ~26 to 27-kDa inact-MLChmix and act-MLChmix was shown by SDS-PAGE with silver staining (**Figure 19A**, lane 1).

MLChmix was subjected to SDS-PAGE; its proteolytic activity was revealed after extraction of proteins from the separated gel slices only in the band corresponding to the MLCh (**Figure 19A** and **B**). Act-MLChmix demonstrated activity in the hydrolysis of MBP (**Figure 19C**, lane 4), while inact-MLChmix had no activity (**Figure 19C**, lane 2). Moreover, in contrast to canonical proteases cleaving all proteins, act-MLChmix hydrolyzes only MBP (**Figure 19C**, lane 4) but no other control proteins (**Figure 19C**, lanes 5–8). All 22 act-MLChs and 5 inact-MLChs showed positive answer with mouse Abs (conjugated with horseradish peroxidase) against light chains of human Abs at Western blotting and positive ELISA response using plates with immobilized MBP.

The RAs in the hydrolysis of four different OPs were analyzed by TLC. **Figure 19(D)** and **(E)** demonstrates several typical examples of the OP19 and OP21 hydrolysis by different MLChs [118]. Initially, we have assumed that every of 22 MLChs corresponds to IgGs to one of four known specific MBP immunodominant sequences and that each MLCh can bind and hydrolyze only one of four OPs. At the same time, unexpected results were obtained. The RAs for 22 MLCh are summarized in **Figure 19(F)**. All 22 MLChs hydrolyzed only three or four OPs and with significantly different efficiency in the case of every OP. Hydrolysis of OP17 MBP was very weak (~1–1.5%) except seven MLChs: 15 ≥ 10 ≥ 12 ≥ 1 ≥ 16 ≥ 20 ≥ 8 (1.6–7.1%) (**Figure 19F**). All MLChs except MLCh-22 hydrolyzed efficiently OP21 and several other OPs, while six other MLChs (8, 9, 10, 12, 13, and 14) demonstrated high activity only in the cleavage of OP21. Several MLChs (1–7, and 11) efficiently hydrolyzed OP19 and OP21, while MLCh-18 and 20 cleaved OP21 and OP25. Four recombinant MLChs (15, 17, 19, and 21) cleaved three OPs with relatively high efficiency, while MLCh-16 hydrolyzed all four OPs (**Figure 19E**). The ratios of the RAs in the hydrolysis of four OPs were specific for every MLCh (**Figure 19E**). OP21 and OP19 were shown to be the best substrates for most MLChs, while 15–22 MLChs better hydrolyzed OP25.

In contrast to MS IgGs [76, 77, 84–87], SLE polyclonal abzymes with MBP-hydrolyzing activity are less sensitive to PMSF than to EDTA. The effect of PMSF and EDTA on the RAs of 22 different MLChs was analyzed [118]. **Figure 20(A)** shows that the 12 MLChs (1, 2, 3, 5, 7, 8, 12, 13, 15, 16, 17, and 19) are metal-dependent proteases; they cannot not remarkably decrease

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus http://dx.doi.org/10.5772/67790 79

observed for polyclonal IgGs from SLE and MS patients in the case of their chromatography

Phage particles eluted from MBP-Sepharose with 0.5 M NaCl (peak 7, **Figure 18A**) were used for preparation of individual colonies. Overall, 72 of 440 individual colonies choosing in a random way were used for study of MBP-hydrolyzing activity. MLChs of 22 of 72 single colonies (~30%) possess MBP-hydrolyzing activity. All 22 recombinant catalytically active MLChs containing a sequence of 6 histidine residues interacting with Ni2+ ions and 5 MLChs without activity were purified by chromatography on charged with Ni2+ ions HiTrap chelating Sepharose and by following FPLC gel filtration. Then a mixture of equal amounts of 22 catalytically active monoclonal MLChs (act-MLChmix) and second mixture of five preparations without activity (inact-MLChmix) were prepared. The electrophoretical homogeneity of ~26 to 27-kDa inact-MLChmix and act-MLChmix was shown by SDS-PAGE with silver staining

MLChmix was subjected to SDS-PAGE; its proteolytic activity was revealed after extraction of proteins from the separated gel slices only in the band corresponding to the MLCh (**Figure 19A** and **B**). Act-MLChmix demonstrated activity in the hydrolysis of MBP (**Figure 19C**, lane 4), while inact-MLChmix had no activity (**Figure 19C**, lane 2). Moreover, in contrast to canonical proteases cleaving all proteins, act-MLChmix hydrolyzes only MBP (**Figure 19C**, lane 4) but no other control proteins (**Figure 19C**, lanes 5–8). All 22 act-MLChs and 5 inact-MLChs showed positive answer with mouse Abs (conjugated with horseradish peroxidase) against light chains of human Abs at Western blotting and positive ELISA response using plates with

The RAs in the hydrolysis of four different OPs were analyzed by TLC. **Figure 19(D)** and **(E)** demonstrates several typical examples of the OP19 and OP21 hydrolysis by different MLChs [118]. Initially, we have assumed that every of 22 MLChs corresponds to IgGs to one of four known specific MBP immunodominant sequences and that each MLCh can bind and hydrolyze only one of four OPs. At the same time, unexpected results were obtained. The RAs for 22 MLCh are summarized in **Figure 19(F)**. All 22 MLChs hydrolyzed only three or four OPs and with significantly different efficiency in the case of every OP. Hydrolysis of OP17 MBP was very weak (~1–1.5%) except seven MLChs: 15 ≥ 10 ≥ 12 ≥ 1 ≥ 16 ≥ 20 ≥ 8 (1.6–7.1%) (**Figure 19F**). All MLChs except MLCh-22 hydrolyzed efficiently OP21 and several other OPs, while six other MLChs (8, 9, 10, 12, 13, and 14) demonstrated high activity only in the cleavage of OP21. Several MLChs (1–7, and 11) efficiently hydrolyzed OP19 and OP21, while MLCh-18 and 20 cleaved OP21 and OP25. Four recombinant MLChs (15, 17, 19, and 21) cleaved three OPs with relatively high efficiency, while MLCh-16 hydrolyzed all four OPs (**Figure 19E**). The ratios of the RAs in the hydrolysis of four OPs were specific for every MLCh (**Figure 19E**). OP21 and OP19 were shown to be the best substrates for most MLChs, while 15–22 MLChs better

In contrast to MS IgGs [76, 77, 84–87], SLE polyclonal abzymes with MBP-hydrolyzing activity are less sensitive to PMSF than to EDTA. The effect of PMSF and EDTA on the RAs of 22 different MLChs was analyzed [118]. **Figure 20(A)** shows that the 12 MLChs (1, 2, 3, 5, 7, 8, 12, 13, 15, 16, 17, and 19) are metal-dependent proteases; they cannot not remarkably decrease

on MBP-Sepharose [76, 77, 86, 87].

78 Lupus

(**Figure 19A**, lane 1).

immobilized MBP.

hydrolyzed OP25.

**Figure 19.** SDS-PAGE analysis of proteolytic activity (A) and homogeneity of act-MLChmix (7 µg) (B, lane 1) using a 5–16% gradient gel with following silver staining; the arrows indicate the positions of protein markers (B, lane 2) [118]. After electrophoresis the gel was incubated using special conditions for renaturation of act-MLChmix. The RAs in the hydrolysis of MBP (%) was determined using the extracts of 2- to 3-mm 22 gel fragments (A). The complete hydrolysis of MBP (0.7 mg/ml) after 24 h of mixture (20 µl) incubation with 15 µl of extracts was taken for 100%. SDS-PAGE analysis of MBP hydrolysis by 30 µg/ml inact-MLChmix (lane 2) and by act-MLChmix (lane 4) for 4 h; MBP incubated alone (lanes 1 and 3). The absence of detectable hydrolysis of control 0.7 mg/ml proteins by act-MLChmix is shown: human serum albumin (lane 6), human milk lactoferrin (lane 8); lanes 5 and 7 correspond to the proteins incubated alone. Lane C corresponds to standard protein markers. TLC analysis of OP19 (D) and OP21 (E) hydrolysis by different MLChs. The 1 mM OPs were incubated at 30°C for 24 h without MLChs (lanes C) or in the presence of 50 µg/ml of various MLChs (MLChs numbers are given on top of the panels) demonstrating relative activities in the hydrolysis of OP19 and OP21. Panel F shows the RAs of 22 various MLChs in the hydrolysis of OP25, OP21, OP19, and OP17.

**Figure 20.** The RAs of 22 MLChs in hydrolysis of MBP after Abzs preincubation with specific inhibitors of proteases of different type. MLChs (0.1 mg/ml) were preincubated without of other components (black bars), with 50 mM EDTA (gray bars) or with 1 mM PMSF (white bars); then aliquots of these mixtures were added to standard reaction mixtures (A and B) [118]. Several examples (C) of the RAs of MLChs having metal-dependent activity (1, 5, 12, 15, and 21) and serine-protease-like activity (4 and 11), demonstrating no iodoacetamide-dependent activity; three MLChs (10, 14, and 18) showing negative response to EDTA and PMSF. MLCh-22 demonstrating positive effects of EDTA and PMSF, but significantly decreasing its activity after preincubation with iodoacetamide. White and gray bars show, respectively, the activity before (control) and after MLChs treatment with iodoacetamide (panel C). The RAs of all 22 MLChs before their treatment with different inhibitors were taken as 100%.

their proteolytic activity after incubation with PMSF, while EDTA significantly suppresses their MBP-hydrolyzing activity.

Four MLChs (4, 6, 9, and 11) demonstrate serine-like proteolytic activity; PMSF suppressed their activity, but there was no noticeable effect of EDTA (**Figure 20B**). PMSF suppressed protease activity of three MLChs (20, 21, and 22) by ~40%, and their inhibition by EDTA was to some extent comparable, 40–60% (**Figure 20B**). Thus, three MLChs (20–22) are characterized to some extent comparable ratios of metal-dependent and serine-like protease activities. A very intriguing situation was observed for three MLChs (18, 14, and 10); EDTA and PMSF do not remarkably decreased their proteolytic activity (**Figure 20B**). No significant suppression (5–15%) of MS and SLE polyclonal MBP-hydrolyzing abzymes by specific inhibitors of thiol proteases was revealed previously [76, 77, 84–87]. However, iodoacetamide inhibited integrase hydrolyzing activities of all polyclonal IgG and IgM preparations from HIV/AIDS patients by 12–99% [97, 98]. Proteolytic activities of three MLChs (18, 14, and 10) not inhibited by EDTA and PMSF were significantly suppressed by iodoacetamide, while there was no effect on the most of MLChs with metal-dependent and serinelike activities (for example, **Figure 20C**). Thus, these three MLChs (18, 14, and 10) are thiol proteases. Interestingly, but iodoacetamide significantly suppressed the activities of MLChs 17 and 12 (**Figure 20C**), which were also significantly inhibited by EDTA (**Figure 20A**). One can suppose that MLChs 17 and 12 may be MLChs, the active sites of which contain amino acid residues corresponding to metal-dependent and thiol proteases. A very surprising data were obtained for MLCh-22; its activity was significantly suppressed not only by EDTA and PMSF (**Figure 20B**), by also iodoacetamide (**Figure 20C**). The relative number of MLChs, which activity depend on iodoacetamide is only approximately 27% of all 22 MLChs, while at the same time, several of them possess metal-dependent and serine-like activities. Therefore, the relative contribution of thiol-like protease activity to a total MBPhydrolyzing activity of polyclonal SLE and MS abzymes may be significantly lower than of Abzs with metal-dependent and serine-like proteolytic activities and, therefore, depending on the patient a relative contribution of thiol-like protease to the total activity may be about 5–15%, as found previously for polyclonal Abzs [76, 77, 84–87]. The effects of various metal ions on the protease activities of 22 MLChs were compared (**Figure 21A** and **B**; B is a continuation of A).

Seven different metal ions did not effect on the activity of MLChs with serine-like (9, 6, and 4) and thiol-dependent (18, 14, and 10) activities. Five MLChs (19, 17, 13, 8, and 2) were only slightly activated by several Me2+ ions, while Ca2+ was the best activator. Two MLCh preparations (5 and 3) were Co2+ dependent, but preparation 15 was better stimulated by Ni2+, MLCh-16 and MLCh-20 were respectively Mn2+- and Zn2+-dependent (**Figure 21**). MLChs 22 and 12 were activated by two different metal ions, Zn2+ and Ca2+. Two MLChs were activated by three different Me2 ions: MLCh-7 (Ca2+ > Zn2+ > Co2+) and MLCh-1 (Ca2+ > Ni2+ > Mg2+). In addition, MLCh-21 was activated by four (Cu2+ > Ca2+ > Co2+ > Zn2+) metal ions. These data show the extreme Me2+-dependence diversity of IgGs from SLE patients and their light chains in the hydrolysis of MBP [118].

All 22 MLCh preparations hydrolyzed efficiently MBP within a wide range of pHs (5.0–10), but in contrast to polyclonal SLE IgGs, they show mainly only one pH optimum [118]. Only the pH profile for preparation 4 demonstrates optimal pH at 5.7–5.9 and pronounced shoulder at pHs 6.5–7.5 (**Figure 21C**). The apparent *k*cat values under optimal conditions for every MLCh were estimated. The data on several characteristics of 22 various MLCh preparations are summarized in **Table 8** [118]. One can see that all MLChs demonstrate very different physicochemical and enzymatic properties.

their proteolytic activity after incubation with PMSF, while EDTA significantly suppresses

**Figure 20.** The RAs of 22 MLChs in hydrolysis of MBP after Abzs preincubation with specific inhibitors of proteases of different type. MLChs (0.1 mg/ml) were preincubated without of other components (black bars), with 50 mM EDTA (gray bars) or with 1 mM PMSF (white bars); then aliquots of these mixtures were added to standard reaction mixtures (A and B) [118]. Several examples (C) of the RAs of MLChs having metal-dependent activity (1, 5, 12, 15, and 21) and serine-protease-like activity (4 and 11), demonstrating no iodoacetamide-dependent activity; three MLChs (10, 14, and 18) showing negative response to EDTA and PMSF. MLCh-22 demonstrating positive effects of EDTA and PMSF, but significantly decreasing its activity after preincubation with iodoacetamide. White and gray bars show, respectively, the activity before (control) and after MLChs treatment with iodoacetamide (panel C). The RAs of all 22 MLChs before

Four MLChs (4, 6, 9, and 11) demonstrate serine-like proteolytic activity; PMSF suppressed their activity, but there was no noticeable effect of EDTA (**Figure 20B**). PMSF suppressed protease activity of three MLChs (20, 21, and 22) by ~40%, and their inhibition by EDTA was to some extent comparable, 40–60% (**Figure 20B**). Thus, three MLChs (20–22) are characterized to some extent comparable ratios of metal-dependent and serine-like protease activities. A very intriguing situation was observed for three MLChs (18, 14, and 10); EDTA and PMSF do not remarkably decreased their proteolytic activity (**Figure 20B**). No significant suppression (5–15%) of MS and SLE polyclonal MBP-hydrolyzing abzymes by specific

their MBP-hydrolyzing activity.

80 Lupus

their treatment with different inhibitors were taken as 100%.

On the next step we analyzed in more detail three additional MLChs (numbers 23–25) corresponding to peak 7 eluted from MBP-Sepharose with 0.5 M NaCl (**Figure 18A**) [119, 120]. These three MLChs were purified and characterized in detail exactly similar to above described 22 preparations [118]. The DNA sequence of NGTA1-Me-pro demonstrated high

**Figure 21.** Effect of various metal ions on the RAs of 22 MLChs in the hydrolysis of MBP (A and B) [118]. Black first bars correspond to the RAs in the presence of EDTA, while white bars to MLChs without external metal ions. The MLChs numbers of and type of Me2+ ions, as well as best activators of various MLChs are shown on panels A and B. Typical examples of the dependences of four MLChs in MBP hydrolysis on pH of reaction mixtures are given (C).

identity to germline VL genes of IgLV8-61\*02, IgLV8-61\*01, and IgLV8-61\*03IGKV1 (90% of identity) [120]. DNA sequence of NGTA2-Me-pro-Tr indicated high identity with germline VL gene IGKJ1\*01 (100%), IGKJ4\*01 (95.7%), IGKJ4\*02 (91.2%), IGKV1-5\*03 (87.9%), IGKV1- 5\*01 (86.2% of identity), and IGKV1-5\*02 (85.6%) [119]. DNA sequence of NGTA3-pro-DNase has similarity with germline DNA sequence of light chains of several IgGs: IGKJ1\*01 (100% of identity), IGKJ4\*01 (95.7%), IGKJ4\*02 (91.2%); IGKV1-5\*03 (79.8% of identity), IGKV1-5\*02 (78.4%), and IGKV1-5\*01 (78.4%) [Timofeeva, Nevinsky, personal communication]. Thus, all three MLChs were shown to be typical light chain of Abs [119, 120, personal communication].

NGTA1-Me-pro was shown to be a specific metalloprotease; only EDTA efficiently inhibits its activity, while specific inhibitors of thiol-, serine-, and acidic-like proteases did not suppress its MBP-hydrolyzing activity (**Figure 22A**) [120].


a For each value, a mean ± S.E. of two/three measurements is reported.

identity to germline VL genes of IgLV8-61\*02, IgLV8-61\*01, and IgLV8-61\*03IGKV1 (90% of identity) [120]. DNA sequence of NGTA2-Me-pro-Tr indicated high identity with germline VL gene IGKJ1\*01 (100%), IGKJ4\*01 (95.7%), IGKJ4\*02 (91.2%), IGKV1-5\*03 (87.9%), IGKV1- 5\*01 (86.2% of identity), and IGKV1-5\*02 (85.6%) [119]. DNA sequence of NGTA3-pro-DNase has similarity with germline DNA sequence of light chains of several IgGs: IGKJ1\*01 (100% of identity), IGKJ4\*01 (95.7%), IGKJ4\*02 (91.2%); IGKV1-5\*03 (79.8% of identity), IGKV1-5\*02 (78.4%), and IGKV1-5\*01 (78.4%) [Timofeeva, Nevinsky, personal communication]. Thus, all three MLChs were shown to be typical light chain of Abs [119, 120, personal communication]. NGTA1-Me-pro was shown to be a specific metalloprotease; only EDTA efficiently inhibits its activity, while specific inhibitors of thiol-, serine-, and acidic-like proteases did not suppress

examples of the dependences of four MLChs in MBP hydrolysis on pH of reaction mixtures are given (C).

**Figure 21.** Effect of various metal ions on the RAs of 22 MLChs in the hydrolysis of MBP (A and B) [118]. Black first bars correspond to the RAs in the presence of EDTA, while white bars to MLChs without external metal ions. The MLChs numbers of and type of Me2+ ions, as well as best activators of various MLChs are shown on panels A and B. Typical

its MBP-hydrolyzing activity (**Figure 22A**) [120].

82 Lupus

b Optimal pH of reaction mixtures and optimal metal cofactor (given in bold) were used for every of MLCh preparations; the apparent *k*cat values under optimal conditions were calculated as *k*cat = *V*max (M/min)/[MLCh] (M). MLChs were used in different concentrations (0.05–0.5 M) depending of their relative activity.

c The best metal activator is given in bold, while alternative cofactors demonstrating relatively high activation are given in parenthesis.

**Table 8.** The optimal pH values, optimal metal cofactors, and apparent *k*cat values for 22 recombinant individual MLChs in the hydrolysis of MBP [118].

Seven various metal ions increase NGTA1-Me-pro activity in the following order: Ca2+ > Mg2+ > Ni2+ ≥ Zn2+ ≥ Co2+ ≥ Mn2+ > Cu2+ (**Figure 22B**). NGTA1-Me-pro demonstrated two different very well expressed pH optima at pH 6.0 and 8.5 (**Figure 22C**). **Figure 22(D)** indicates that at pH 6.0 MLCh has optimum at ~6 mM, when at pH 8.5 at 1 mM CaCl<sup>2</sup> . The apparent values of *K*m and *k*cat for MBP in the presence of optimal CaCl<sup>2</sup> concentration at pH 6.0 (20 ± 2 µM;

**Figure 22.** The RAs of NGTA1-Me-pro in the hydrolysis of MBP before and after its preincubation with specific inhibitors of various type proteases [120]. MLCh (0.1 mg/ml) was preincubated without other components (control), or the presence of EDTA, PMSF, pepstatin, and iodoacetamide; 1.0 µl of the mixtures were added to 29 µl of MBPcontaining standard reaction mixtures (A). The RA before NGTA1-Me-pro preincubation with various inhibitors was taken as 100%. Effects of different Me2+ ions (2 mM) and EDTA on the RAs of MLCh are shown (B). Dependence of the RA upon pH of reaction mixture is shown (C). Dependence of NGTA1-Me-pro activity on CaCl2 concentration at pHs 6.0 and 8.5 (D).

0.22 ± 0.02 min−1; 6.0 mM CaCl<sup>2</sup> ) and pH 8.5 (40 ± 3 µM; 0.07 ± 0.005 min−1; 0.7 mM CaCl<sup>2</sup> ) were different. All data obtained unequivocally testified that NGTA1-Me-pro has two independent metal-dependent active centers [120].

MLCh NGTA2-Me-pro-Tr demonstrated two different activities: trypsin-like and metalloprotease. **Figure 23(A)** shows that NGTA2-Me-pro-Tr is not sensitive to pepstatin and iodoacetamide [119]. Preincubation of this MLCh with specific inhibitor of serine-like proteases results in a decrease of its activity for 42 ± 4%.

**Figure 23.** The RAs of the activity of NGTA2-Me-pro-Tr in the hydrolysis of MBP after its preincubation with specific inhibitors of different type proteases (A) [119]. MLCh (0.3 mg/ml) was preincubated alone (control), or the presence of iodoacetamide, PMSF, pepstatin, or EDTA; 1.0 µl of these mixture was added to 29 µl of MBP-containing standard reaction mixtures (A). The relative activity of NGTA2-Me-pro-Tr after preincubation with without different inhibitors (control) was taken for 100%. Effects of 10 mM EDTA and various Me2+ ions (2 mM) on the RAs of MLCh are shown (B). Dependences of the relative proteolytic activity of NGTA2-Me-pro-Tr before and after its treatment with PMSF and EDTA upon pH of reaction mixtures are shown (C). Dependence of the MBP-hydrolyzing activity on concentration of CaCl2 at pHs 6.0 and 8.5 (D).

Intact polyclonal Abs interact with various metal ions and they do not lose completely intrinsically bound ions during the standard purification procedures [121]. Addition of EDTA to NGTA2-Me-pro-Tr containing only intrinsically bound Me2+-ions results in a decrease in its activity for 58 ± 5% (**Figure 23A**) [119]. Average serine-like activity of NGTA2-Me-pro-Tr containing only intrinsically bound Me2+ ions was ~1.4-fold lower than its Me2+-dependent protease activity. Seven various external metal ions activate this MLCh in the following order: Ca2+ ≥ Mn2+ ≥ Mg2+ > Co2+> Ni2+ ≥ Cu2+ ≥ Zn2+ (**Figure 23B**). After NGTA2-Me-pro-Tr treatment with PMSF, its metalloprotease activity demonstrated pH optimum at 6.5–6.6 (**Figure 23C**). After dialysis of this MLCh against EDTA or in the presence of EDTA, serine-like protease activity showed pH optimum at 7.4–7.5. **Figure 23(D)** demonstrates that the increase in PMSF concentration results in a complete suppression of the activity at pH 7.5 in the presence of 50 mM EDTA, conditions corresponding to serine-like activity. NGTA2-Me-pro-Tr containing

0.22 ± 0.02 min−1; 6.0 mM CaCl<sup>2</sup>

6.0 and 8.5 (D).

84 Lupus

metal-dependent active centers [120].

in a decrease of its activity for 42 ± 4%.

) and pH 8.5 (40 ± 3 µM; 0.07 ± 0.005 min−1; 0.7 mM CaCl<sup>2</sup>

different. All data obtained unequivocally testified that NGTA1-Me-pro has two independent

**Figure 22.** The RAs of NGTA1-Me-pro in the hydrolysis of MBP before and after its preincubation with specific inhibitors of various type proteases [120]. MLCh (0.1 mg/ml) was preincubated without other components (control), or the presence of EDTA, PMSF, pepstatin, and iodoacetamide; 1.0 µl of the mixtures were added to 29 µl of MBPcontaining standard reaction mixtures (A). The RA before NGTA1-Me-pro preincubation with various inhibitors was taken as 100%. Effects of different Me2+ ions (2 mM) and EDTA on the RAs of MLCh are shown (B). Dependence of the RA upon pH of reaction mixture is shown (C). Dependence of NGTA1-Me-pro activity on CaCl2 concentration at pHs

MLCh NGTA2-Me-pro-Tr demonstrated two different activities: trypsin-like and metalloprotease. **Figure 23(A)** shows that NGTA2-Me-pro-Tr is not sensitive to pepstatin and iodoacetamide [119]. Preincubation of this MLCh with specific inhibitor of serine-like proteases results

) were

no intrinsic metal ions demonstrated in the absence of external metal ions at pH 7.5 *K*m and *k*cat (9.0 ± 1.0 µM, 8 ± 0.6 min−1) different as in the presence of CaCl<sup>2</sup> at pH 6.5 (24.0 ± 2.0 µM, 15.2 ± 1.1 min−1) [119]. Thus, NGTA2-Me-pro-Tr is the first example of recombinant MLCh having two combined serine-like and metalloprotease activities.

It should be emphasized that all recombinant MLChs were obtained using affinity chromatography of phage particles on MBP-Sepharose and all electrophoretically homogeneous preparations of MLChs have affinity for MBP-Sepharose; similar to phage particles homogeneous MLChs were eluded from the sorbet by 0.5 M NaCl. Taking this into account, a very unexpected result was obtained from the analysis of enzymatic activities of NGTA3-pro-DNase [Timofeeva and Nevinsky, personal communication].

The homogeneity of ~26–27-kDa NGTA3-pro-DNase was confirmed using SDS-PAGE with following silver staining (**Figure 24B**, lane 1). NGTA3-pro-DNase demonstrated positive answer with horseradish peroxidase conjugated with mouse IgGs against human Abs light chains at Western blotting and positive ELISA answer using plates with immobilized MBP and DNA.

After SDS-PAGE, MBP-hydrolyzing activity was revealed only in the band corresponding to the light chains in the presence of CaCl2 (o) and in the absence of external metal ions (□); the positions of proteolytic (o, □) and DNase (x) activities of MLCh are coincided (**Figure 24A**). NGTA3-pro-DNase hydrolyzed specifically only MBP and not hydrolyzed foreign control proteins (**Figure 24C**).

Only one (NGTA3-pro-DNase) of 25 recombinant MLChs analyzed by us efficiently hydrolyzed not only MBP, but also DNA (for example, **Figure 24D**). DNase activity of NGTA3 pro-DNase was determined *in situ* after separation of proteins using SDS-PAGE gels copolymerized with calf thymus DNA (**Figure 24E**). Ethidium bromide staining of the gels after the electrophoresis of the NGTA3-pro-DNase revealed sharp dark bands against a fluorescent background of DNA in the gel zone corresponding only to the MLCh and there were no other peaks of proteins or DNase activity (**Figure 24E**).

NGTA3-pro-DNase containing intrinsic metal ions was not sensitive to treatment with iodoacetamide and pepstatin, while its preincubation with PMSF led to decrease in the activity for 67 ± 5% (**Figure 25A**).

The dialysis of NGTA3-pro-DNase containing only intrinsically bound Me2+ ions against EDTA or addition of EDTA to reaction mixture led to a decrease in its activity for 33 ± 3% (**Figure 25A**). And average Me2+-dependent protease activity of MLCh containing only intrinsically bound Me2+ ions was approximately 2.0-fold lower (**Figure 25A**), but after addition of external Ca2+ ions became to be 2.2-fold higher than its serine-like activity (**Figure 25B**). Seven various external metal ions activate NGTA3-pro-DNase in the following order: Ca2+ ≥ Ni2+ > Co2+ ~ Mn2+ ≥ Cu2+ ~ Zn2+ ≥ Mg2+ (**Figure 25B**). An optimal concentration of CaCl2 , which is the best activator of this MLCh, was 3 mM. NGTA3-pro-DNase demonstrates two different optimal pHs (**Figure 25C**). After treatment of MLCh with PMSF, its metalloprotease activity was maximal at pH 8.6, while in the presence of EDTA, serine-like protease activity demonstrated pH optimum at 7.0 (**Figure 25B**). NGTA3-pro-DNase treated with PMSF in the presence of 3 mM CaCl<sup>2</sup> (pH 7.0) demonstrated *K*m for intact MBP (15 ± 1.1 µM) and *k*cat value 0.4 ± 0.03 min−1, while in the presence of EDTA at pH 8.6, *K*m and *k*cat values were different (45 ± 1.1 µM and 0.2 ± 0.04 min−1).

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus http://dx.doi.org/10.5772/67790 87

no intrinsic metal ions demonstrated in the absence of external metal ions at pH 7.5 *K*m and *k*cat

± 1.1 min−1) [119]. Thus, NGTA2-Me-pro-Tr is the first example of recombinant MLCh having

It should be emphasized that all recombinant MLChs were obtained using affinity chromatography of phage particles on MBP-Sepharose and all electrophoretically homogeneous preparations of MLChs have affinity for MBP-Sepharose; similar to phage particles homogeneous MLChs were eluded from the sorbet by 0.5 M NaCl. Taking this into account, a very unexpected result was obtained from the analysis of enzymatic activities of NGTA3-pro-DNase

The homogeneity of ~26–27-kDa NGTA3-pro-DNase was confirmed using SDS-PAGE with following silver staining (**Figure 24B**, lane 1). NGTA3-pro-DNase demonstrated positive answer with horseradish peroxidase conjugated with mouse IgGs against human Abs light chains at Western blotting and positive ELISA answer using plates with immobilized MBP and DNA. After SDS-PAGE, MBP-hydrolyzing activity was revealed only in the band corresponding to the

proteolytic (o, □) and DNase (x) activities of MLCh are coincided (**Figure 24A**). NGTA3-pro-DNase hydrolyzed specifically only MBP and not hydrolyzed foreign control proteins (**Figure 24C**).

Only one (NGTA3-pro-DNase) of 25 recombinant MLChs analyzed by us efficiently hydrolyzed not only MBP, but also DNA (for example, **Figure 24D**). DNase activity of NGTA3 pro-DNase was determined *in situ* after separation of proteins using SDS-PAGE gels copolymerized with calf thymus DNA (**Figure 24E**). Ethidium bromide staining of the gels after the electrophoresis of the NGTA3-pro-DNase revealed sharp dark bands against a fluorescent background of DNA in the gel zone corresponding only to the MLCh and there were

NGTA3-pro-DNase containing intrinsic metal ions was not sensitive to treatment with iodoacetamide and pepstatin, while its preincubation with PMSF led to decrease in the activity for

The dialysis of NGTA3-pro-DNase containing only intrinsically bound Me2+ ions against EDTA or addition of EDTA to reaction mixture led to a decrease in its activity for 33 ± 3% (**Figure 25A**). And average Me2+-dependent protease activity of MLCh containing only intrinsically bound Me2+ ions was approximately 2.0-fold lower (**Figure 25A**), but after addition of external Ca2+ ions became to be 2.2-fold higher than its serine-like activity (**Figure 25B**). Seven various external metal ions activate NGTA3-pro-DNase in the following order: Ca2+ ≥ Ni2+ > Co2+ ~ Mn2+ ≥

tor of this MLCh, was 3 mM. NGTA3-pro-DNase demonstrates two different optimal pHs (**Figure 25C**). After treatment of MLCh with PMSF, its metalloprotease activity was maximal at pH 8.6, while in the presence of EDTA, serine-like protease activity demonstrated pH optimum at 7.0 (**Figure 25B**). NGTA3-pro-DNase treated with PMSF in the presence of 3 mM CaCl<sup>2</sup>

7.0) demonstrated *K*m for intact MBP (15 ± 1.1 µM) and *k*cat value 0.4 ± 0.03 min−1, while in the presence of EDTA at pH 8.6, *K*m and *k*cat values were different (45 ± 1.1 µM and 0.2 ± 0.04 min−1).

(o) and in the absence of external metal ions (□); the positions of

at pH 6.5 (24.0 ± 2.0 µM, 15.2

, which is the best activa-

(pH

(9.0 ± 1.0 µM, 8 ± 0.6 min−1) different as in the presence of CaCl<sup>2</sup>

two combined serine-like and metalloprotease activities.

[Timofeeva and Nevinsky, personal communication].

no other peaks of proteins or DNase activity (**Figure 24E**).

Cu2+ ~ Zn2+ ≥ Mg2+ (**Figure 25B**). An optimal concentration of CaCl2

light chains in the presence of CaCl2

86 Lupus

67 ± 5% (**Figure 25A**).

**Figure 24.** SDS-PAGE analysis of MBP- and DNA-hydrolyzing activities (A) and homogeneity of NGTA3-pro-DNase (7 µg) using a reducing 5–16% gradient gel followed by silver staining (B, lane 1); the arrows (B, lane 2) indicate the positions of protein markers. After SDS-PAGE the gel was incubated using conditions for renaturation of NGTA3 pro-DNase. The relative MBP- and DNA-hydrolyzing activity (%) was revealed using the extracts of 2- to 3-mm gel fragments (A). The activity of NGTA3-pro-DNase corresponding to a complete hydrolysis of 0.5 mg/ml MBP (or 18 nM scDNA) after 24 h of incubation of 25 µl reaction mixture containing 10 µl of the gel extracts was taken for 100%. SDS-PAGE analysis of hydrolysis of MBP by inact-MLChmix (lane 1) or NGTA3-pro-DNase (lanes 2 and 3, different time of incubation) (C). Hydrolysis of control proteins (0.5 mg/ml) by inact-MLChmix and NGTA3-pro-DNase was analyzed: human albumin (lanes 4 and 5) and lactoferrin from human milk (lanes 6 and 7) (C). The mixtures were incubated for 6 h with inact-MLChmix (lanes 4 and 6), or NGTA3-pro-DNase (lanes 5 and 7). All lanes C correspond to different proteins incubated alone without MLChs, while lane C1- to standard protein markers. DNase activity of NGTA3-pro-DNase and two control MLCh1 and MLCh2 (10 nM) was analyzed in the presence of 5 mM MgCl2 (D); lane C corresponds to scDNA incubated alone. *In situ* assay of DNase activity of the NGTA3-pro-DNase (8 µg) after treatment with DTT (lane A) (E). DNase activity was revealed by ethidium bromide staining as a dark band on the fluorescent background. A part of the gel was stained with Coomassie R250 to show the position of the SLE IgGs before (lane 1) and after incubation with DTT (lane 2), as well as NGTA3-pro-DNase (lane 3) (E). MLCh was analyzed by Western blotting to a nitrocellulose membrane using mouse IgGs against light chains of human Abs conjugated with horsedish peroxidase (lane WB) (E).

**Figure 25.** The RAs of MBP-hydrolyzing activity of NGTA3-pro-DNase after its preincubation with specific inhibitors of different types proteases (A). MLCh (0.1 mg/ml) was preincubated alone (control), in the presence of iodoacetamide, PMSF, pepstatin, or EDTA, and then 1.5 µl added to 29 µl of standard reaction mixture (A). The RA of NGTA1-Me-pro before its preincubation with various inhibitors was taken as 100%. Effect of EDTA and different metal ions (2 mM) on the RA of MLCh is shown (B). Dependence of RA of MBP-hydrolyzing activity of NGTA1-Me-pro on pH of reaction mixture before and after its treatment with EDTA and PMSF is given (C).

It is known that Mg2+ (10 mM) is optimal cofactor of DNase I, while other Me2+ ions very weakly activate DNase I [109, 110]. Optimal concentration for Mn2+, Mg2+, and Ni2+ in activation NGTA3-pro-DNase was ~4–5 mM, for Ca2+ and Zn2+ 2 mM, while Co2+ and Cu2+ activate MLCh up to 10 mM concentration. DNase activity increased in the presence of metal ions in the following order: Mn2+ ≈ Co2+ ≥ Mg2+ > Cu2+ ≈ Ni2+ ≥ Ca2+ > Zn2+), which is completely different in comparing with that for DNases I and other recombinant MLChs analyzed.

DNase activity for NGTA3-pro-DNase in the presence of Mg2+ or Mn2+ at fixed concentration (5 mM) was increased at optimal concentrations of NaCl or KCl (30–40 mM) for only 27–28%. In optimal conditions, NGTA3-pro-DNase demonstrated well expressed optima at pH 6.5–6.6. The *K*m (2 ± 0.2 nM) and *k*cat (1.1 ± 0.1) × 10–3 min−1 values for scDNA were estimated. The affinity of NGTA3-pro-DNase for supercoiled DNA is about 3.5 orders of magnitude higher than affinity of scDNA for DNase I (*K*m = 46–58 µM [122].

#### **8. Conclusion**

It is known that Mg2+ (10 mM) is optimal cofactor of DNase I, while other Me2+ ions very weakly activate DNase I [109, 110]. Optimal concentration for Mn2+, Mg2+, and Ni2+ in activation NGTA3-pro-DNase was ~4–5 mM, for Ca2+ and Zn2+ 2 mM, while Co2+ and Cu2+ activate MLCh up to 10 mM concentration. DNase activity increased in the presence of metal ions in the following order: Mn2+ ≈ Co2+ ≥ Mg2+ > Cu2+ ≈ Ni2+ ≥ Ca2+ > Zn2+), which is completely different in comparing with that for DNases I and other recombinant MLChs

mixture before and after its treatment with EDTA and PMSF is given (C).

**Figure 25.** The RAs of MBP-hydrolyzing activity of NGTA3-pro-DNase after its preincubation with specific inhibitors of different types proteases (A). MLCh (0.1 mg/ml) was preincubated alone (control), in the presence of iodoacetamide, PMSF, pepstatin, or EDTA, and then 1.5 µl added to 29 µl of standard reaction mixture (A). The RA of NGTA1-Me-pro before its preincubation with various inhibitors was taken as 100%. Effect of EDTA and different metal ions (2 mM) on the RA of MLCh is shown (B). Dependence of RA of MBP-hydrolyzing activity of NGTA1-Me-pro on pH of reaction

DNase activity for NGTA3-pro-DNase in the presence of Mg2+ or Mn2+ at fixed concentration (5 mM) was increased at optimal concentrations of NaCl or KCl (30–40 mM) for only 27–28%.

analyzed.

88 Lupus

In several articles, it was demonstrated that polyclonal RNA-, DNA-, MBP- integrase-, and oligosaccharide-hydrolyzing antibodies of different classes and subclasses from patients with SLE, MS, AIDS, and other diseases are very catalytically heterogeneous. These abzymes can contain lambda- and kappa- types of light chains, may be of different subclasses (IgG1–IgG4), can demonstrate different affinity for specific sorbents and free antigens-substrates, very different pH optima, and may be independent or dependent on metal ions. Different abzymes can catalyze the hydrolysis of MBP, HIV integrase, and other proteins as serine-, thiol-, and acidic-like or metalloproteases. Various IgGs of four subclasses (IgG1–IgG4) and/or IgAs and IgMs from the sera of patients with autoimmune and viral diseases are catalytically active in the hydrolysis of RNA, DNA, oligosaccharides, and various proteins with their different contribution to the total activity of the Abs in the hydrolysis of these substrates in the case of every individual patient.

At the same time, the analysis of polyclonal antibodies does not allow to obtain detail characteristics of monoclonal abzymes entering to small pools of polyclonal antibodies separated by affinity chromatography on sorbents with different immobilized antigens-substrates. As it was shown on the example of polyclonal IgGs with DNase and MBP-hydrolyzing activities from sera of SLE and MS patients, elution of Abs by a NaCl concentration gradient leads to their distribution all over the chromatography profiles. In this case, each eluted Ab fraction contains abzymes with comparable affinity for immobilized ligand, but demonstrating a significant diversity of various enzymatic properties described above. These data are strong evidence of exceptional diversity abzymes in the blood of some patients with SLE, MS, and other diseases. In this regard, it should be mentioned that theoretically immune system of human can produce up to 106 different Abs against one antigen. It is evident that all theoretically possible variants of antibodies are in reality not realized and much less than one million. However, in the case of some patients, a possible number of abzymes can be very large. In our studies, we used a cDNA library only kappa light chains of Abs from three patients with SLE [111, 112, 118–120]. We have analyzed only 45 of 451 single of colonies corresponding one peak eluted from DNA-cellulose with 0.5 M NaCl and 33 of 687 colonies of peak eluded with acidic buffer. In the first case 15 of 45 (~33%) [111] and in the second 19 of 33 MLChs (58%) demonstrated DNase activity [112]. For analysis of MBP-hydrolyzing activity, we have used 72 of 440 individual colonies corresponding to phage particles eluted from MBP-Sepharose with 0.5 M NaCl; 25 of 72 MLChs (~35%) effectively hydrolyzed MBP [118–120]. Since we analyzed abzymes corresponding only one or two of ≥10 phage particles, it is obvious that the number of MLChs with DNase and MBP-hydrolyzing activity with very different enzymatic properties may be at least ≥ 500–1000.

The question is why many abzyme with nuclease and protease activities exist in SLE and other AI patients. First, immunization of autoimmune mice leads to a dramatically higher incidence of Abzs with a higher activity comparing to conventionally used normal mouse strains [51, 52]. The immune response to RNA and DNA and their complexes with histones and other proteins only partially depends on the length and sequence of nucleic acid [123, 124]. In addition, antiidiotypic Abs against the active centres of different DNA- and RNA-dependent enzymes can also possess catalytic activity. We have shown that polyclonal nuclease abzymes of autoimmune patients are usually different cocktails of Abzs against DNA and RNA and their complexes with proteins as well as antiidiotypic Abzs to active centers of various DNA- and RNA-hydrolyzing enzymes [13–22].

It is possible to explain to some extent in a similar way the exceptional diversity of abzymes hydrolyzing MBP and other proteins. At the same time, possible ways of production of monoclonal abzymes having two or even three different catalytic centers have a special interest. It should be noted that the known antigenic determinants of different proteins are usually relatively long and the active centers of some abzymes with two activities can correspond at once to variable parts of the antibodies to different contiguous parts of these determinants. One cannot exclude that metal-dependent active centers may be against specific part of protein antigenic determinants bound with one or several metal ions.

The second question is why NGTA3-pro-DNase against MBP can hydrolyze DNA. It is believed that MBP and anti-MBP Abs cannot interact with DNA or RNA. However, it was recently shown that anti-MBP IgGs can efficiently interact with nucleic acids [125]. Using quenching of MBP tryptophan fluorescence emission, we have shown that MBP bind oligonucleotides showing two *K*d values: 65 ± 5 and 250 ± 20 µM [Timofeeva and Nevinsky, personal communication]. Therefore, it is possible to suggest that 24 of 25 MLChs interacting only with MBP correspond to Abzs directly against this protein, while NGTA3-pro-DNase may be against the complex of MBP with DNA. In the latter case, it is impossible to exclude possibility of a formation of the chimeric MLChs possessing affinity for MBP and for DNA and also hydrolyzing these absolutely different substrates.

As mentioned above, DNA-hydrolyzing Bence-Jones proteins [60] and DNase abzymes of patients with SLE [59] and MS [16] are dangerous since they are cytotoxic, can penetrate to cell nuclear, and hydrolyze nuclear DNA resulting in cell apoptosis. Abzymes against vasoactive peptide are harmful since they decrease in the concentration of the peptide and have an important negative role in pathogenesis of patients with asthma [126]. RAs of DNase abzymes of patients with Hashimoto thyroiditis well correlate with different immunological and biochemical indices of this disease including a concentration of thyroid hormones, while decrease in their activity is related to decrease in thyroid gland damage and improvement of the clinical status [105]. Protease IgGs of patients with sepsis participate in the control of disseminated microvascular thrombosis and play important role in recovery from the disease [127]. Thus, various abzymes can play both a negative and positive role in the pathogenesis of SLE and other autoimmune or viral diseases. Meanwhile, it should be mentioned that in the later stages of SLE, MS, and other diseases, the blood of these patients contains abzymes not only with DNase and MBP-hydrolyzing activities, but also hydrolyzing other proteins, oligosaccharides, and lipids [13–22].

As it was shown in the example of Hashimoto thyroiditis production of harmful abzymes can be suppressed by using therapy with suppressing immune system drug plaquenil [102]. In MS and SLE, anti-MBP abzymes with proteolytic activity can attack MBP of the myelin-proteolipid sheath of axons. The established MS drug Copaxone was shown to be a specific inhibitor of abzymes with MBP-hydrolyzing activity [128]. One cannot exclude that the same drugs can be used for the treatment of SLE and other autoimmune diseases, which characterized by high level of abzymes with nuclease and MBP-hydrolyzing activities.

The presence of anti-DNA Abs is known as the main important diagnostic index for SLE. High-affinity anti-DNA Abs was recently shown to be major component of the intrathecal IgG in cerebrospinal fluid and brain of MS patients [48]. Moreover, DNase abzymes from SLE and MS patients are cytotoxic and induce cell death by apoptosis [16, 59]. The sera of patients with SLE and MS patients contain different free light chains [61, 62]. Therefore, we propose that exceptional diverse of intact antibodies and their free light chains hydrolyzing DNA, MBP, nucleotides, and polysaccharides may cooperatively all together promote important neuropathologic pathogenic mechanisms in SLE and MS.

Our data on the study of abzymes production in SLE patients associated with the change in profile differentiation of brain stem cells seem to be very important for understanding possible mechanisms of various autoimmune diseases development.

#### **Acknowledgements**

The question is why many abzyme with nuclease and protease activities exist in SLE and other AI patients. First, immunization of autoimmune mice leads to a dramatically higher incidence of Abzs with a higher activity comparing to conventionally used normal mouse strains [51, 52]. The immune response to RNA and DNA and their complexes with histones and other proteins only partially depends on the length and sequence of nucleic acid [123, 124]. In addition, antiidiotypic Abs against the active centres of different DNA- and RNA-dependent enzymes can also possess catalytic activity. We have shown that polyclonal nuclease abzymes of autoimmune patients are usually different cocktails of Abzs against DNA and RNA and their complexes with proteins as well as antiidiotypic Abzs to active

It is possible to explain to some extent in a similar way the exceptional diversity of abzymes hydrolyzing MBP and other proteins. At the same time, possible ways of production of monoclonal abzymes having two or even three different catalytic centers have a special interest. It should be noted that the known antigenic determinants of different proteins are usually relatively long and the active centers of some abzymes with two activities can correspond at once to variable parts of the antibodies to different contiguous parts of these determinants. One cannot exclude that metal-dependent active centers may be against specific part of pro-

The second question is why NGTA3-pro-DNase against MBP can hydrolyze DNA. It is believed that MBP and anti-MBP Abs cannot interact with DNA or RNA. However, it was recently shown that anti-MBP IgGs can efficiently interact with nucleic acids [125]. Using quenching of MBP tryptophan fluorescence emission, we have shown that MBP bind oligonucleotides showing two *K*d values: 65 ± 5 and 250 ± 20 µM [Timofeeva and Nevinsky, personal communication]. Therefore, it is possible to suggest that 24 of 25 MLChs interacting only with MBP correspond to Abzs directly against this protein, while NGTA3-pro-DNase may be against the complex of MBP with DNA. In the latter case, it is impossible to exclude possibility of a formation of the chimeric MLChs possessing affinity for MBP and for DNA

As mentioned above, DNA-hydrolyzing Bence-Jones proteins [60] and DNase abzymes of patients with SLE [59] and MS [16] are dangerous since they are cytotoxic, can penetrate to cell nuclear, and hydrolyze nuclear DNA resulting in cell apoptosis. Abzymes against vasoactive peptide are harmful since they decrease in the concentration of the peptide and have an important negative role in pathogenesis of patients with asthma [126]. RAs of DNase abzymes of patients with Hashimoto thyroiditis well correlate with different immunological and biochemical indices of this disease including a concentration of thyroid hormones, while decrease in their activity is related to decrease in thyroid gland damage and improvement of the clinical status [105]. Protease IgGs of patients with sepsis participate in the control of disseminated microvascular thrombosis and play important role in recovery from the disease [127]. Thus, various abzymes can play both a negative and positive role in the pathogenesis of SLE and other autoimmune or viral diseases. Meanwhile, it should be mentioned that in the later stages of SLE, MS, and other diseases, the blood of these patients contains abzymes not only with DNase and MBP-hydrolyzing activities, but also hydrolyzing other proteins,

centers of various DNA- and RNA-hydrolyzing enzymes [13–22].

90 Lupus

tein antigenic determinants bound with one or several metal ions.

and also hydrolyzing these absolutely different substrates.

oligosaccharides, and lipids [13–22].

This research was made possible by grant from the Russian Science Foundation (no 16-15- 10103 to G.A. Nevinsky).

#### **Abbreviations**



#### **Author details**

Georgy A. Nevinsky

Address all correspondence to: nevinsky@niboch.nsc.ru

Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia

#### **References**


**Author details**

92 Lupus

Georgy A. Nevinsky

**References**

Address all correspondence to: nevinsky@niboch.nsc.ru

MS Multiple sclerosis m-BSA Methylated BSA

OP Oligopeptide

RA Relative activity

Academy of Sciences, Novosibirsk, Russia

1986;**234**:1570–1573.

1986;**83**:6736–6740.

1993;**10**:229–240.

2005. 586 p.

Cell Biol. 1999;**9**:24–28.

Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian

OP-17, OP-19, OP-21, and OP-25 17–25mer oligopeptides corresponding to four known

nat-DNA and den-DNA Native and denatured DNA, respectively

SDS-PAGE SDS-polyacrylamide gel electrophoresis

SLE Systemic lupus erythematosus

MBP cleavage sites

[1] Pollack SJ, Jacobs JW, Schultz PG. Selective chemical catalysis by an antibody. Science.

[2] Tramontano A, Janda KD, Lerner RA. Catalytic antibodies. Science. 1986;**234**:1566–1570. [3] Tramontano A, Janda KD, Lerner RA. Chemical reactivity at an antibody binding site elicited by mechanistic design of a synthetic antigen. Proc Natl Acad Sci USA.

[4] Lerner RA, Tramontano A. Antibodies as enzymes. Trends Biochem Sci. 1987;**12**:427–438. [5] Stewart JD, Benkovic SJ. Recent developments in catalytic antibodies. Int Rev Immunol.

[6] Martin AB, Schultz PG. Opportunities at the interface of chemistry and biology. Trends

[7] Nevinsky GA, Semenov DV, Buneva VN. Catalytic antibodies (abzymes) induced by

[8] Keinan EE, editor. Catalytic antibodies. Germany: Wiley-VCH Verlag GmbH and Co;

[9] Paul S, Volle DJ, Beach CM, Johnson DR, Powell MJ, Massey RJ. Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody. Science. 1989;**244**:1158–1162.

stable transition-state analogs. Biochemistry (Moscow). 2000;**65**:1233–1244.


[38] Pisetsky D. Immune response to DNA in systemic lupus erythematosus. Isr Med Ass J. 2001;**3**:850–853.

[25] Hu R, Xie GY, Zhang X, Guo ZQ, Jin S. roduction and characterization of monoclonal anti-idiotypic antibody exhibiting a catalytic activity similar to carboxypeptidase A.

[26] Friboulet A, Izadyar L, Avalle B, Roseto A, Thomas D. Abzyme generation using an anti-idiotypic antibody as the "internal image" of an enzyme active site. Appl Biochem

[27] Debat H, Avalle B, Chose O, Sarde C-O, Friboulet A, Thomas D. Overpassing an aberrant V(kappa) gene to sequence an anti-idiotypic abzyme with (beta)-lactamase-like activity

[28] Hifumi E, Morihara F, Hatiuchi K, Okuda T, Nishizono A, Uda T. Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori. J Biol

[29] Andryushkova AA, Kuznetsova IA, Orlovskaya IA, Buneva VN, Nevinsky GA. Antibodies with amylase activity from the sera of autoimmune-prone MRL/MpJ-lpr

[30] Andryushkova AS, Kuznetsova IA, Orlovskaya IA, Buneva VN, Nevinsky GA. Nucleotide-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr

[31] Andryushkova AS, Kuznetsova IA, Buneva VN, Toporkova LB, Sakhno LV, Tichonova MA, Chernykh ER, Orlovskaya IA, Nevinsky GA. Formation of different abzymes in autoimmune-prone MRL-lpr/lpr mice is associated with changes in colony formation of

[32] Doronin VB, Parkhomenko TA, Korablev A, Toporkova LB, Lopatnikova JA, Alshevskaja AA, Sennikov SV, Buneva VN, Budde T, Meuth SG, Orlovskaya IA, Popova NA, Nevinsky GA. Changes in different parameters, lymphocyte proliferation, and hematopoietic progenitor colony formation in EAE mice treated with myelin oligodendrocyte

[33] Wentworth P, Liu Y, Wentworth AD, Fan P, Foley MJ, Janda KD. A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis. Proc Natl

[34] Tellier C. Exploiting antibodies as catalysts: potential therapeutic applications. Transfus

[35] Zhou YX, Karle S, Taguchi P, Planque S, Nishiyama Y, Paul S. Prospects for immuno-

[36] Zouali M. B cell tolerance to self in systemic autoimmunity. Arch Immunol Ther Exp

[37] Gabibov AG, Ponomarenko NA, Tretyak EB, Paltsev MA, Suchkov SV. Catalytic autoantibodies in clinical autoimmunity and modern medicine. Autoimmun Rev. 2006;**5**:324–330.

therapeutic proteolytic antibodies. J Immunol Meth. 2002;**269**:257–268.

haematopoetic progenitors. J Cell Mol Med. 2007;**11**:531–551.

that could have a linkage with autoimmune diseases. FASEB J. 2001;**15**:815–822.

J Biotechnol. 1998;**61**:109–115.

94 Lupus

Biotechnol. 1994;**47**:229–237.

Chem. 2008;**283**:899–907.

mice. FEBS Lett. 2006;**580**:5089–5095.

mice. Int Immunol. 2009;**21**:935–945.

glycoprotein. J Cell Mol Med. 2015;**20**:81–94.

Acad Sci USA. 1998;**95**:5971–5975.

Clin Biol. 2002;**9**:1–8.

(Warsz). 2001;**49**:361–365.


Avalle B, Tornatore P, Karavanov A, Morse HC 3rd, Thomas D, Friboulet A, Gabibov AG. Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen. Proc Natl Acad Sci USA. 2006;**103**:281–286.

[67] Parkhomenko TA, Doronin VB, Castellazzi M, Padroni M, Pastore M, Buneva VN, Granieri E, Nevinsky GA. Comparison of DNA-hydrolyzing antibodies from the cerebrospinal fluid and serum of patients with multiple sclerosis. PLoS One. 2014;**9**:e93001.

[52] Tawfik DS, Chap R, Green BS, Sela M, Eshhar Z. Unexpectedly high occurrence of catalytic antibodies in MRL/lpr and SJL mice immunized with a transition-state analog:

is there a linkage to autoimmunity?. Proc Natl Acad Sci USA. 2009;**92**:2145–2149.

the sera of patients with autoimmune diseases. FEBS Lett. 1992;**314**:259–263.

internal image method. CR Acad Sci III. 1994;**317**:819–823.

2008;**21**:233–242.

96 Lupus

Immunol. 2009;**21**:349–360.

Biotechnol. 1998;**75**:45–61.

Biol (Moscow). 1995;**29**:727–749.

8341–8344.

[53] Bronshtein IB, Shuster AM, Gololobov GV, Gromova II, Kvashuk OA, Belostotskaya K M, Alekberova ZS, Prokaeva TB, Gabibov AG. DNA-specific antiidiotypic antibodies in

[54] Crespeau H, Laouar A, Rochu D. Polyclonal DNase abzyme produced by anti-idiotypic

[55] Krasnorutskii MA, Buneva VN, Nevinsky GA. Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities. J Mol Recognit.

[56] Krasnorutskii MA, Buneva VN, Nevinsky GA. Anti-RNase antibodies against pancreatic

[57] Krasnorutskii MA, Buneva VN, Nevinsky GA. Immunization of rabbits with DNase II leads to formation of polyclonal antibodies with DNase and RNase activities. Int

[59] Kozyr AV, Kolesnikov AV, Aleksandrova ES, Sashchenko LP, Gnuchev NV, Favorov PV, Kotelnikov MA, Iakhnina EI, Astsaturov IA, Prokaeva TB, Alekberova ZS, Suchkov SV, Gabibov AG. Novel functional activities of anti-DNA autoantibodies by proteases from sera of patients with lymphoproliferative and autoimmune diseases. Appl Biochem

[60] Sinohara H, Matsuura K. Does catalytic activity of Bence-Jones proteins contribute to the pathogenesis of multiple myeloma?. Appl Biochem Biotechnol. 2000;**83**:85–94.

[61] Boiko AN, Favorova OO. Multiple sclerosis: molecular and cellular mechanisms. Mol

[63] Ikehara S, Kawamura M, Takao F. Organ-specific and systemic autoimmune diseases originate from defects in hematopoietic stem cells. Proc Natl Acad Sci USA. 1990;**87**:

[64] Dubrovskaya VV, Andryushkova AS, Kuznetsova IA, Toporkova LB, Buneva VN, Orlovskaya IA, Nevinsky GA. DNA-hydrolyzing antibodies from sera of autoimmune-

[65] Kuznetsova IA, Orlovskaya IA, Buneva VN, Nevinsky GA. Activation of DNAhydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by differ-

[66] Ponomarenko NA, Durova OM, Vorobiev II, Belogurov AA, Kurkova IN, Petrenko AG, Telegin GB, Suchkov SV, Kiselev SL, Lagarkova MA, Govorun VM, Serebryakova MV,

[62] Gusev EI, Demina TL, Boiko AN. Multiple Sclerosis. Moscow: Oil and Gas; 1997.

prone MRL/MpJ-lpr mice. Biochem (Moscow). 2003;**68**:1081–1088.

ent metal ions. Biochim Biophys Acta. 2007;**1774**:884–896.

ribonuclease A hydrolyze RNA and DNA. Int Immunol. 2008;**20**:1031–1040.

[58] Gottieb AA, Shwartz RH. Antigen-RNA interactions. Cell Immunol. 1972;**5**:341–362.


[90] Savel'ev AN, Kulminskaya AA, Ivanen DR, Nevinsky GA, Neustroev KN. Human antibodies with amylolytic activity. Trends Glycosci Glycotechnol. 2004;**16**:17–31.

[78] Bezuglova AM, Dmitrenok PS, Konenkova LP, Buneva VN, Nevinsky GA. Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with

[79] Timofeeva AM, Dmitrenok PS, Konenkova LP, Buneva VN, Nevinsky GA. Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with

[80] Baranovskii AG, Kanyshkova TG, Mogelnitskii AS, Naumov VA, Buneva VN, Gusev EI, Boiko AN, Zargarova TA, Favorova OO, Nevinsky GA. Polyclonal antibodies from blood and cerebrospinal fluid of patients with multiple sclerosis effectively hydrolyze

[81] Baranovskii AG, Ershova NA, Buneva VN, Kanyshkova TG, Mogelnitskii AS, Doronin BM, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Catalytic heterogeneity of polyclonal DNA- hydrolyzing antibodies from the sera of patients with multiple sclerosis.

[82] Baranovskii AG, Buneva VN, Doronin BM, Nevinsky GA. Innunoglobulins from blood of patients with multiple sclerosis like catalytic heterogeneous nucleases. Russian J

[83] Polosukhina DI, Garmashova NV, Tyshkevich OB, Doronin BM, Buneva VN, Nevinskii GA. Autoantibodies to myelin basic protein in patients with multiple sclerosis. Int J

[84] Polosukhina DI, Kanyshkova T, Doronin BM, Tyshkevich OB, Buneva VN, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis. J Cell Mol Med. 2004;**8**:359–368.

[85] Polosukhina DI, Buneva VN, Doronin BM, Tyshkevich OB, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Hydrolysis of myelin basic protein by IgM and IgA antibodies from the sera of patients with multiple sclerosis. Med Sci Monit. 2005;**11**:BR266–BR272.

[86] Polosukhina DI, Buneva VN, Doronin BM, Tyshkevich OB, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis. Immunol Lett. 2006;**103**:75–81.

[87] Legostaeva GA, Polosukhina DI, Bezuglova AM, Doronin BM, Buneva VN, Nevinsky GA. Affinity and catalytic heterogeneity of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with multiple sclerosis. J Cell Mol Med. 2010;**14**:699–709.

[88] Savel'ev AN, Eneyskaya EV, Shabalin KA, Filatov MV, Neustroev KN. Antibodies with

[89] Ivanen DR, Kulminskaya AA, Ershova NA, Eneyskaya EV, Shabalin KA, Savel'ev AN, Kanyshkova TG, Buneva VN, Nevinsky GA, Neustroev KN. Human autoantibodies

systemic lupus erythematosus. Peptides. 2012;**37**:69–78.

systemic lupus erythematosus. PLoS One. 2013;**8**:e51600.

DNA and RNA. Biochemistry (Moscow). 1998;**63**:1239–1248.

Immunol Lett. 2001;**76**:163–167 .

Immunorehabilatation. 2009;**11**:10–18.

amylolytic activity. Prot Pept Lett. 1999;**6**:179–184.

with amylolytic activity. Biologia. 2002;**11**:253–260.

Immunol. 2008;**2**:405–419.

98 Lupus


[118] Timofeeva AM, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein. J Mol Recognit. 2015;**28**:614–627.

[103] Ermakov EA, Smirnova LP, Parkhomenko TA, Dmitrenok PS, Krotenko NM, Fattakhov NS, Bokhan NA, Semke AV, Ivanova SA, Buneva VN, Nevinsky GA. DNA-hydrolysing activity of IgG antibodies from the sera of patients with schizophrenia. Open Biol.

[104] Akagi K, Murai K, Hirao N, Yamanaka M. Purification and properties of alkaline ribo-

[105] Blank A, Dekker CA. Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-poly-

[106] Sierakowska H, Shugar D. Mammalian nucleolytic enzymes. Prog Nucleic Acid Res

[107] Andrievskaia OA, Kanyshkova TG, Iamkovoi VI, Buneva VN, Nevinskii GA. Monoclonal antibodies to DNA hydrolyze RNA better than DNA. Dokl Akad Nauk

[108] Love JD, Hewitt RR. The relationship between human serum and human pancreatic

[110] Parkhomenko TA, Legostaeva GA, Doronin BM, Buneva VN, Nevinsky GA. IgGs containing light chains of the kappa and lambda type and of all subclasses (IgG1- IgG4) from sera of patients with multiple sclerosis hydrolyze DNA. J Mol Recognit.

[111] Kostrikina IA, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: molecular cloning of fourteen recombinant DNase monoclonal kappa light chains with different

[112] Botvinovskaya AV, Kostrikina IA, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light

[113] Archelos JJ, Storch MK, Hartung HP. The role of B cells and autoantibodies in multiple

[114] Hemmer B, Archelos JJ, Hartung HP. New concepts in the immunopathogenesis of

[115] Cross AH, Trotter JL, Lyons, J. B cells and antibodies in CNS demyelinating disease.

[116] Kalaga R, Li L, O'Dell JR, Paul S. Unexpected presence of polyreactive catalytic antibodies in IgG from unimmunized donors and decreased levels in rheumatoid arthritis.

[117] Odintsova ES, Buneva VN, Nevinsky GA. Casein-hydrolyzing activity of sIgA antibod-

chains with different catalytic properties. J Mol Recognit. 2013;**24**:450–460.

nuclease from human serum. Biochim Biophys Acta. 1976;**442**:368–378.

acrylamide gels. Biochemistry. 1981;**20**:2261–2267.

DNase I. J Biol Chem. 1979;**254**:12588–12594.

sclerosis. Ann Neurol. 2000;**47**:694–706.

J Neuroimmunol. 2001;**112**:1–14.

J Immunol. 1995;**155**:2695–2702.

multiple sclerosis. Nat Rev Neurosci. 2002;**3**:291–301.

ies from human milk. J Mol Recognit. 2005;**18**:413–421.

[109] Suck D. DNA recognition by DNase I. J Mol Recognit. 1994;**7**:65–70.

catalytic properties. Biochim Biophys Acta. 2014;**1840**:1725–1737.

2015;**5**:150064.

100 Lupus

Mol Biol. 1977;**20**:59–130.

(Russian). 1997;**355**:401–403.

2010;**23**:486–494.


## **Elimination of Nucleoproteins in Systemic Lupus Erythematosus and Antinuclear Autoantibodies Production**

Andrei S. Trofimenko

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68496

#### **Abstract**

The distinctive feature of systemic lupus erythematosus (SLE) is an immune reaction directed to diverse spectrum of autoantigens, which tends to change along with the disease spreading. The most common targets of the autoantibodies are protein and nucleoprotein components of cell nuclei: dsDNA, histones, nucleosomes, Sm antigen, and Ro and La antigens. Considering that the exact causes of this tolerance loss are unknown, a certain number of hypotheses are now discussed. One of the most promising is "waste disposal" concept, which makes a link between broken elimination of cellular debris, mononuclear phagocyte system dysfunction, and initiation of autoimmunity by the antigen presenting cells in SLE. This chapter concerns the ways nuclear antigens release from cells, necrosis, and apoptosis, as well as the key molecular mechanisms of transport and elimination of these antigens, its disturbances in SLE, and connection with innate immunity by mononuclear cells. Special attention is paid to nucleosomes and DNA degradation process, its principal factors (DNase I, C1q, SAP), blood DNA transportation by immune complexes, and immune stimulating action of DNA in SLE. Current pros and cons for the waste disposal concept and existing research trends in this field are discussed.

**Keywords:** systemic lupus erythematosus, autoantigens, DNA, nucleoproteins, DNase I, antigen cleavage

#### **1. Introduction**

Systemic lupus erythematosus (SLE) is a prototypic diffuse autoimmune disease of connective tissue with multiple organ involvement. The history of its exploration is not so long, compared with some other rheumatic diseases, such as osteoarthritis and gout. But, there is

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

surprisingly few breakthrough advances in its basic conception since the 1950s, when this condition was established as a separate autoimmune disease and glucocorticoids became a groundwork in its treatment. The absence of integral and fully consistent theory of SLE etiopathogenesis appears to be the main problem for researchers, trying to improve the treatment mainly by empirical approach.

SLE etiology and pathogenesis are generally interpreted now as a multifactorially driven autoimmune process [1]. According to this conception, SLE is induced by multiple interactions of immunological, genetic, hormonal, microbial, and environmental factors. Meanwhile, first three ones apparently play the lead [2]. Genetic predisposition to SLE is suggested to be constituted mainly by definite HLA alleles, especially DR2 and DR3, by congenital deficiency of early complement components (C1, C2, C4) and by other genetic associations, including TNF, TCR, IL-6, and other genes [3]. There are genes of C-reactive protein (CRP), C1q, Fcγreceptors, DNase I, serum amyloid P (SAP), and PDCD1 within seven loci, which are strongly linked with SLE [4]. Moreover, knocking out of these genes in mice induces autoimmune condition with glomerulonephritis [5, 6].

SLE occurs predominantly in women of childbearing age and, to a lesser extent, in prepuberta or menopause, whereupon a contribution of sex hormones could be assumed. Both men and women with SLE have high estrogen levels, the men also have low testosterone and high luteinizing hormone concentrations [7]. The connection between these deviations and SLE could be explained considering their influence on immune system cells, in particular, promotion of B cell proliferation and antibody synthesis under high estrogen levels [8].

Among all the events that can be proposed to initiate SLE onset, the leading one is suggested to be virus infection [9]. Although this "trigger agent" is not definitely identified, a wide spectrum of viruses, including Epstein-Barr virus, retroviruses, and herpesviruses, could make a substantial contribution [10]. Other influencing factors are insolation, drugs, and some pollutants.

The prominent immunological feature of SLE is the production of autoantibodies directed to a wide spectrum of self-antigens. According to Sherer et al. [11], more than 100 autoantigens, which could react with SLE-related antibodies, were mentioned in previously reported researches. However, antibodies to chromatin and its particular elements, nucleosomes, dsDNA, histones, components of DNA replication, and transcription apparatus are most representative for SLE. The second important cluster of antigens involves ribonucleoproteins and its constituents: RNA, small nuclear ribonucleoprotein (snRNP), Sm antigen, and Ro (SS-A) and La (SS-B) antigens. The third group, antiphospholipid antibodies, is common in SLE as well. Anti-DNA antibodies, and specifically anti-double-stranded DNA (dsDNA) antibodies, are thought to have most pathogenetic and diagnostic importance in SLE [12]. Their titers correlate with disease activity, and participation of anti-dsDNA antibodies in lupus nephritis is well established [13, 14].

The realization of anti-dsDNA pathogenic potential can occur by several ways. The most important contribution to systemic inflammation is generally attributed to the formation of immune complexes (IC), with both circulating and tissue-fixed antigens [15]. Nephritogenic action of ICs is mediated primarily by interaction with Fc receptors and Toll-like receptors, and, to a lesser extent, through classical pathway of complement activation [16]. In addition, autoantibodies could interfere in functioning of circulating, membrane, or even intracellular molecules [17].

However, pathogenic action is not an overall feature of anti-DNA antibodies. Both healthy individuals and SLE patients have at least two types of serum anti-DNA antibodies, unrelated directly with autoimmunity. First, there are low affinity antibodies, directed mainly against single-stranded DNA, which can be attributed to natural autoantibodies repertoire [18]. Another type consists of antibodies that are highly specific to microbial single-stranded DNA [19]. The essence of differences, influencing the pathogenic potential of these three types, is given in **Table 1**.

surprisingly few breakthrough advances in its basic conception since the 1950s, when this condition was established as a separate autoimmune disease and glucocorticoids became a groundwork in its treatment. The absence of integral and fully consistent theory of SLE etiopathogenesis appears to be the main problem for researchers, trying to improve the treatment

SLE etiology and pathogenesis are generally interpreted now as a multifactorially driven autoimmune process [1]. According to this conception, SLE is induced by multiple interactions of immunological, genetic, hormonal, microbial, and environmental factors. Meanwhile, first three ones apparently play the lead [2]. Genetic predisposition to SLE is suggested to be constituted mainly by definite HLA alleles, especially DR2 and DR3, by congenital deficiency of early complement components (C1, C2, C4) and by other genetic associations, including TNF, TCR, IL-6, and other genes [3]. There are genes of C-reactive protein (CRP), C1q, Fcγreceptors, DNase I, serum amyloid P (SAP), and PDCD1 within seven loci, which are strongly linked with SLE [4]. Moreover, knocking out of these genes in mice induces autoimmune

SLE occurs predominantly in women of childbearing age and, to a lesser extent, in prepuberta or menopause, whereupon a contribution of sex hormones could be assumed. Both men and women with SLE have high estrogen levels, the men also have low testosterone and high luteinizing hormone concentrations [7]. The connection between these deviations and SLE could be explained considering their influence on immune system cells, in particular, promo-

Among all the events that can be proposed to initiate SLE onset, the leading one is suggested to be virus infection [9]. Although this "trigger agent" is not definitely identified, a wide spectrum of viruses, including Epstein-Barr virus, retroviruses, and herpesviruses, could make a substantial contribution [10]. Other influencing factors are insolation, drugs, and some pollutants. The prominent immunological feature of SLE is the production of autoantibodies directed to a wide spectrum of self-antigens. According to Sherer et al. [11], more than 100 autoantigens, which could react with SLE-related antibodies, were mentioned in previously reported researches. However, antibodies to chromatin and its particular elements, nucleosomes, dsDNA, histones, components of DNA replication, and transcription apparatus are most representative for SLE. The second important cluster of antigens involves ribonucleoproteins and its constituents: RNA, small nuclear ribonucleoprotein (snRNP), Sm antigen, and Ro (SS-A) and La (SS-B) antigens. The third group, antiphospholipid antibodies, is common in SLE as well. Anti-DNA antibodies, and specifically anti-double-stranded DNA (dsDNA) antibodies, are thought to have most pathogenetic and diagnostic importance in SLE [12]. Their titers correlate with disease activity, and participation of anti-dsDNA antibodies in lupus nephritis is

The realization of anti-dsDNA pathogenic potential can occur by several ways. The most important contribution to systemic inflammation is generally attributed to the formation of immune complexes (IC), with both circulating and tissue-fixed antigens [15]. Nephritogenic action of ICs is mediated primarily by interaction with Fc receptors and Toll-like receptors, and, to a lesser extent, through classical pathway of complement activation [16]. In addition, autoantibodies could interfere in functioning of circulating, membrane, or even intracellular molecules [17].

tion of B cell proliferation and antibody synthesis under high estrogen levels [8].

mainly by empirical approach.

104 Lupus

condition with glomerulonephritis [5, 6].

well established [13, 14].

The way pathogenic anti-DNA antibodies appear in SLE is not well established until now. Several conjectures were made for explaining disturbed tolerance to autologous DNA. One of the hypotheses is implication of molecular mimicry, when immune response to autoantigens is induced by exogenous molecules with similar epitopes [24]. Epitope spreading mechanism may also participate in it, subsequently producing antibodies to hidden epitopes after initial reaction to major epitope [25]. Disturbance of T and/or B cellular function is third possible cause of it. Th2-polarization of CD4+ T-cellular response and predominance of Th2-associated cytokines generally distinguish SLE [26]. In addition, there is low content of regulatory CD4+ CD25+ T cells that restrict effector functions of CD4<sup>+</sup> and CD8+ T cells and diminished suppressor activity of CD8+ T cells [27, 28]. Circulating B cells are usually low, mainly due to decrease of resting subpopulations, naïve and memory B cells, being in possible connection with high levels of mature plasmocytes in bone marrow [29]. Causes and mechanisms of the lymphocyte imbalance in SLE are incompletely disclosed now, as well as its pathogenetic relevance.

For the reviewing problem, information about structure of anti-dsDNA V genes, obtained from the mouse models and SLE patients, is of particular importance. Compared to their progenitors, mature genes were found to have multiple somatic hypermutations, which lead to very high avidity of these anti-dsDNA IgG [30]. Increase of arginine, asparagine, and lysine


**Table 1.** Tentative differences of pathogenic anti-DNA antibodies in SLE [20–23].

in the Complementarity-determining regions (CDR) due to hypermutations results in high isoelectric point of the antibodies, named cationic because of it [31]. Cationic anti-dsDNAs are more nephritogenic apparently through interaction with either negatively charged elements of glomerular basement membrane or DNA-containing antigens *in situ* [32].

Both somatic hypermutations and isotype switching are distinctive features of antigendependent B cell selection by T helper cells. High avidity of these autoantibodies points out the similarity of epitopes of the relevant autoantigen to dsDNA. Meanwhile, purified homologic DNA have been considered to be poorly immunogenic in health and in SLE models for a long time [33]. In view of this contradiction, there is emerging attention to different classes of endogenous nucleoproteins as anti-dsDNA inductors in SLE.

Besides anti-DNA antibodies, anti-nucleosome antibodies are also attributed to have a special pathogenetic significance in SLE [34]. Priority of anti-nucleosome immune response compared to anti-DNA and anti-histone ones is indirectly confirmed by revelation of earlier subtype of anti-nucleosome antibodies that do not interact with both DNA and histones [35]. There is close association of these antibodies with SLE activity and the kidney involvement [36]. But, unlike anti-dsDNA, anti-nucleosome antibodies do not develop glomerular deposits in the absence of nucleosomal antigens; further perfusion of nucleosome-containing ICs through the kidneys results in appearance of linear immunoglobulin deposits along glomerular basement membrane [37]. In addition, after interaction with antinuclear antibodies, nucleosomecontaining apoptotic bodies, deposited on glomerular basement membrane or in mesangial space, turn into so-called electron-dense deposits, an attribute of IC-mediated nephritis. There is no immunoglobulin fixation in the kidneys outside these deposits [38].

In most SLE cases, serum anti-dsDNA and anti-nucleosome antibodies are presented at the same time [39]. Furthermore, chromatin immunization induces not only anti-nucleosome but also anti-dsDNA and anti-nucleosome antibodies, possibly through epitope spreading [40]. High avidity of anti-nucleosome antibodies is achieved by the same somatic hypermutations, as for anti-dsDNA production; reversion of these mutations to the initial sequence results in the loss of capability to interact with nucleoproteins and, interestingly, in obtaining antiphospholipid activity [41].

Altogether, increasing research data suggest that nucleosomes are just the best candidate antigen to induce and/or maintain production of anti-chromatin autoantibodies and to influence pathogenicity of preexisting immunoglobulins. In view of it, efficient elimination of endogenous nucleoproteins in SLE seems to be an important factor that counteracts the disease spreading.

#### **2. Normal generation and clearance of extracellular DNA**

Normal extracellular DNA concentrations are usually quite low, but the values may substantially differ depending on the detection approach and contamination of plasma with leukocytic DNA [42]. Circulating DNA is found to be not in free state but mainly as a part of mono- and oligonucleosomes; this conclusion is based upon its particular molecular weight and binding with histones [43]. Nucleosomes can release from cells during several physiological and pathological processes, namely apoptosis, necrosis, and formation of extracellular traps.

in the Complementarity-determining regions (CDR) due to hypermutations results in high isoelectric point of the antibodies, named cationic because of it [31]. Cationic anti-dsDNAs are more nephritogenic apparently through interaction with either negatively charged elements

Both somatic hypermutations and isotype switching are distinctive features of antigendependent B cell selection by T helper cells. High avidity of these autoantibodies points out the similarity of epitopes of the relevant autoantigen to dsDNA. Meanwhile, purified homologic DNA have been considered to be poorly immunogenic in health and in SLE models for a long time [33]. In view of this contradiction, there is emerging attention to different classes

Besides anti-DNA antibodies, anti-nucleosome antibodies are also attributed to have a special pathogenetic significance in SLE [34]. Priority of anti-nucleosome immune response compared to anti-DNA and anti-histone ones is indirectly confirmed by revelation of earlier subtype of anti-nucleosome antibodies that do not interact with both DNA and histones [35]. There is close association of these antibodies with SLE activity and the kidney involvement [36]. But, unlike anti-dsDNA, anti-nucleosome antibodies do not develop glomerular deposits in the absence of nucleosomal antigens; further perfusion of nucleosome-containing ICs through the kidneys results in appearance of linear immunoglobulin deposits along glomerular basement membrane [37]. In addition, after interaction with antinuclear antibodies, nucleosomecontaining apoptotic bodies, deposited on glomerular basement membrane or in mesangial space, turn into so-called electron-dense deposits, an attribute of IC-mediated nephritis. There

In most SLE cases, serum anti-dsDNA and anti-nucleosome antibodies are presented at the same time [39]. Furthermore, chromatin immunization induces not only anti-nucleosome but also anti-dsDNA and anti-nucleosome antibodies, possibly through epitope spreading [40]. High avidity of anti-nucleosome antibodies is achieved by the same somatic hypermutations, as for anti-dsDNA production; reversion of these mutations to the initial sequence results in the loss of capability to interact with nucleoproteins and, interestingly, in obtaining antiphos-

Altogether, increasing research data suggest that nucleosomes are just the best candidate antigen to induce and/or maintain production of anti-chromatin autoantibodies and to influence pathogenicity of preexisting immunoglobulins. In view of it, efficient elimination of endogenous nucleoproteins in SLE seems to be an important factor that counteracts the disease

Normal extracellular DNA concentrations are usually quite low, but the values may substantially differ depending on the detection approach and contamination of plasma with leukocytic DNA [42]. Circulating DNA is found to be not in free state but mainly as a part of mono- and

of glomerular basement membrane or DNA-containing antigens *in situ* [32].

of endogenous nucleoproteins as anti-dsDNA inductors in SLE.

is no immunoglobulin fixation in the kidneys outside these deposits [38].

**2. Normal generation and clearance of extracellular DNA**

pholipid activity [41].

spreading.

106 Lupus

Apoptosis is considered to be predetermined death followed by the removal of damaged or unnecessary cells that is genetically, morphologically, and biochemically standalone of other kinds of cell destruction [44]. An essential condition for normal course of apoptosis is cleavage and utilization of chromosomal DNA. Internucleosomal fragmentation of chromatin is performed by specific apoptotic nucleases during early phase of the process [45]. Nuclear antigens, including nucleosomes, moved then to little bulbs of cell membrane, so-called apoptotic bodies [46]. Interestingly, in some virus infections, endogenous nucleoproteins are bundled together with virions and, thereby, can be jointly presented in apoptotic bodies [47].

The next phase includes transition of aminophospholipids, phosphatidylserine, and phosphatidylethanolamine to external side of cell membrane, and their opsonization by serum proteins, especially by C-reactive protein, C1q, and serum amyloid P (SAP) [48, 49]. This complex becomes a signal to mononuclear phagocytes for recognition and uptake. Interaction of phosphatidylserine and its circulating cofactors (C1q, β<sup>2</sup> -glycoprotein I) with C1q receptor and Mer receptor of phosphatidylserine, expressed on macrophage surface, probably plays the lead in this complicated and insufficiently explored process [50, 51]. The ultimate destruction of engulfed nucleoproteins is provided by lysosomal enzymes, primarily by DNase II and cathepsins D, B, and L [52]. This way of clearance, which is supposed to be a major one, allows to keep the continuity of cell membrane as its distinctive feature and, thus, enables to prevent full-scale release of intracellular compounds to interstitial space [53]. Another peculiarity is the production of proinflammatory cytokines (TGF-β and IL-10), inhibiting antigen presentation by dendritic cells [54].

Appearance of circulating oligonucleosomes in apoptosis depends, to a large extent, on the activity of phagocytes [55]. Functional blocking of these cells in mice *in vivo* with clodronate is demonstrated to abolish plasma DNA spike after loading by apoptotic or necrotic cells [56]. Additional factor of substantial influence on DNA release is sex hormone balance, so far as above-mentioned DNA spike is much more higher in female mice compared to males and spays [57]. The causes of partial dissipation of DNA-containing substance during phagocytosis are now unsure. Tentative persistence of apoptotic cells, until their secondary necrosis and membrane disruption begin, is an alternative way of DNA release if elimination potential of mononuclear phagocytes is insufficient.

The second important source of extracellular DNA is cell necrosis. Unlike apoptosis, it is characterized by early cell membrane, proinflammatory effect as a result of different influences, and induction of dendritic cell maturation [58]. In necrosis, DNA is degraded at a later stage compared to apoptosis, with DNase I playing a considerable part in it [59].

The newly discovered and promising phenomenon, characterized by DNA release out of its natural compartment, is a formation of so-called extracellular traps. They were first found in neutrophils, thus being named neutrophil extracellular traps (NETs) [60]. NET are unusual extracellular structures, which are suggested to be a spare defense mechanism, activating when there are pathogens or particles, too big to be englobed by phagocytes [61]. In this case, large fibers, consisting of chromatin, serve as an external scaffold for immobilized enzymes, antimicrobial peptides, and ion chelators with locally high levels [62]. The components of NET, including dsDNA, histones, nucleosomes, and ribonucleoproteins, become bound to exogenous molecules when NET eliminates its target and thus may obtain new antigenic features.

In general, there is sustained release of nucleoproteins to extracellular space in health, and its rate can be considerably increased under certain conditions. Efficiency of its elimination strongly depends on circulating cofactor molecules, such as C1q, CRP, SAP, as well as DNase I and IgM [63]. They opsonize chromatin and keep it soluble, thus promoting digestion of long chromatin segments, transportation through circulation, and further recognition by macrophages [64]. The terminal points of this transfer are mononuclear phagocyte cells, primarily in the liver and spleen [65]. Overall efficiency of this elimination mechanism is quite high, since after injection of considerable amount of exogenous DNA, or after spontaneous release of endogenous nucleoproteins during hemodialysis, half-life of the DNA in circulation is within 4–15 min [66].

An alternative pathway of DNA elimination, that is just a subsidiary one in the absence of SLE, carries out by means of circulating immune complexes (CIC). Their clearance is determined principally by the activity of complement system. Binding of C1q with CIC results in the restriction of its further growth, prevention of precipitation, and induction of C3b and C4b occurrence [67]. Coupling of these molecules with CIC allows it to interact with CR1 complement receptor (CD35) of red blood cells [68]. Normal CIC transfer to macrophages of the spleen and liver presumably goes on in connection with erythrocytes, probably for prevention of CIC outflow from circulation, and the binding is more tight when CIC contains high molecular DNA (nearly 6000 kDa), then in case of shorter DNA segments (200–600 kDa) [69]. Both CIC and DNA, complexed with circulating opsonins, are captured by macrophages through Fcγ-receptors, the former alongside with CR1 cleavage [70]. However, elimination of DNA by means of CIC is much slower compared to CRP-SAP-linked DNA [71].

Apart from the elimination, binding of circulating ligands with DNA makes an obstacle for access of immune cells to nucleosome etitopes. This is especially important in view of chromatin immunology. It is generally considered that pure extracellular DNA have limited immunogenicity unless CpG motifs [72]. On the contrary, conjugation of protein with oligodeoxyribonucleotide can strongly promote interaction of the protein portion with antigen presenting cells, enhance antibody production, and presumably induce Th2-polarization [73]. From the other side, protein could serve as a carrier for oligonucleotide hapten. Circulating DNA ligands might also interfere in reaction of preexisting autoantibodies with apoptotic debris [74]. In light of all mentioned above, endogenous DNA elimination pathway, especially serum clearance mediators and mononuclear phagocytes, should be regarded in SLE.

#### **3. DNA elimination pathway in SLE**

Extracellular DNA levels in SLE patients tend to be appreciably elevated, their circulating DNA have predominantly low molecular weight and contain only human sequences [75]. It is also almost completely double-stranded and mainly included in oligonucleosomes, linked with serum proteins and immunoglobulins [76]. High plasma DNA concentration is usually associated with SLE flares and vascular involvement, being inversely correlated with antidsDNA titers, and decreases after efficient SLE treatment [77].

when there are pathogens or particles, too big to be englobed by phagocytes [61]. In this case, large fibers, consisting of chromatin, serve as an external scaffold for immobilized enzymes, antimicrobial peptides, and ion chelators with locally high levels [62]. The components of NET, including dsDNA, histones, nucleosomes, and ribonucleoproteins, become bound to exogenous molecules when NET eliminates its target and thus may obtain new antigenic features. In general, there is sustained release of nucleoproteins to extracellular space in health, and its rate can be considerably increased under certain conditions. Efficiency of its elimination strongly depends on circulating cofactor molecules, such as C1q, CRP, SAP, as well as DNase I and IgM [63]. They opsonize chromatin and keep it soluble, thus promoting digestion of long chromatin segments, transportation through circulation, and further recognition by macrophages [64]. The terminal points of this transfer are mononuclear phagocyte cells, primarily in the liver and spleen [65]. Overall efficiency of this elimination mechanism is quite high, since after injection of considerable amount of exogenous DNA, or after spontaneous release of endogenous nucleoproteins during hemodialysis, half-life of the DNA in circulation is

An alternative pathway of DNA elimination, that is just a subsidiary one in the absence of SLE, carries out by means of circulating immune complexes (CIC). Their clearance is determined principally by the activity of complement system. Binding of C1q with CIC results in the restriction of its further growth, prevention of precipitation, and induction of C3b and C4b occurrence [67]. Coupling of these molecules with CIC allows it to interact with CR1 complement receptor (CD35) of red blood cells [68]. Normal CIC transfer to macrophages of the spleen and liver presumably goes on in connection with erythrocytes, probably for prevention of CIC outflow from circulation, and the binding is more tight when CIC contains high molecular DNA (nearly 6000 kDa), then in case of shorter DNA segments (200–600 kDa) [69]. Both CIC and DNA, complexed with circulating opsonins, are captured by macrophages through Fcγ-receptors, the former alongside with CR1 cleavage [70]. However, elimination of

Apart from the elimination, binding of circulating ligands with DNA makes an obstacle for access of immune cells to nucleosome etitopes. This is especially important in view of chromatin immunology. It is generally considered that pure extracellular DNA have limited immunogenicity unless CpG motifs [72]. On the contrary, conjugation of protein with oligodeoxyribonucleotide can strongly promote interaction of the protein portion with antigen presenting cells, enhance antibody production, and presumably induce Th2-polarization [73]. From the other side, protein could serve as a carrier for oligonucleotide hapten. Circulating DNA ligands might also interfere in reaction of preexisting autoantibodies with apoptotic debris [74]. In light of all mentioned above, endogenous DNA elimination pathway, especially serum clearance mediators and mononuclear phagocytes, should be regarded in SLE.

Extracellular DNA levels in SLE patients tend to be appreciably elevated, their circulating DNA have predominantly low molecular weight and contain only human sequences [75]. It

DNA by means of CIC is much slower compared to CRP-SAP-linked DNA [71].

**3. DNA elimination pathway in SLE**

within 4–15 min [66].

108 Lupus

Functioning of the clearance mediators in SLE has some differences. Increase of disease activity does not generally combine with substantial elevation of plasma SAP and CRP levels; SAP molecular weight as well as its affinity to nucleosomes and heparin are also changeless [78]. Moreover, SAP-linked DNA levels are substantially decreased in SLE, despite elevation of total extracellular DNA; they reversely correlate with anti-dsDNA and disease activity [79]. On the contrary, plasma C1q concentrations tend to be lower in high SLE activity and in lupus nephritis, also directly correlating with CIC-linked DNA levels [80]. These changes taken one with another can be accounted for reallocation of plasma DNA pool to CIC in the presence of high-avidity anti-dsDNA. As C1q binds with both CIC and CRP-SAP-chromatin complex and participates in elimination of every type, simultaneous decrease of C1q and CIC-linked DNA is supposed to be a result of joint tissue deposition [81]. Some evidences were indeed revealed after analysis of DNA-containing CIC in SLE.

Compared to normal individuals, SLE patients commonly have elevated CIC-linked DNA concentration, which further increases along with disease activity, but its decrease is more inherent in extreme SLE flares and overt nephritis [82]. DNA from SLE CIC is double-stranded and mainly consists of fragments, which correspond to oligonucleosomes in their length, 150– 250 and 370–460 bp, compared to 20 and 30–40 bp in normal controls [83]. It is revealed in SLE that in this DNA pool CpG motifs are 5–6 times more frequent than in human genome [84]. Apart from DNA and immunoglobulins, SLE CICs contain CRP, C1q, C3b, and C4b [85].

Clearance of CIC is reduced in SLE, and their half-life negatively correlates with SLE activity and extent of lupus nephritis manifestation [86]. This might be due to either impairment of CIC transportation or disturbance of phagocytosis. Furthermore, active SLE is known to have C3/C4 hypocomplementemia and low CR1 on red blood cells, probably because of its consumption [87]. It leads to persistence of CIC mostly out of erythrocytic pool, both free and connected with other blood cells [88]. This circumstance may be the cause of increased uptake of CIC by the liver macrophages and decreased one in the spleen, revealed by injection of labeled ICs to SLE patients [89]. Another unexpected finding from this experiment is substantial reversed release of partially digested ICs outside of phagocytes, which begins 40–60 min after the injection, coinciding with internalization period [90]. The causes and mechanisms of this phenomenon are now unknown. There is single publication about tentative disturbance of interaction between Fcγ receptors and intermediate filaments of mononuclear cells in SLE, what might affect internalization [91]. It is also known that knocking out of Axl/mer/tyro3 tyrosin kinase gene in Merkd mice is followed by disturbance of apoptotic debris internalization together with development of spontaneous autoimmunity. [92].

Delivery of endogenous nucleoproteins to the resident liver and spleen macrophages is thus realized in SLE presumably by way of CIC, while circulating protein mediators are responsible for this function in health. Pathogenetic importance of this shift is not restricted only to extravasation and tissue deposition of "free" DNA-containing CIC. Apart from phagocytosis, contact of CIC with macrophage Fcγ receptors initiates synthesis of proinflammatory signals, which can induce and maintain autoimmune responses [93]. Conversely, CRP-SAP-linked DNA promotes release of cytokines and chemokines, which suppress inflammation and autoimmunity as well as raise activation threshold of dendritic cells [94].

It is supposed that immune stimulating action of DNA-containing CIC in SLE is mediated by TLR9 Toll-like receptors, together with Fcγ receptors. After CIC internalization by phagocyte, TLR9 move from endoplasmic reticulum to phagosomes and then bind with CpG motifs of DNA-IgG-FcγRII complex [95]. According to the data reported by Lövgren et al. [96] and Means et al. [97], DNA-containing IC obtained from SLE patients promote macrophages and dendritic cells *in vitro* by means of TLR9 to produce α and γ interferons, IL-8, IL-1β, IL-6, IL-18, IL-12p40, TNF, and to generate chemokine signals to peripheral mononuclear cells, immature dendritic cells, T and NK cells. IC derived from patients with rheumatoid arthritis, Sjogren's disease, and DNA-lacking IC from SLE patients does not demonstrate these effects. Treatment of the IC from SLE patients with DNase I makes cytokine and chemokine induction down by 90–100% [98]. One may conclude that abundance of "free" DNA-containing CIC could amplify inflammation in SLE both directly and indirectly.

Using gene knockout approach, a possible relation between disturbance of cell debris removal and autoantibody synthesis is managed to establish. Mice with disabled SAP, C1q, Mer, secreted IgM genes develop spontaneous autoimmune disease with glomerular lesion and production of antinuclear antibodies [99]. This connection could also appear in human SLE.

As follows from the above, additional factors, that could digest extracellular DNA, mainly DNase I, become of special importance in SLE, when ordinary clearance pathway is disabled. Results of DNase I gene knockout had been published in 2000 [100], and since then the enzyme is considered to be a mediator of DNA clearance. Earlier data about low serum DNase I activity in SLE [101, 102] made this factor even more challenging for exploration of immunological tolerance to autologous DNA.

#### **4. The DNase I riddle**

DNase I is a DNA-specific endonuclease, which participates in DNA destruction in the presence of Mg2+ or Mn2+ cations. DNase I is able to destruct single-stranded, double-stranded, and protein-bound DNA; in the latter case, DNA breakdown is performed presumably in segments, free from protein, for example, in internucleosomal connectors of chromatin or in DNA segments where expression is going on [103]. Serum DNase I is usually supposed to be synthesized in gastrointestinal tract, and normal serum nuclease activity is provided almost completely by its function [104]. Proteases enhance DNase I effect on chromatin DNA, possibly due to removal of histones or liberation of basic amino acids, histidine, arginine, and lysine, which are known to be DNase I activators [105]. In general, little is known about physiological DNase I activators, including those, by which serum DNase I activity become significantly increased shortly after injection of purified DNA *in vivo* [106]. G-actin is widely considered to be a predominant physiological DNase I inhibitor [107].

Despite extensive examination, our knowledge about DNase I functions is quite superficial. Its digestive function as a participant of pancreatic secretion is the only universally recognized one. Other possible roles, including apoptotic chromatin degradation, cellular debris removal after necrosis, destruction of DNA genome viruses and some other, need to be fully established [108–110]. An important aspect of DNase I action is the loss of antigenic properties; it can be achieved for nucleoproteins and ICs, both circulating and *in situ* [111].

The rise of interest to DNase I in SLE became after the research performed by Napirei and colleagues had been published [100]. DNase I knockout in mice led to anti-dsDNA production, glomerular IC deposition, and lupus-like glomerulonephritis pathology. SLE patients and NZB/NZW F1 lupus mice models were found to have low serum DNase I activity [112, 113]. Subsequently, it was shown with some preanalytic corrections that change of serum DNase I activity in SLE was bidirectional, with only about 30% of low enzyme activity, while other patients had moderately increased serum DNase I activity [114].

The origin of these changes is now unknown. The attempt to connect low DNase I activity with high serum actin concentrations was then rejected [114]. Numerous efforts to identify genetic changes, which can influence on the enzyme activity, resulted in very rare incidence of functionally significant gene alterations, about two per 1000 sequenced SLE patients [115–117]. Other important information, provided by geneticists, was markedly increased expression of DNase I gene in SLE [115]. One consistent explanation for it, enhanced DNase I inhibition in SLE, was challenged by Prince and colleagues [118]. Another hypothesis can be the inhibition of DNase I by specific autoantibodies, which were found by Yeh and colleagues [119]. Several factors are more likely to influence DNase I activity in SLE, as it was later shown, with about 50% cases of predominant inhibition by autoantibodies and/or actin, and the other half, impacted by unknown factor [114]. Without extensive research, this riddle is now difficult to solve.

#### **5. Conclusion**

which can induce and maintain autoimmune responses [93]. Conversely, CRP-SAP-linked DNA promotes release of cytokines and chemokines, which suppress inflammation and auto-

It is supposed that immune stimulating action of DNA-containing CIC in SLE is mediated by TLR9 Toll-like receptors, together with Fcγ receptors. After CIC internalization by phagocyte, TLR9 move from endoplasmic reticulum to phagosomes and then bind with CpG motifs of DNA-IgG-FcγRII complex [95]. According to the data reported by Lövgren et al. [96] and Means et al. [97], DNA-containing IC obtained from SLE patients promote macrophages and dendritic cells *in vitro* by means of TLR9 to produce α and γ interferons, IL-8, IL-1β, IL-6, IL-18, IL-12p40, TNF, and to generate chemokine signals to peripheral mononuclear cells, immature dendritic cells, T and NK cells. IC derived from patients with rheumatoid arthritis, Sjogren's disease, and DNA-lacking IC from SLE patients does not demonstrate these effects. Treatment of the IC from SLE patients with DNase I makes cytokine and chemokine induction down by 90–100% [98]. One may conclude that abundance of "free" DNA-containing CIC

Using gene knockout approach, a possible relation between disturbance of cell debris removal and autoantibody synthesis is managed to establish. Mice with disabled SAP, C1q, Mer, secreted IgM genes develop spontaneous autoimmune disease with glomerular lesion and production of antinuclear antibodies [99]. This connection could also appear in human SLE.

As follows from the above, additional factors, that could digest extracellular DNA, mainly DNase I, become of special importance in SLE, when ordinary clearance pathway is disabled. Results of DNase I gene knockout had been published in 2000 [100], and since then the enzyme is considered to be a mediator of DNA clearance. Earlier data about low serum DNase I activity in SLE [101, 102] made this factor even more challenging for exploration of

DNase I is a DNA-specific endonuclease, which participates in DNA destruction in the presence of Mg2+ or Mn2+ cations. DNase I is able to destruct single-stranded, double-stranded, and protein-bound DNA; in the latter case, DNA breakdown is performed presumably in segments, free from protein, for example, in internucleosomal connectors of chromatin or in DNA segments where expression is going on [103]. Serum DNase I is usually supposed to be synthesized in gastrointestinal tract, and normal serum nuclease activity is provided almost completely by its function [104]. Proteases enhance DNase I effect on chromatin DNA, possibly due to removal of histones or liberation of basic amino acids, histidine, arginine, and lysine, which are known to be DNase I activators [105]. In general, little is known about physiological DNase I activators, including those, by which serum DNase I activity become significantly increased shortly after injection of purified DNA *in vivo* [106]. G-actin is widely

considered to be a predominant physiological DNase I inhibitor [107].

immunity as well as raise activation threshold of dendritic cells [94].

could amplify inflammation in SLE both directly and indirectly.

immunological tolerance to autologous DNA.

**4. The DNase I riddle**

110 Lupus

If we could try to bring together all the facts, mentioned above, to puzzle them all into a single reasonable explanation, we will inevitably create so-called waste disposal hypothesis first published by Walport [120]. This concept defines that in SLE the most likely source of autoantigens and also leading autoimmunity inductor could be apoptotic bodies on the surface of apoptotic cells, containing almost all characteristic SLE antigens, or, as an alternative, necrotic cell debris. Another obligate condition for autoimmunity induction is postulated to be impaired clearance of the cellular "waste" and, as a consequence, antigen uptake by immature dendritic cells and their activation [121]. Several different impairments of the clearance pathway are proposed to induce SLE. Although this hypothesis seems to be consistent, and accounts for many clinical peculiarities and controversies of SLE, it has some weak points. There is no good inducible SLE model based on this concept. There is no explanation of late SLE onset, especially long after pregnancy, within this theory. The cases of spontaneous remission without glucocorticoid treatment are quite rare, despite obvious variability of "waste" generation rate. Results of treatment with DNase I are generally discouraging. An enthusiast can, however, object to it that any correct theory usually has multiple discordances at the beginning of its life. So we shall wait a little and collect pros and contras for the final assessment of this hypothesis.

#### **Author details**

Andrei S. Trofimenko

Address all correspondence to: a.s.trofimenko@mail.ru

1 Research Institute for Clinical and Experimental Rheumatology, Volgograd, Russia

2 Volgograd State Medical University, Volgograd, Russia

#### **References**


[9] Nelson P, Rylance P, Roden D, Trela M, Tugnet N. Viruses as potential pathogenic agents in systemic lupus erythematosus. Lupus. 2014;**23**(6):596–605. DOI: 10.1177/09612 03314531637

Results of treatment with DNase I are generally discouraging. An enthusiast can, however, object to it that any correct theory usually has multiple discordances at the beginning of its life. So we shall wait a little and collect pros and contras for the final assessment of this hypothesis.

1 Research Institute for Clinical and Experimental Rheumatology, Volgograd, Russia

Rheumatology. 10th ed. Philadelphia: Elsevier; 2017. pp. 1329–1344

[1] Crow MK. Etiology and pathogenesis of systemic lupus erythematosus. In: Firestein GS, Budd RC, Gabriel SE, McInnes IB, O'Dell JR, editors. Kelley and Firestein's Textbook of

[2] Ferretti C, La Cava A. Overview of the pathogenesis of systemic lupus erythematosus. In: Tsokos GC, editor. Systemic Lupus Erythematosus: Basic, Applied and Clinical Aspects.

[3] Deng Y, Tsao BP. Genes and genetics in human systemic lupus erythematosus. In: Tsokos GC, editor. Systemic Lupus Erythematosus: Basic, Applied and Clinical Aspects.

[4] Ceccarelli F, Perricone C, Borgiani P, et al. Genetic factors in systemic lupus erythematosus: Contribution to disease phenotype. Journal of Immunology Research.

[5] Botto M, Dell'Agnola C, Bygrave AE, et al. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nature Genetics. 1998;**19**(1):56–

[6] Scott RS, McMahon EJ, Pop SM, et al. Phagocytosis and clearance of apoptotic cells is

[7] Zen M, Ghirardello A, Iaccarino L, et al. Hormones, immune response, and pregnancy in healthy women and SLE patients. Swiss Medical Weekly. 2010;**140**(13–14):187–201

[8] Hughes GC, Choubey D. Modulation of autoimmune rheumatic diseases by oestrogen and progesterone. Nature Reviews Rheumatology. 2014;**10**(12):740–751. DOI: 10.1038/

mediated by MER. Nature. 2001;**411**(6834):207–211. DOI: 10.1038/35075603

Philadelphia: Elsevier; 2016. pp. 55–62. DOI: 10.1016/B978-0-12-801917-7.00008-5

Philadelphia: Elsevier; 2016. pp. 69–76. DOI: 10.1016/B978-0-12-801917-7.00010-3

**Author details**

112 Lupus

**References**

Andrei S. Trofimenko

Address all correspondence to: a.s.trofimenko@mail.ru

2 Volgograd State Medical University, Volgograd, Russia

2015;**2015**:745647. DOI: 10.1155/2015/745647

59. DOI: 10.1038/ng0598-56

nrrheum.2014.144


[35] Li T, Prokopec SD, Morrison S, et al. Anti-nucleosome antibodies outperform traditional biomarkers as longitudinal indicators of disease activity in systemic lupus erythematosus. Rheumatology (Oxford). 2015;**54**(3):449–457. DOI: 10.1093/rheumatology/keu326

[22] Goilav B, Putterman C. The role of anti-DNA antibodies in the development of lupus nephritis: A complementary, or alternative, viewpoint? Seminars in Nephrology. 2015;**35**

[23] Witte T. IgM antibodies against dsDNA in SLE. Clinical Reviews in Allergy & Immu-

[24] Aas-Hanssen K, Thompson KM, Bogen B, Munthe LA. Systemic lupus erythematosus: Molecular mimicry between anti-dsDNA CDR3 Idiotype, microbial and selfpeptides as antigens for Th cells. Frontiers in Immunology. 2015;**6**:382. DOI: 10.3389/

[25] Rigante D, Esposito S. Infections and systemic lupus erythematosus: Binding or sparring partners?. International Journal of Molecular Sciences. 2015;**16**(8):17331–17343. DOI:

[26] Talaat RM, Mohamed SF, Bassyouni IH, Raouf AA. Th1/Th2/Th17/Treg cytokine imbalance in systemic lupus erythematosus (SLE) patients: Correlation with disease activity.

[27] Barreto M, Ferreira RC, Lourenço L, et al. Low frequency of CD4+CD25+ Treg in SLE patients: A heritable trait associated with CTLA4 and TGF-beta gene variants. BMC

[28] Mak A, Kow NY. The pathology of T cells in systemic lupus erythematosus. Journal of

[29] Zhao L, Ye Y, Zhang X. B cells biology in systemic lupus erythematosus-from bench to bedside. Science China Life Sciences. 2015;**58**(11):1111–1125. DOI: 10.1007/s11427-015-4953-x

[30] Bobeck MJ, Cleary J, Beckingham JA, et al. Effect of somatic mutation on DNA binding properties of anti-DNA autoantibodies. Biopolymers. 2007;**85**(5–6):471–480. DOI:

[31] Guo W, Smith D, Aviszus K, et al. Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity. Journal of Experimental

[32] Kohro-Kawata J, Wang P, Kawata Y, et al. Highly cationic anti-DNA antibodies in patients with lupus nephritis analyzed by two-dimensional electrophoresis and immunoblotting. Electrophoresis. 1998;**19**(8–9):1511–1555. DOI: 10.1002/elps.1150190849 [33] Al Arfaj AS, Chowdhary AR, Khalil N, Ali R. Immunogenicity of singlet oxygen modified human DNA: Implications for anti-DNA antibodies in systemic lupus erythemato-

sus. Clinical Immunology. 2007;**124**(1):83–89. DOI: 10.1016/j.clim.2007.03.548

Molecular Sciences. 2015;**16**(4):7917–7931. DOI: 10.3390/ijms16047917

[34] Yap DY, Lai KN. Pathogenesis of renal disease in systemic lupus erythematosus: The role of autoantibodies and lymphocytes subset abnormalities. International Journal of

nology. 2008;**34**(3):345–347. DOI: 10.1007/s12016-007-8046-x

Cytokine. 2015;**72**(2):146–153. DOI: 10.1016/j.cyto.2014.12.027

Immunology Research. 2014;**2014**:419029. DOI: 10.1155/2014/419029

Medicine. 2010;**207**(10):2225–2237. DOI: 10.1084/jem.20092712

Immunology. 2009;**10**:5. DOI: 10.1186/1471-2172-10-5

(5):439–443

114 Lupus

fimmu.2015.00382

10.1002/bip.20691

10.3390/ijms160817331


[64] Gregory CD, Pound JD. Microenvironmental influences of apoptosis *in vivo* and *in vitro*. Apoptosis. 2010;**15**(9):1029–1049. DOI: 10.1007/s10495-010-0485-9

[49] Kinchen JM. A model to die for: Signaling to apoptotic cell removal in worm, fly and

[50] Peter C, Wesselborg S, Herrmann M, Lauber K. Dangerous attraction: Phagocyte recruitment and danger signals of apoptotic and necrotic cells. Apoptosis. 2010;**15**(9):1007–

[51] Biermann MH, Maueröder C, Brauner JM, et al. Surface code – biophysical signals for apoptotic cell clearance. Physical Biology. 2013;**10**(6):065007. DOI: 10.1088/1478-3975/

[52] Kawane K, Nagata S. Nucleases in programmed cell death. Methods in Enzymology.

[53] Poon IK, Lucas CD, Rossi AG, Ravichandran KS. Apoptotic cell clearance: Basic biology and therapeutic potential. Nature Reviews Immunology. 2014;**14**(3):166–180. DOI:

[54] Biermann MH, Veissi S, Maueröder C. The role of dead cell clearance in the etiology and pathogenesis of systemic lupus erythematosus: Dendritic cells as potential targets. Expert Review of Clinical Immunology. 2014;**10**(9):1151–1164. DOI:

[55] Arandjelovic S, Ravichandran KS. Phagocytosis of apoptotic cells in homeostasis. Nature

[56] Jiang N, Reich CF 3rd, Pisetsky DS. Role of macrophages in the generation of circulating blood nucleosomes from dead and dying cells. Blood. 2003;**102**(6):2243–2250

[57] Pisetsky DS, Jiang N. The generation of extracellular DNA in SLE: The role of death and sex. Scandinavian Journal of Immunology. 2006;**64**(3):200–204. DOI: 10.1111/j.1365-3083.

[58] Podolska MJ, Biermann MH, Maueröder C. Inflammatory etiopathogenesis of systemic lupus erythematosus: An update. Journal of Inflammation Research. 2015;**8**:161–171.

[59] Krysko DV, D'Herde K, Vandenabeele P. Clearance of apoptotic and necrotic cells and

[60] Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacte-

[61] Zawrotniak M, Rapala-Kozik M. Neutrophil extracellular traps (NETs) - formation and

[62] Gupta S, Kaplan MJ. The role of neutrophils and NETosis in autoimmune and renal diseases. Nature Reviews Nephrology. 2016;**12**(7):402–413. DOI: 10.1038/nrneph.2016.71

[63] Park B, Lee J, Moon H, et al. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells. Cell Death & Disease. 2015;**6**:e1772. DOI: 10.1038/

its immunological consequences. Apoptosis. 2006;**11**(10):1709–1726

implications. Acta Biochimica Polonica. 2013;**60**(3):277–284

mouse. Apoptosis. 2010;**15**(9):998–1006. DOI: 10.1007/s10495-010-0509-5

1028. DOI: 10.1007/s10495-010-0472-1

2008;**442**:271–287. DOI: 10.1016/S0076-6879(08)01414-6

Immunology. 2015;**16**(9):907–917. DOI: 10.1038/ni.3253

10/6/065007

116 Lupus

10.1038/nri3607

2006.01822.x

cddis.2015.140

DOI: 10.2147/JIR.S70325

ria. Science. 2004;**303**:1532–1535

10.1586/1744666X.2014.944162


[91] Vázquez-Doval J, Sánchez-Ibarrola A. Defective mononuclear phagocyte function in systemic lupus erythematosus: Relationship of FcRII (CD32) with intermediate cytoskeletal filaments. Journal of Investigational Allergology and Clinical Immunology. 1993;**3**(2):86–91

[77] Zborovskaya IA, Trofimenko AS, Gontar IP, et al. Prospects of extracorporeal biological therapy of systemic lupus erythematosus using the composite adsorbents. Kremlevskaya

[78] Firooz N, Albert DA, Wallace DJ, et al. High-sensitivity C-reactive protein and erythrocyte sedimentation rate in systemic lupus erythematosus. Lupus. 2011;**20**(6):588–597

[79] Voss A, Nielsen EH, Svehag SE, Junker P. Serum amyloid P component-DNA complexes are decreased in systemic lupus erythematosus: Inverse association with anti-dsDNA

[80] Fenton K. The effect of cell death in the initiation of lupus nephritis. Clinical & Experi-

[81] Truedsson L, Bengtsson AA, Sturfelt G. Complement deficiencies and systemic lupus erythematosus. Autoimmunity. 2007;**40**(8):560–566. DOI: 10.1080/08916930701510673

[82] Nezlin R. A quantitative approach to the determination of antigen in immune com-

[83] Nezlin R, Alarcón-Segovia D, Shoenfeld Y. Immunochemical determination of DNA in immune complexes present in the circulation of patients with systemic lupus erythema-

[84] Sano H, Takai O, Harata N, et al. Binding properties of human anti-DNA antibodies to cloned human DNA fragments. Scandinavian Journal of Immunology. 1989;**30**(1):51–63

[85] Pradhan V, Rajadhyaksha A, Mahant G, et al. Anti-C1q antibodies and their association with complement components in Indian systemic lupus erythematosus patients. Indian

[86] Kavai M, Szegedi G. Immune complex clearance by monocytes and macrophages in

[87] Julkunen H, Ekblom-Kullberg S, Miettinen A. Nonrenal and renal activity of systemic lupus erythematosus: A comparison of two anti-C1q and five anti-dsDNA assays and complement C3 and C4. Rheumatology International. 2012;**32**(8):2445–2451. DOI:

[88] Elkon KB, Santer DM. Complement, interferon and lupus. Current Opinion in Immu-

[89] Davies KA, Peters AM, Beynon HL, Walport MJ. Immune complex processing in patients with systemic lupus erythematosus. *In vivo* imaging and clearance studies. Journal of

[90] Davies KA, Robson MG, Peters AM, et al. Defective Fc-dependent processing of immune complexes in patients with systemic lupus erythematosus. Arthritis & Rheumatology.

Journal of Nephrology. 2012;**22**(5):353–357. DOI: 10.4103/0971-4065.103911

systemic lupus erythematosus. Autoimmunity Reviews. 2007;**6**(7):497–502

nology. 2012;**24**(6):665–670. DOI: 10.1016/j.coi.2012.08.004

Clinical Investigation. 1992;**90**(5):2075–2083. DOI: 10.1172/JCI116090

tosus. Journal of Autoimmunity. 1998;**11**(5):489–493. DOI: 10.1006/jaut.1998.0231

antibodies. Journal of Rheumatology. 2008;**35**(4):625–630

mental Immunology. 2015;**179**(1):11–16. DOI: 10.1111/cei.12417

plexes. Journal of Immunological Methods. 2000;**237**(1–2):1–17

medicina. 2013;**3**:85–89 [in Russian]

118 Lupus

10.1007/s00296-011-1962-3

2002;**46**(4):1028–1038


[117] Bodaño A, González A, Ferreiros-Vidal I, et al. Association of a non-synonymous single-nucleotide polymorphism of DNASEI with SLE susceptibility. Rheumatology (Oxford). 2006;45(7):819–823. DOI: 10.1093/rheumatology/kel019

[104] Fujihara J, Yasuda T, Ueki M, et al. Comparative biochemical properties of vertebrate deoxyribonuclease I. Comparative Biochemistry and Physiology Part B: Biochemistry

and Molecular Biology. 2012;163(3–4):263–273. DOI: 10.1016/j.cbpb.2012.07.002

[106] Mazurik VK, Moskaliova EU. Aspartate carbamoyltransferase, DNA polymerase, and DNase activities in rat hemopoietic tissues after single DNA injection. Bulletin of

[107] Yokota E. Isolation of actin and actin-binding proteins. Methods in Molecular Biology.

[108] Nikiforov ND, Mamontov SG, Ilnitsky Yu A, et al. Treatment of acute hepatitis B with

[109] Peer V, Abu Hamad R, Berman S, Efrati S. Renoprotective effects of DNAse-I treatment in a rat model of ischemia/reperfusion-induced acute kidney injury. American Journal

[110] Koyama R, Arai T, Kijima M, et al. DNase γ, DNase I and caspase-activated DNase cooperate to degrade dead cells. Genes to Cells. 2016;21(11):1150–1163. DOI: 10.1111/

[111] Lefkowith JB, Kiehl M, Rubenstein J, et al. Heterogeneity and clinical significance of glomerular-binding antibodies in systemic lupus erythematosus. Journal of Clinical

[112] Macanovic M, Lachmann PJ. Measurement of deoxyribonuclease I (DNase) in the serum and urine of systemic lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial enzyme diffusion assay. Clinical & Experimental Immunology.

[113] Sallai K, Nagy E, Derfalvy B, et al. Antinucleosome antibodies and decreased deoxyribonuclease activity in sera of patients with systemic lupus erythematosus. Clinical and Diagnostic Laboratory Immunology. 2005;12(1):56–59. DOI: 10.1128/

[114] Trofimenko AS, Gontar IP, Zborovsky AB, Paramonova OV. Anti-DNase I antibodies in systemic lupus erythematosus: Diagnostic value and share in the enzyme inhibition. Rheumatology International. 2016;36(4):521–529. DOI: 10.1007/s00296-016-3437-z [115] Feng XB, Shen N, Qian J, et al. Single nucleotide polymorphisms of deoxyribonucle-

[116] Yasutomo K, Horiuchi T, Kagami S, et al. Mutation of DNASE1 in people with systemic lupus erythematosus. Nature Genetics. 2001;28(4):313–314. DOI: 10.1038/91070

ase I and their expression in Chinese systemic lupus erythematosus patients. Chinese

[105] Shapot VS. Nucleases. Moscow: Meditsina Publishers; 1968. p. 212 [in Russian]

Experimental Biology and Medicine. 1974;77(2):32–35 [in Russian]

deoxyribonuclease. Sovetskaia Meditsina. 1990;7:82–83 [in Russian]

of Nephrology. 2016;43(3):195–205. DOI: 10.1159/000445546

gtc.12433

120 Lupus

1997;108(2):220–226

CDLI.12.1.56-59.2005

Investigation. 1996;98(6):1373–1380

Medical Journal (England). 2004;**117**(11):1670–1676

2017;1511:291–299. DOI: 10.1007/978-1-4939-6533-5\_23


## **T Regulatory Cells in Systemic Lupus Erythematosus: Current Knowledge and Future Prospects**

Konstantinos Tselios, Alexandros Sarantopoulos, Ioannis Gkougkourelas and Panagiota Boura

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68479

#### **Abstract**

Systemic lupus erythematosus (SLE) is one of the most diverse autoimmune diseases, regarding clinical manifestations and therapeutic management. Visceral involvement is often and is generally associated with increased mortality and/or permanent disability. Thus, a reliable assessment of disease activity is required in order to follow‐up disease activity and apply appropriate therapy. Several serological indexes have been studied due to their competence in assessing disease activity in SLE. Apart from conventional and currently assessed serological indexes, regulatory T cells (Tregs), a CD4+ cellular population of the acquired immune compartment with homeostatic phenotype, are cur‐ rently under intense investigation in SLE. In this chapter, Tregs ontogenesis and sub‐ populations are discussed focusing on their implications in immunopathophysiology of SLE. The authors present data indicating that this CD4+ population is highly associated with disease activity and response to treatment, concluding that Tregs are a promising biomarker in SLE. Future prospective includes Tregs implication in SLE therapeutic interventions.

**Keywords:** regulatory T cells, systemic lupus erythematosus, SLE immunopathphysiology, Treg therapy

#### **1. Introduction**

Systemic lupus erythematosus represents the prototype of autoimmune diseases and is characterized by an unparalleled variety of clinical and laboratory manifestations. From a pathogenetic perspective, a breakdown of immune tolerance will lead to the proliferation and functional differentiation of certain effector cells of the innate and adaptive immunity, such as

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

neutrophils, dendritic cells (DCs), macrophages, and auto‐reactive lymphocytes [1, 2]. The net result will be the production of pro‐inflammatory cytokines and autoantibodies, formation of immune‐complexes and, eventually, tissue damage driven by the deposition of these com‐ plexes onto certain tissues and the activation of the complement cascade; other mechanisms have also been described, such as autoantibody‐ and cell‐mediated toxicity. Tissue damage will, in turn, provide the substrate for neo‐epitope formation or the revealing of cryptic epit‐ opes; this will further amplify the autoimmune response. Given the clinical diversity of SLE, several studies investigating the molecular mechanisms of the disease have yielded contra‐ dictory findings regarding multiple cellular subpopulations or soluble mediators. These find‐ ings seem to be influenced by disease duration, global disease activity, therapeutic variables, and other confounders [2]. Among them, an impairment of the mechanisms of the peripheral immune tolerance, mainly represented by the T regulatory cells (Tregs), has been universally documented in SLE and considered to be a crucial factor in disease pathogenesis.

#### **1.1. T regulatory cells**

Tregs represent a subpopulation of the CD4+ T lymphocytes which were first described in the 1970s [3] as suppressor cells since their primary function was the suppression of the immune response [4]. At that time, the term 'infectious tolerance' was introduced to describe the acqui‐ sition of suppressive capacity of non‐suppressor cells from Tregs with an, as yet, unknown mechanism [5]. The study of this cellular subpopulation was initially abandoned due to tech‐ nical difficulties with regard to the isolation and analysis of these cells because of the lack of specific surface markers [6, 7].

Research interest in suppressor T cells re‐emerged in 1995, when Sakaguchi et al. described the intense expression of the α chain of the IL‐2 receptor (IL‐2Rα, CD25) on their surface [8]. These cells were then called regulatory T cells since their function was the multifaceted regu‐ lation of the immune response and the maintenance of immune homeostasis [9]. During the next few years, several investigators showed that these cells are characterized by a unique functional phenotype, which is marked, not only by the over‐expression of the CD25, but also from decreased responsiveness after polyclonal stimulation of their T cell receptor (TCR) [10, 11]. These studies suggested that their regulatory/suppressive capacity against the effector T cells was irrespective of the antigen that generated the initial activation of the effector cells (non‐antigen specific and, thus, non‐MHC restricted).

The demonstration of the high surface expression of the CD25 molecule led to the character‐ ization and distinction of Tregs from other subsets of T lymphocytes, as well as to the dis‐ covery of their thymic origin and initial functional differentiation [12]. However, it was later shown that CD25 is not exclusively expressed in Tregs. Other recently activated T lympho‐ cytes and all T cells with regulatory function *in vitro* were also expressing this molecule [13]. As might be expected, Tregs do express the highest levels of CD25 (CD25high) as compared to the conventional CD4+ T cells, in which its expression is transient and of low intensity. Based on flow cytometric analysis, it has been shown that, among CD4+CD25+ cells, only those at the upper 2% of CD25 expression possess suppressive capacity [14].

In 2001, the gene FOΧP3 (Forkhead Box P3) was discovered in mice; its mutations were leading to the spontaneous development of autoimmune phenomena [15]. Mutations of the human FOXP3 have been associated with two distinct systemic autoimmune syndromes, namely the IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X‐linked syndrome) and XLAAD (X‐linked, autoimmunity, allergy, dysregulation) [16–18]. In 2003, it was proven that FOΧP3 is the master regulator for the functional differentiation of Tregs and is required for their proliferation [19]. It is found in the Χ chromosome (Xq11.23‐Xq13.3) and consists of 11 exons that code a 48‐kDa protein with 431 amino acids [18]. FOXP3 is mainly expressed in the T lymphocytes (mainly those that bear the αβTCR), whereas it is hardly detectable in B cells, γδ T cells, NK, macrophages, and dendritic cells. It is considered the lineage‐specification fac‐ tor of the natural T regulatory cells (nTregs).

neutrophils, dendritic cells (DCs), macrophages, and auto‐reactive lymphocytes [1, 2]. The net result will be the production of pro‐inflammatory cytokines and autoantibodies, formation of immune‐complexes and, eventually, tissue damage driven by the deposition of these com‐ plexes onto certain tissues and the activation of the complement cascade; other mechanisms have also been described, such as autoantibody‐ and cell‐mediated toxicity. Tissue damage will, in turn, provide the substrate for neo‐epitope formation or the revealing of cryptic epit‐ opes; this will further amplify the autoimmune response. Given the clinical diversity of SLE, several studies investigating the molecular mechanisms of the disease have yielded contra‐ dictory findings regarding multiple cellular subpopulations or soluble mediators. These find‐ ings seem to be influenced by disease duration, global disease activity, therapeutic variables, and other confounders [2]. Among them, an impairment of the mechanisms of the peripheral immune tolerance, mainly represented by the T regulatory cells (Tregs), has been universally

documented in SLE and considered to be a crucial factor in disease pathogenesis.

Tregs represent a subpopulation of the CD4+ T lymphocytes which were first described in the 1970s [3] as suppressor cells since their primary function was the suppression of the immune response [4]. At that time, the term 'infectious tolerance' was introduced to describe the acqui‐ sition of suppressive capacity of non‐suppressor cells from Tregs with an, as yet, unknown mechanism [5]. The study of this cellular subpopulation was initially abandoned due to tech‐ nical difficulties with regard to the isolation and analysis of these cells because of the lack of

Research interest in suppressor T cells re‐emerged in 1995, when Sakaguchi et al. described the intense expression of the α chain of the IL‐2 receptor (IL‐2Rα, CD25) on their surface [8]. These cells were then called regulatory T cells since their function was the multifaceted regu‐ lation of the immune response and the maintenance of immune homeostasis [9]. During the next few years, several investigators showed that these cells are characterized by a unique functional phenotype, which is marked, not only by the over‐expression of the CD25, but also from decreased responsiveness after polyclonal stimulation of their T cell receptor (TCR) [10, 11]. These studies suggested that their regulatory/suppressive capacity against the effector T cells was irrespective of the antigen that generated the initial activation of the effector cells

The demonstration of the high surface expression of the CD25 molecule led to the character‐ ization and distinction of Tregs from other subsets of T lymphocytes, as well as to the dis‐ covery of their thymic origin and initial functional differentiation [12]. However, it was later shown that CD25 is not exclusively expressed in Tregs. Other recently activated T lympho‐ cytes and all T cells with regulatory function *in vitro* were also expressing this molecule [13]. As might be expected, Tregs do express the highest levels of CD25 (CD25high) as compared to the conventional CD4+ T cells, in which its expression is transient and of low intensity. Based on flow cytometric analysis, it has been shown that, among CD4+CD25+ cells, only those at

In 2001, the gene FOΧP3 (Forkhead Box P3) was discovered in mice; its mutations were leading to the spontaneous development of autoimmune phenomena [15]. Mutations of the human

**1.1. T regulatory cells**

124 Lupus

specific surface markers [6, 7].

(non‐antigen specific and, thus, non‐MHC restricted).

the upper 2% of CD25 expression possess suppressive capacity [14].

The respective transcription factor FOXP3 is highly expressed in the CD4+CD25high T cells, while its early activation in the naive T cells drives their differentiation toward a regulatory phenotype. This is particularly detected under inflammatory conditions where CD4+CD25‐ T cells overexpress FOXP3, which in turn leads to the increased surface expression of other molecules, such as CTLA‐4 (cytotoxic T lymphocyte‐associated antigen 4, CD152) and GITR (glucocorticoid‐induced TNF receptor‐a family‐related protein) [20, 21]. These cells now pos‐ sess suppressive capacity; they secrete less IL‐2 and proliferate slowly.

Further research revealed that, like CD25, the expression of FOXP3 is not confined to natu‐ rally occurring Tregs; actually, it could be induced in recently activated cells (in low intensity) and CD4+ T cells that acquire suppressive properties afterward [22]. However, based on its critical importance, all cells bearing the FOXP3 key regulator are considered to be regulatory in function. FOXP3+ Tregs are divided in natural and inducible cells, according to their origin (thymus and/or periphery, respectively). The most well‐studied subgroups of the inducible Tregs (iTregs) are the Tr1, Th3, and CD8+ Tregs (**Figure 1**).

**Figure 1.** Natural (thymus derived) and inducible (peripheral) Tregs.

#### **1.2. Natural Tregs (nTregs)**

Thymic‐derived Tregs or natural Tregs are characterized by the CD4+CD25highFOXP3+ phe‐ notype and range between 1 and 3% of the peripheral CD4+ T lymphocytes [13, 23]. They are considered to maintain an anergic state (based on the findings of decreased responsiveness to antigen stimulation and limited proliferation capacity), nTregs have remarkable prolifera‐ tive potential, both *in vitro* and *in vivo*, upon antigen stimulation in the presence of dendritic cells [24]. Reciprocally, Tregs are able to induce tolerogenic DCs, further complicating their interactions with these cells [7, 12].

nTregs express the same αβTCR as the conventional T lymphocytes but they comprise a dis‐ tinct clone [25]. They derive from pluripotent stem cells and differentiate in the thymic cortex through a positive selection process after the linkage of their TCR with self‐antigens with intermediate affinity [26, 27]. These antigens are presented through MHC II molecules from the thymic cortical cells [28]. Co‐stimulation via the CD28 molecule induces the FOXP3 pro‐ moter either directly or through other genes that increase its activation [29]. It seems that the selection of these CD25+ cells occurs according to a predefined ratio to the respective CD25− cells, which have been generated earlier. They are long‐lived cells capable of producing anti‐ apoptotic molecules that protect them from the process of negative selection [26, 27].

Upon migration to the periphery, nTregs maintain their regulatory phenotype and suppres‐ sive capacity, which are mediated through cell‐to‐cell contact. This mechanism involves certain surface molecules, and it is independent of secreted cytokines [13, 26]. Survival in the periphery is facilitated by CD28 and its respective ligands (CD80, CD86), transforming growth factor β (TGF‐β) and IL‐2 [12].

Several surface molecules have been considered to allow the laboratory isolation from other cellular subpopulations and are crucial for their function. The most important such molecules are the CD4, CD25high, CD127low, CD45RO and CD45RBlow, providing a phenotype of activated memory cells [30].

Moreover, nTregs express other activation markers, such as CD28, CTLA‐4, GITR and che‐ mokine receptors, which are implicated in their migration and trafficking [30, 31]. They also express several Toll‐like receptors (TLRs), TGF‐β, neuropilin‐1, perforin and granzymes, L‐selectin (CD62L), LAG‐3 (lymphocyte activation gene‐3, CD223) and the folate receptor FR4 [32–35]. Multiple adhesion molecules are also abundantly expressed on their surface, such as CD11a, CD44, CD54, and CD103 [36]. All the aforementioned markers have been described in other cell types, which are not exclusively expressed in nTregs and cannot be used as differentiation markers.

Other markers that are thought to be highly specific for nTregs were discovered from the Ikaros gene family; the respective transcription factor is called Helios and is preferentially expressed in nTregs as compared to other CD4+ T cells [37].

Recently, it has been demonstrated that certain epigenetic mechanisms are implicated in the regulation and maintenance of FOXP3 expression [38]. In this regard, the methylation state of the Treg‐specific demethylated region (TSDR, a conserved non‐coding sequence in the CNS2 region of the FOXP3 gene) plays a crucial role. Current isolation techniques require this method since only CD4+CD25+FOXP3+ T cells with demethylated TSDR were capable of strongly and permanently expressing FOXP3 and suppressing effector cells [39]. TSDR is incompletely hypomethylated in Tregs that are induced in the periphery and completely methylated in all other CD4+CD25+ T cells [38]. Helios+FOXP3+ Tregs have increased sup‐ pressive potential and are fully demethylated at the TSDR region [37].

#### **1.3. Inducible or adaptive Tregs (iTregs)**

**1.2. Natural Tregs (nTregs)**

126 Lupus

interactions with these cells [7, 12].

growth factor β (TGF‐β) and IL‐2 [12].

memory cells [30].

differentiation markers.

Thymic‐derived Tregs or natural Tregs are characterized by the CD4+CD25highFOXP3+ phe‐ notype and range between 1 and 3% of the peripheral CD4+ T lymphocytes [13, 23]. They are considered to maintain an anergic state (based on the findings of decreased responsiveness to antigen stimulation and limited proliferation capacity), nTregs have remarkable prolifera‐ tive potential, both *in vitro* and *in vivo*, upon antigen stimulation in the presence of dendritic cells [24]. Reciprocally, Tregs are able to induce tolerogenic DCs, further complicating their

nTregs express the same αβTCR as the conventional T lymphocytes but they comprise a dis‐ tinct clone [25]. They derive from pluripotent stem cells and differentiate in the thymic cortex through a positive selection process after the linkage of their TCR with self‐antigens with intermediate affinity [26, 27]. These antigens are presented through MHC II molecules from the thymic cortical cells [28]. Co‐stimulation via the CD28 molecule induces the FOXP3 pro‐ moter either directly or through other genes that increase its activation [29]. It seems that the selection of these CD25+ cells occurs according to a predefined ratio to the respective CD25− cells, which have been generated earlier. They are long‐lived cells capable of producing anti‐

apoptotic molecules that protect them from the process of negative selection [26, 27].

Upon migration to the periphery, nTregs maintain their regulatory phenotype and suppres‐ sive capacity, which are mediated through cell‐to‐cell contact. This mechanism involves certain surface molecules, and it is independent of secreted cytokines [13, 26]. Survival in the periphery is facilitated by CD28 and its respective ligands (CD80, CD86), transforming

Several surface molecules have been considered to allow the laboratory isolation from other cellular subpopulations and are crucial for their function. The most important such molecules are the CD4, CD25high, CD127low, CD45RO and CD45RBlow, providing a phenotype of activated

Moreover, nTregs express other activation markers, such as CD28, CTLA‐4, GITR and che‐ mokine receptors, which are implicated in their migration and trafficking [30, 31]. They also express several Toll‐like receptors (TLRs), TGF‐β, neuropilin‐1, perforin and granzymes, L‐selectin (CD62L), LAG‐3 (lymphocyte activation gene‐3, CD223) and the folate receptor FR4 [32–35]. Multiple adhesion molecules are also abundantly expressed on their surface, such as CD11a, CD44, CD54, and CD103 [36]. All the aforementioned markers have been described in other cell types, which are not exclusively expressed in nTregs and cannot be used as

Other markers that are thought to be highly specific for nTregs were discovered from the Ikaros gene family; the respective transcription factor is called Helios and is preferentially

Recently, it has been demonstrated that certain epigenetic mechanisms are implicated in the regulation and maintenance of FOXP3 expression [38]. In this regard, the methylation state of the Treg‐specific demethylated region (TSDR, a conserved non‐coding sequence in the

expressed in nTregs as compared to other CD4+ T cells [37].

These Tregs subgroups are not derived from the thymus but they are induced from naive CD4+ T cells in the periphery in response to the occasional micro‐environmental conditions, **Figure 1** [40]. Inducible Tregs regulate the immune response against self and non‐self antigens and are implicated in the pathophysiology of infections, neoplasias and organ transplantation. Their mechanism of action is usually dependent on the secreted cytokines and not on direct cellular contact. Their characterization is based on the aforementioned surface markers (CD25, CD127, CTLA‐4, GITR, etc.), the intensity of intracellular FOXP3 expression as well as their suppressive capacity [13]. As mentioned above, their TSDR is incompletely hypomethylated; thus, FOXP3 expression is transient and unstable. The most important subgroups include the Tr1 and Th3 lymphocytes, the CD4+CD25+ Tregs that are induced from the CD4+CD25− acti‐ vated T cells, CD103+ Tregs, CD8+ Tregs and the double negative Tregs (CD4−CD8−DN).

Tr1 cells are antigen‐specific CD4+ T regulatory lymphocytes that are induced in the presence of IL‐10 [41]. They derive from CD4+CD25− naive T lymphocytes after antigenic stimulation with certain costimulatory molecules, such as CD3 and CD46 [42, 43]. Apart from the epi‐ genetic differences, they are phenotypically indistinguishable from natural Tregs, but they secrete large amounts of IL‐10. The intensity of the surface expression of CD25 and intracel‐ lular FOXP3 is lower than that of nTregs; however, their suppressive capacity is as intense [44]. Their regulatory function is mediated mainly through IL‐10 and, secondarily, through TGF‐β. They play a major role in the pathophysiology of certain infections and the regulation of allergic reactions [45].

Th3 lymphocytes are CD4+ Tregs that were called helper T cells (T helper 3), although their func‐ tion is primarily suppressive [46]. Their cardinal characteristic is the secretion of large amounts of TGF‐β [47]. The *ex vivo* expansion of the Th3 cells was one of the first reports of clonal expan‐ sion of Tregs using an orally administered antigen in mice [48]. Th3 cells are generated and activated through an antigen‐specific process but their suppressive function is not specific and mediated through TGF‐β. Even in the absence of inflammation, TGF‐β secretion induces the expression of FOXP3 in the activated T cells and maintains Tregs' survival in the periphery [49].

Other types of Tregs include the CD4+CD25+ Tregs deriving from CD4+CD25− T lymphocytes under specific conditions, the CD103+ Tregs (expressing integrin alpha‐E beta‐7), the CD8+CD28− Tregs and the CD4−CD8− double negative Tregs [13]. All these subpopulations express FOXP3 upon activation and are able to suppress immune responses in a non‐antigen specific fashion.

Further research using certain surface markers revealed the existence of novel subpopula‐ tions of iTregs, including the CD4+CD25−FOXP3+ T cells, the CD4+CD45RA+FOXP3+ Tregs, the CD4+CD161+ Tregs, and the CD4+CXCR5+FOXP3+ Tregs [50–52]. Although the CD25− FOXP3+ T cells could suppress effector cells *in vitro*, it is still uncertain if they represent dysfunctional Tregs or, simply, activated T cells. The CD161+ Tregs represent an excellent paradigm of T cell plasticity, as they are capable of producing pro‐inflammatory cytokines such as IL‐2, IFN‐γ, and IL‐17, behaving like Th1 or Th17 cells under proper cytokine micro‐ environment [53]. In spite of their cytokine‐producing properties, these cells also retain their regulatory functions and have the already mentioned demethylated TSDR in the FOXP3 locus, like the nTregs. Finally, the CXCR5+ Tregs are follicular T cells, which are able to gain access into the germinal centres (through the CXCR5) and directly suppress the B cells that undergo hypermutation and isotype switch at those sites. These cells are decreased in active and new onset SLE, seemingly allowing for the activation of B cells [54].

#### **2. Mechanisms of action**

The mechanisms of action of Tregs have been studied mostly in *in vitro* systems. Thus, it is not clear how accurately these studies may reproduce Treg activity *in vivo.* Tregs delete auto‐ reactive T cells and induce tolerance and dampen inflammation, while their cellular targets include CD4+CD25− T cells, CD8+ T cells, B cells, monocytes, DCs, and NK cells [55]. These cells appear to inhibit the target cells via IL‐2 deprivation, cell‐to‐cell contact and cytolysis, secretion of inhibitory cytokines, metabolic disruption and modulation of DC maturation and function [56–60], **Figure 2**.

**Figure 2.** Treg mechanisms of action.

IL‐2 is not required for the thymic development of nTregs; however, in the periphery, it acts as a growth factor, essential for their survival and functional integrity. Tregs have more requirements in IL‐2 than conventional T cells. IL‐2 drives the production of IL‐10, CTLA‐4, TGF‐β, and the activation of FOXP3 [61]. Simultaneously, CD25 expression is induced, further amplifying the affinity of Tregs for IL‐2. In co‐cultures of Tregs and T effector cells, addition of exogenous IL‐2 led to active proliferation and activation of Tregs [62]. In addition, Tregs inhibit the function of other T helper cells or cytotoxic cells by deprivation of other cytokines that share the common γ chain, such as IL‐4 and IL‐7; this leads to apoptosis of the effector cells [63]. Moreover, the prioritized usage of IL‐2 may modify the function of Tregs by the increased IL‐10 production [64].

the CD4+CD161+ Tregs, and the CD4+CXCR5+FOXP3+ Tregs [50–52]. Although the CD25− FOXP3+ T cells could suppress effector cells *in vitro*, it is still uncertain if they represent dysfunctional Tregs or, simply, activated T cells. The CD161+ Tregs represent an excellent paradigm of T cell plasticity, as they are capable of producing pro‐inflammatory cytokines such as IL‐2, IFN‐γ, and IL‐17, behaving like Th1 or Th17 cells under proper cytokine micro‐ environment [53]. In spite of their cytokine‐producing properties, these cells also retain their regulatory functions and have the already mentioned demethylated TSDR in the FOXP3 locus, like the nTregs. Finally, the CXCR5+ Tregs are follicular T cells, which are able to gain access into the germinal centres (through the CXCR5) and directly suppress the B cells that undergo hypermutation and isotype switch at those sites. These cells are decreased in active

The mechanisms of action of Tregs have been studied mostly in *in vitro* systems. Thus, it is not clear how accurately these studies may reproduce Treg activity *in vivo.* Tregs delete auto‐ reactive T cells and induce tolerance and dampen inflammation, while their cellular targets include CD4+CD25− T cells, CD8+ T cells, B cells, monocytes, DCs, and NK cells [55]. These cells appear to inhibit the target cells via IL‐2 deprivation, cell‐to‐cell contact and cytolysis, secretion of inhibitory cytokines, metabolic disruption and modulation of DC maturation and

and new onset SLE, seemingly allowing for the activation of B cells [54].

**2. Mechanisms of action**

128 Lupus

function [56–60], **Figure 2**.

**Figure 2.** Treg mechanisms of action.

The suppressive function of nTregs is mediated through direct cell‐to‐cell contact and is not dependent on the presence of inhibitory cytokines like IL‐10 and TGF‐β [13, 56, 57]. Surface molecules that are involved in this process include CTLA‐4 [65, 66], membrane‐bound TGF‐β [67, 68], LAG‐3 (lymphocyte activation gene‐3, CD223) [69], GITR (glucocorticoid‐induced TNFR‐a family‐related protein) [70, 71], PD‐1 (programmed death‐1, CD279) [72] and perfo‐ rin and granzymes, which lead to cytolysis of the target cell [73].

Granzyme B, in particular, has been implicated as an effector mechanism in Treg‐mediated suppression, since its up‐regulation was associated with the killing of target cells in a gran‐ zyme B‐dependent, perforin‐independent manner [73]. Granzyme B‐deficient Tregs display reduced suppressive activity [74]. Other studies proved that the activated Tregs could lyse CD4+, CD8+ T cells and B cells through granzyme A and perforin [56–58].

The intracellular signal transduction pathways have not been elucidated yet; however, it has been demonstrated that CTLA‐4 induces the expression of ICER (inducible cAMP early repressor) and, subsequently, inhibition of IL‐2 signals to target cells [75]. Membrane‐bound TGF‐β activates the Smad proteins and inhibits genes that are required for the functional dif‐ ferentiation of the effector cells [68].

Suppression by inhibitory cytokines is an important mode of action utilized by iTregs. The most important cytokines with regulatory/suppressive capacity are TGF‐β [76, 77], primar‐ ily produced by the Th3 cells and IL‐10 [78, 79] by the Tr1 cells. IL‐10 activates the JAK/STAT intracellular pathway and the MAP kinases [78]. The net result is the inhibition of genes that control the synthesis and secretion of pro‐inflammatory cytokines. Another regulatory cyto‐ kine that is produced from Tregs is the IL‐35 [80]. This cytokine is assembled by two chains, IL‐12α and EBI3, and is required for the suppressive capacity of Tregs *in vivo*, since inabil‐ ity to express these chains results in uncontrolled expansion of T effector cells in systemic autoimmune diseases [81]. Other anti‐inflammatory cytokines that have been implicated in the mechanisms of action of Tregs include IL‐27 and IL‐37 [82]; more recently, it has been shown that fibrinogen‐like protein 2 is also secreted by Tregs and mediates immune sup‐ pression [38].

Metabolic disruption of the target cells is another mechanism utilized by Tregs to regulate immune responses. nTregs possess large amounts of cAMP (cyclic adenosine monophos‐ phate), which exerts potent inhibitory action against the proliferation and differentiation of the effector T cells and the expression of genes that control the synthesis of IL‐2 and IFN‐γ [83]. Gene expression is inhibited through the suppression of the protein kinase A of NF‐κB or through activation of ICER. Tregs induce intracellular cAMP within the effector T cells with cell‐to‐cell cAMP transfer through the gap junctions. Neutralization of cAMP or block‐ age of the gap junctions led to significant weakening of Tregs' suppressive function [83]. In addition, co‐expression of CD39 and CD73 on the surface of Tregs induces the secretion of large amounts of adenosine that suppresses T effectors [84]. The linkage of adenosine to its receptor A2A on Tregs induces TGF‐β secretion and inhibits IL‐6, generating appropriate circumstances for new Treg development [85, 86].

Tregs seem capable of limiting the capacity of DCs to stimulate effector cells. In this context, their interaction with the dendritic cells and the inhibition of their maturation is of particular importance [87, 88]. Tregs induce the production of regulatory molecules from DC, such as indoleamine‐2,3‐dioxygenase (IDO), IL‐10 and TGF‐β, through interactions between CTLA‐4 and CD80/CD86 [89, 90]. The same investigators showed that Tregs reduce the expression of the costimulatory molecules CD80 and CD86 on DCs. Moreover, the catabolism of tryptophan and arginine through IDO leads to Tregs activation and induction of regulatory phenotype in naive T cells and T effector cell apoptosis [91].

#### **3. Homeostasis of Tregs**

#### **3.1. Functional differentiation of Tregs in the periphery**

Certain transcriptional factors are implicated in the process of maturation and functional spe‐ cialization of the CD4+ T cells. The most important factors are T‐bet for Th1 cells, GATA‐3 for Th2 cells, RORγt for Th17 cells, Bcl6 for follicular T helper cells (Tfh), and FOXP3 for Tregs [92]. For Tregs, in particular, their functional integrity depends on the dynamic interaction of differ‐ ent transcriptional regulators, which is shaped by the occasional micro‐environmental circum‐ stances. These regulators include members of the nuclear factor of activated T (NFAT) cell family, the NF‐κB, the activator protein‐1 (AP‐1) and STAT5 [93]. Furthermore, functional specialization of Tregs has been documented; for instance, these cells are using Th‐related transcription fac‐ tors during Th1, Th2 or Th17 immune responses. In this context, T‐bet+ Tregs migrate into the inflamed tissue in cases of Th‐1‐mediated inflammation and suppress the Th1 effectors [94]. Accordingly, the expression of IRF4 in Tregs is required for the suppression of Th2 responses, while the deletion of STAT3 is linked to uncontrolled Th17 responses [95]. The precise mecha‐ nisms by which these transcription factors control Tregs differentiation are unknown. However, the experimental inhibition of these factors was associated with impaired expression of certain surface chemokine receptors, such as CXCR3 (for Th1), CCR8 (for Th2) and CCR6 (for Th17 immune response) [96]. Moreover, deletion of the respective genes of these chemokine receptors led to decreased Tregs activity and renders Tregs incapable of migrating into the site of Th‐1‐ mediated inflammation [97]. Based on these data, it seems possible that phenotypically and functionally distinct Tregs may be active against different effector arms of the immune response.

#### **3.2. Clonal expansion of Tregs**

the effector T cells and the expression of genes that control the synthesis of IL‐2 and IFN‐γ [83]. Gene expression is inhibited through the suppression of the protein kinase A of NF‐κB or through activation of ICER. Tregs induce intracellular cAMP within the effector T cells with cell‐to‐cell cAMP transfer through the gap junctions. Neutralization of cAMP or block‐ age of the gap junctions led to significant weakening of Tregs' suppressive function [83]. In addition, co‐expression of CD39 and CD73 on the surface of Tregs induces the secretion of large amounts of adenosine that suppresses T effectors [84]. The linkage of adenosine to its receptor A2A on Tregs induces TGF‐β secretion and inhibits IL‐6, generating appropriate

Tregs seem capable of limiting the capacity of DCs to stimulate effector cells. In this context, their interaction with the dendritic cells and the inhibition of their maturation is of particular importance [87, 88]. Tregs induce the production of regulatory molecules from DC, such as indoleamine‐2,3‐dioxygenase (IDO), IL‐10 and TGF‐β, through interactions between CTLA‐4 and CD80/CD86 [89, 90]. The same investigators showed that Tregs reduce the expression of the costimulatory molecules CD80 and CD86 on DCs. Moreover, the catabolism of tryptophan and arginine through IDO leads to Tregs activation and induction of regulatory phenotype in

Certain transcriptional factors are implicated in the process of maturation and functional spe‐ cialization of the CD4+ T cells. The most important factors are T‐bet for Th1 cells, GATA‐3 for Th2 cells, RORγt for Th17 cells, Bcl6 for follicular T helper cells (Tfh), and FOXP3 for Tregs [92]. For Tregs, in particular, their functional integrity depends on the dynamic interaction of differ‐ ent transcriptional regulators, which is shaped by the occasional micro‐environmental circum‐ stances. These regulators include members of the nuclear factor of activated T (NFAT) cell family, the NF‐κB, the activator protein‐1 (AP‐1) and STAT5 [93]. Furthermore, functional specialization of Tregs has been documented; for instance, these cells are using Th‐related transcription fac‐ tors during Th1, Th2 or Th17 immune responses. In this context, T‐bet+ Tregs migrate into the inflamed tissue in cases of Th‐1‐mediated inflammation and suppress the Th1 effectors [94]. Accordingly, the expression of IRF4 in Tregs is required for the suppression of Th2 responses, while the deletion of STAT3 is linked to uncontrolled Th17 responses [95]. The precise mecha‐ nisms by which these transcription factors control Tregs differentiation are unknown. However, the experimental inhibition of these factors was associated with impaired expression of certain surface chemokine receptors, such as CXCR3 (for Th1), CCR8 (for Th2) and CCR6 (for Th17 immune response) [96]. Moreover, deletion of the respective genes of these chemokine receptors led to decreased Tregs activity and renders Tregs incapable of migrating into the site of Th‐1‐ mediated inflammation [97]. Based on these data, it seems possible that phenotypically and functionally distinct Tregs may be active against different effector arms of the immune response.

circumstances for new Treg development [85, 86].

naive T cells and T effector cell apoptosis [91].

**3.1. Functional differentiation of Tregs in the periphery**

**3. Homeostasis of Tregs**

130 Lupus

The question if nTregs numbers remain stable through life or if their pool is constantly enriched with new cells was based on the findings of stable numbers of CD4+CD25+ T cells in mice from the age of 2 weeks up to 1 year. In thymectomized mice with no T cells, adoptive transfer of Tregs was followed by an expansion of these cells to the extent of the nonthymec‐ tomized animals of similar age [98].

In humans, it has demonstrated that nTregs, after they leave thymus, are constantly prolif‐ erating after cytokine (TGF‐β, IL‐2, IL‐10) stimulation and in the presence of tissue antigens. DCs can also induce Tregs in the presence of IL‐2 [99].

#### **3.3. Tregs recruitment at the site of inflammation**

Natural Tregs are generated in the thymus and migrate into the periphery where their population will be enriched with inducible Tregs. The precise site of their clonal expansion (peripheral lym‐ phoid organs or the site of inflammation) is not known. Apart from the thymus, Tregs have been found in the bone marrow, lymph nodes, intestine, liver, synovial fluid, skin, vessel wall, etc.

More than 25% of the total CD4+ T cells residing in the bone marrow have regulatory pheno‐ type and properties [100]. In this regard, the bone marrow acts as a reservoir for Tregs that is able to release them upon inflammation. Bone marrow Tregs express CXCR4 (the CXCL12 receptor), which is crucial for their migration and their return to the bone marrow after sup‐ pression [100]. Integrins are also implicated in their migration; intense expression of CD62L and CCR7 along with poor expression of CD103 (integrin αEβ7) allows for penetration into the lymph nodes. On the contrary, strong expression of CD103 is required for migrating into the inflamed tissues [94].

Integrins are crucial for the homeostasis of iTregs; integrin α4β7 tissue expression (usually in the mucosal vessels) attracts Tr1 cells, whereas the α4β1 (on the endothelium of inflamed tis‐ sues) engages the Th3 cells [101]. Furthermore, it has been showed that when Tregs migrate to the T‐zone of lymph nodes, they utilize the CCL19/CCR7 ligation, while when they migrate to the B‐zone, they utilize the CXCL13/CXCR9 interaction [102].

#### **3.4. How effector cells escape Treg‐mediated suppression**

Tregs are also regulated by the immune system in a fashion that allows the control of their action either through negative feedback or through the development of escape mechanisms for the effector cells [103, 104]. The negative feedback is maintained through various mechanisms, such as TLR activation on DCs [104] and the direct regulation by cytokines like IL‐21, IL‐7, IL‐15, TNF‐α and IL‐6. In particular, IL‐21 increases the resistance of the effector T cells against Tregs in experimental diabetes [105]. DC‐derived IL‐6 renders CD4+ T cells resistant to Tregs suppression [106]. Additional mechanisms include the amplification of co‐stimulatory molecule expression on the surface of T effectors and DCs, such as the CD80 and CD86 molecules, CD28, NFATc1, c2, c3 and TRAF6, which protect the integrity of intracellular signal transduction [10].

### **4. Tregs in systemic lupus erythematosus**

#### **4.1. A matter of numbers and function?**

SLE is characterized by the breakdown of immune tolerance against self‐antigens. The net result is the induction and proliferation of auto‐reactive lymphocytes, the production pro‐ inflammatory soluble mediators, the formation of pathogenic autoantibodies and immuno‐ complexes that cause tissue damage [1]. Tregs are thought to play a critical role before and during this pathophysiological process. Most studies in lupus‐prone mice and humans dem‐ onstrated quantitative and/or qualitative defects of these cells [14, 107–113]. Other reports present insignificant variations in Tregs numbers between lupus patients and healthy controls [50, 114, 115] or even higher numbers [116], probably as a result of significant differences in protocol designs. With regard to the functional capacity of these cells, studies are again con‐ troversial with reportedly defective [110, 111] or normal [14, 114, 115] function. In the latter case, T effectors showed decreased sensitivity to the suppressive function of Tregs [117].

In a seminal paper, Miyara et al. described the characteristics of Tregs kinetics and the strongly inverse the correlation with SLE disease activity [14]. They found that Tregs (CD4+CD25bright) were globally depleted from the periphery of active lupus patients. They provided evidence that these cells do not accumulate in involved organs (by kidney biopsies) or lymphoid tissue (by lymph node biopsies). In fact, Tregs were found to be more sensitive to Fas‐mediated apoptosis although they were still functionally intact. Moreover, they were the first to show that Tregs are increased after the successful treatment of disease flare. FOXP3 expression was found in 85.6% of the CD4+CD25high compartment.

The issue of functional integrity of Tregs within the lupus inflammatory microenvironment was questioned later by the findings of Valencia et al. [110] and Lyssouk et al. [111]. These groups reported that CD4+CD25high Tregs were defective in terms of both proliferation and suppression against CD4+ and CD8+ T effector cells. They also showed that FOXP3 expres‐ sion was decreased in Tregs from active lupus patients, generating doubt about the appropri‐ ate immunophenotype that should be used for cell isolation and study. These findings were confirmed later by using the mean fluorescence intensity (MFI) in newly diagnosed, untreated lupus patients [118].

At the same time, Barath et al. were the first to utilize the CD4+CD25highFOXP3+ immunophe‐ notype for Tregs characterization [112]. They found these cells in significantly lower levels as compared to healthy controls, whereas the inducible CD4+IL‐10+ Tregs did not display any significant quantitative differences. At the tissue level, CD4+CD25+FOXP3+ Tregs were found in decreased numbers in the skin lesions of active cutaneous lupus as compared to other inflammatory skin diseases, such as psoriasis, atopic dermatitis, and lichen planus [119].

In 2008, a new subpopulation was described, namely the CD4+CD25−FOXP3+ T cells [50]. These cells were found in increased numbers in newly diagnosed SLE patients and were asso‐ ciated with other indices of disease activity, such as low complement C3 and C4 [51]. Their function was primarily regulatory as they were able to suppress the effector T cell prolifera‐ tion but not IFN‐γ production [51]. Other investigators opposed these findings by performing the measurements in untreated lupus patients, reaching the conclusion that not all FOXP3 expressing T cells are Tregs [120].

**4. Tregs in systemic lupus erythematosus**

found in 85.6% of the CD4+CD25high compartment.

lupus patients [118].

SLE is characterized by the breakdown of immune tolerance against self‐antigens. The net result is the induction and proliferation of auto‐reactive lymphocytes, the production pro‐ inflammatory soluble mediators, the formation of pathogenic autoantibodies and immuno‐ complexes that cause tissue damage [1]. Tregs are thought to play a critical role before and during this pathophysiological process. Most studies in lupus‐prone mice and humans dem‐ onstrated quantitative and/or qualitative defects of these cells [14, 107–113]. Other reports present insignificant variations in Tregs numbers between lupus patients and healthy controls [50, 114, 115] or even higher numbers [116], probably as a result of significant differences in protocol designs. With regard to the functional capacity of these cells, studies are again con‐ troversial with reportedly defective [110, 111] or normal [14, 114, 115] function. In the latter case, T effectors showed decreased sensitivity to the suppressive function of Tregs [117].

In a seminal paper, Miyara et al. described the characteristics of Tregs kinetics and the strongly inverse the correlation with SLE disease activity [14]. They found that Tregs (CD4+CD25bright) were globally depleted from the periphery of active lupus patients. They provided evidence that these cells do not accumulate in involved organs (by kidney biopsies) or lymphoid tissue (by lymph node biopsies). In fact, Tregs were found to be more sensitive to Fas‐mediated apoptosis although they were still functionally intact. Moreover, they were the first to show that Tregs are increased after the successful treatment of disease flare. FOXP3 expression was

The issue of functional integrity of Tregs within the lupus inflammatory microenvironment was questioned later by the findings of Valencia et al. [110] and Lyssouk et al. [111]. These groups reported that CD4+CD25high Tregs were defective in terms of both proliferation and suppression against CD4+ and CD8+ T effector cells. They also showed that FOXP3 expres‐ sion was decreased in Tregs from active lupus patients, generating doubt about the appropri‐ ate immunophenotype that should be used for cell isolation and study. These findings were confirmed later by using the mean fluorescence intensity (MFI) in newly diagnosed, untreated

At the same time, Barath et al. were the first to utilize the CD4+CD25highFOXP3+ immunophe‐ notype for Tregs characterization [112]. They found these cells in significantly lower levels as compared to healthy controls, whereas the inducible CD4+IL‐10+ Tregs did not display any significant quantitative differences. At the tissue level, CD4+CD25+FOXP3+ Tregs were found in decreased numbers in the skin lesions of active cutaneous lupus as compared to other inflammatory skin diseases, such as psoriasis, atopic dermatitis, and lichen planus [119].

In 2008, a new subpopulation was described, namely the CD4+CD25−FOXP3+ T cells [50]. These cells were found in increased numbers in newly diagnosed SLE patients and were asso‐ ciated with other indices of disease activity, such as low complement C3 and C4 [51]. Their function was primarily regulatory as they were able to suppress the effector T cell prolifera‐ tion but not IFN‐γ production [51]. Other investigators opposed these findings by performing

**4.1. A matter of numbers and function?**

132 Lupus

The association of Tregs with SLE disease activity was tested in several studies with a small number of patients. Most of them reported a strongly inverse correlation [14, 113], whereas others found insignificant correlations [109, 112].

In the first large‐scale (*n* = 100 patients), longitudinal (mean follow up of 5 years) study of CD4+CD25highFOXP3+ Tregs as a biomarker of disease activity, we found that these cells are gradually decreased from healthy controls to patients with inactive, mild, or severe dis‐ ease [113]. Moreover, we observed inverse alterations in cases of changing disease activity; these cells were reduced during disease flare and increased upon remission, while numbers remained stable during stable disease activity. Their sensitivity and specificity to assess a clinically significant change in global disease activity was 88 and 74%, respectively. Their positive and negative predictive values were 85 and 79%, respectively. In the same study, we reported decreased Tregs numbers in active lupus nephritis and active neuropsychiatric involvement, whereas no differences were observed in patients with active antiphospholipid syndrome (APS). The ability of CD4+CD25highFOXP3+ Tregs to predict disease flares was low (positive predictive value 17%).

Concerning the influence of certain therapeutic approaches, we prospectively showed that Tregs' numerical restoration after treatment is independent of the occasional medication administered [121]. In that study, patients achieved remission after administration of various immunomodulatory agents, including glucocorticoids (oral and intravenous), cyclophospha‐ mide, intravenous immuno globulins, azathioprine and hydroxy chloroquxine. In all cases, a significant increase of CD4+CD25highFOXP3+ Tregs was observed. That restoration was faster with the intravenous regimens as compared to oral therapies. Cyclophosphamide pulse therapy, in particular, led to a significant increase of Tregs after the fourth pulse in patients with active lupus nephritis and/or neuropsychiatric involvement [122]. Of note, an even faster response (shortly after the first infusion) was documented after treatment with intravenous tocilizumab in patients with rheumatoid arthritis [123].

#### **4.2. Novel theories for Tregs in the pro‐inflammatory environment of SLE**

As mentioned above, most studies on Tregs have been conducted *in vitro*; thus, their reliabil‐ ity and accuracy pertaining to the actual function of these cells *in vivo* are questionable. After the discovery that a fully demethylated TSDR is required for intense and sustained expression of FOXP3, the hallmark of regulatory function, many beliefs have been revised [39]. In this context, Helios+FOXP3+ T cells, with a fully demethylated TSDR, were found in increased numbers in active lupus patients; their function was intact [124]. It is not yet known if Helios represents a unique marker for Tregs; however, the epigenetic change is believed to differenti‐ ate between natural and inducible Tregs. Nevertheless, there is disequilibrium between Tregs and effector cells that is more prominent in the pro‐inflammatory environment of SLE.

Several studies have reported on altered ratios between Tregs and T effectors in lupus patients [125]. The most striking feature, among other findings, was that there is plasticity between Tregs and Th17 populations, and the latter may derive from the former under certain circum‐ stances [126]. In this regard, the presence of TGF‐β alone will drive naive CD4+ T cells towards Tregs differentiation, while the simultaneous presence of IL‐6 will lead to Th17 proliferation [127]. Other transitions have also been described between Th1 and Th17 cells, based on the presence of IL‐12 receptors on the surface of Th17 cells; upon activation with IL‐12, these cells are capable of producing IFN‐γ [125, 127]. Of note, it has been documented that some Tregs down‐regulate FOXP3 expression and act as effectors, promoting inflammation through the secretion of IL‐17 and IFN‐α [39]. These cells are called ex Tregs and believed to derive from the Tregs lineage prior to natural Tregs commitment. They acquire pro‐inflammatory charac‐ teristics in the periphery, probably in the context of a generalized immune response.

The concept of Tregs/Th17 imbalance, in particular, seems of paramount importance in SLE. It has been demonstrated that disease relapses may occur as a consequence of an impaired Tregs/Th17 ratio, in favour of the latter, in animal models [128]. Those findings were later confirmed in lupus patients, in whom the altered Tregs/Th17 ratio was documented even in clinically quiescent disease; this may represent a hallmark of SLE [129, 130]. In this context, it is believed that the sole targeting of the Th17 arm of the immune response will not render meaningful results; approaches aiming at the restoration of the Tregs/Th17 balance will be more likely to exert beneficial effects [125, 131].

The disturbed balance between T effectors (Th1, Th2, Th17) and Tregs is driven by a relative IL‐2 deficiency in SLE [132]. Treatment with low doses of IL‐2 re‐established the equilibrium between Tregs and T effectors in animal models of the disease; accordingly, IL‐2 neutraliza‐ tion or CD25 depletion accelerated disease onset [39]. Studies in lupus patients showed that FOXP3+Helios+ Tregs were capable of proliferating despite the reduced IL‐2 levels; however, the integrity of their suppressive function has not been confirmed [124]. Apart from IL‐2, other cytokines, such as IL‐6, IL‐21 and IFN‐α may inhibit the Tregs function and/or render T effectors resistant to regulation. All these cytokines are found in abundance in SLE and are positively correlated to disease activity. On the other hand, the main regulatory cytokine, TGF‐β, is lower in active disease, generating hypotheses that the cytokine disequilibrium drives the imbalance of Tregs and T effectors. The exact mechanisms by which these cytokines increase the T effectors' resistance to Tregs suppression have not been elucidated yet [39].

#### **4.3. Epigenetics and Tregs**

Latest studies revealed that certain epigenetic mechanisms, such as methylation, histone modification and miRNAs, play a significant role in Tregs biology [38]. In this regard, the methylation status of the TSDR is of paramount importance for the sustained expression of FOXP3 and, hence, the intensity of Treg suppressive function. Histone modification is another mechanism involved in Tregs functional differentiation. The acetylation of his‐ tones H3 and H4 has been shown to reliably differentiate Tregs than FOXP3+ effectors [133]. Modification of the FOXP3 promoter by other genes influenced dramatically the rate of dif‐ ferentiation of iTregs in the periphery [38]. Finally, miRNA‐155 is associated with less Tregs, though functionally intact, in mice, while miRNA‐126 up‐regulates Tregs and enhances their function [134].

Epigenetic regulation of the FOXP3 gene has been reported in lupus patients. In this con‐ text, decreased peripheral Tregs were associated with hypermethylation of the promoter of the FOXP3 gene [135]. Genome‐wide studies have shown that virtually all immune cells in SLE, including Tregs, had severe hypomethylation in interferon‐type I‐related genes [38]. Treatment with a histone modification inhibitor the enhanced Tregs number and function in lupus‐prone mice [136]; the same results were reached with an inhibitor of the protein kinase IV [137]. It is believed that these approaches will soon be tested in lupus patients [38].

#### **5. Tregs‐based therapeutic approaches in SLE**

#### **5.1. Adoptive transfer of** *ex vivo* **expanded Tregs**

Tregs and Th17 populations, and the latter may derive from the former under certain circum‐ stances [126]. In this regard, the presence of TGF‐β alone will drive naive CD4+ T cells towards Tregs differentiation, while the simultaneous presence of IL‐6 will lead to Th17 proliferation [127]. Other transitions have also been described between Th1 and Th17 cells, based on the presence of IL‐12 receptors on the surface of Th17 cells; upon activation with IL‐12, these cells are capable of producing IFN‐γ [125, 127]. Of note, it has been documented that some Tregs down‐regulate FOXP3 expression and act as effectors, promoting inflammation through the secretion of IL‐17 and IFN‐α [39]. These cells are called ex Tregs and believed to derive from the Tregs lineage prior to natural Tregs commitment. They acquire pro‐inflammatory charac‐

teristics in the periphery, probably in the context of a generalized immune response.

more likely to exert beneficial effects [125, 131].

**4.3. Epigenetics and Tregs**

134 Lupus

function [134].

The concept of Tregs/Th17 imbalance, in particular, seems of paramount importance in SLE. It has been demonstrated that disease relapses may occur as a consequence of an impaired Tregs/Th17 ratio, in favour of the latter, in animal models [128]. Those findings were later confirmed in lupus patients, in whom the altered Tregs/Th17 ratio was documented even in clinically quiescent disease; this may represent a hallmark of SLE [129, 130]. In this context, it is believed that the sole targeting of the Th17 arm of the immune response will not render meaningful results; approaches aiming at the restoration of the Tregs/Th17 balance will be

The disturbed balance between T effectors (Th1, Th2, Th17) and Tregs is driven by a relative IL‐2 deficiency in SLE [132]. Treatment with low doses of IL‐2 re‐established the equilibrium between Tregs and T effectors in animal models of the disease; accordingly, IL‐2 neutraliza‐ tion or CD25 depletion accelerated disease onset [39]. Studies in lupus patients showed that FOXP3+Helios+ Tregs were capable of proliferating despite the reduced IL‐2 levels; however, the integrity of their suppressive function has not been confirmed [124]. Apart from IL‐2, other cytokines, such as IL‐6, IL‐21 and IFN‐α may inhibit the Tregs function and/or render T effectors resistant to regulation. All these cytokines are found in abundance in SLE and are positively correlated to disease activity. On the other hand, the main regulatory cytokine, TGF‐β, is lower in active disease, generating hypotheses that the cytokine disequilibrium drives the imbalance of Tregs and T effectors. The exact mechanisms by which these cytokines increase the T effectors' resistance to Tregs suppression have not been elucidated yet [39].

Latest studies revealed that certain epigenetic mechanisms, such as methylation, histone modification and miRNAs, play a significant role in Tregs biology [38]. In this regard, the methylation status of the TSDR is of paramount importance for the sustained expression of FOXP3 and, hence, the intensity of Treg suppressive function. Histone modification is another mechanism involved in Tregs functional differentiation. The acetylation of his‐ tones H3 and H4 has been shown to reliably differentiate Tregs than FOXP3+ effectors [133]. Modification of the FOXP3 promoter by other genes influenced dramatically the rate of dif‐ ferentiation of iTregs in the periphery [38]. Finally, miRNA‐155 is associated with less Tregs, though functionally intact, in mice, while miRNA‐126 up‐regulates Tregs and enhances their Based on the aforementioned data, Tregs may represent a promising target in SLE therapeutics. Several groups have tried to manipulate this cellular subpopulation in order to restore the defective immune tolerance that is a crucial component of disease pathophysiology [138]. Early experiments in mice models showed that adoptive transfer of *ex vivo* expanded Tregs was capable of ameliorating the disease [139, 140]. In the first experiment, T cells treated with IL‐2 and TGF‐β lost their ability to induce a graft‐versus‐host disease and prevented other effector T cells from activating B cells [139]. In addition, when transferred to animals with high titers of anti‐dsDNA antibodies, they led to a significant reduction of their titers and doubled survival. In New Zealand black/New Zealand white mice, a well‐studied lupus model, transfer of thymic Tregs (CD4+CD25+CD62Lhigh) decreased the rate of glomerulone‐ phritis, the severity of proteinuria and improved survival [140]. The precise mechanism by which these Tregs suppress the autoimmune response has not been elucidated; however, it was demonstrated that the induction of tolerogenic DCs plays a critical role [141]. These DCs were also able to expand the recipient's CD4+FOXP3+ Tregs (infectious tolerance).

Studies in humans also showed that *in vitro* expanded Tregs, both polyclonal [142] and anti‐ gen‐specific [143] may display enhanced regulatory activity. These encouraging results led to the implementation of this strategy in phase I and II clinical trials in other autoimmune diseases. Seminal studies in type 1 diabetes (T1D) proved the feasibility of generating puri‐ fied iTregs for therapeutic purposes [144, 145]. Bluestone et al. demonstrated that Tregs could survive for more than 1 year after infusion in 14 patients with T1D; although there were no significant reactions to infusion, from an efficacy standpoint there was no significant improve‐ ment in C‐peptide levels and HbA1c [144]. In another study of 12 children with T1D, adoptive transfer of Tregs led to significant reduction in exogenous insulin needs and improvement in C‐peptide levels. Of note, two children were insulin independent after 12 months [145]. In chronic graft‐versus‐host disease (GVHD), adoptive transfer of Tregs ameliorated symptoms in two out of five patients, while the remaining patients did not show any deterioration after 21 months of follow‐up [146]. In another study, where umbilical cord‐derived Tregs were used in 11 patients, there was a significant reduction in the rate of severe acute GVHD, whereas chronic GVHD at 1 year was 0% in Treg‐treated patients and 14% in patients who received the conventional therapy with immunosuppressives (sirolimus and mycophenolate mofetil) [147]. All the aforementioned studies reported a purity of approximately 90%, demonstrat‐ ing that this approach is feasible; on the other hand, survival of Tregs *in vivo* (after infusion) was limited with a dramatic decline after 14 days from infusion. There are several currently ongoing clinical trials based on adoptive Treg transfer mainly in solid organ transplantation [147]. Such therapeutic approaches have not been published yet in lupus patients; one phase I clinical trial aiming to assess Treg efficacy in cutaneous lupus started in 2015 [148].

#### **5.2. Hematopoietic and mesenchymal stem cell transplantation**

Hematopoietic and mesenchymal stem cell transplantation (HSCT and MSCT, respectively) aim at immune reconstitution after intensive chemotherapy and have been implemented in cases with refractory autoimmune diseases.

In the context of SLE, HSCT has been shown to induce long‐term remission for approximately 5 years in half patients, whereas relapse was usually mild [149, 150]. On the other hand, MSCT exerts potent immunosuppressive capacity since mesenchymal stem cells do not require MHC (major histocompatibility complex) restriction for their function [151]. The effects of these ther‐ apeutic approaches on Tregs numbers and function have a critical role with respect to their efficacy. Zhang et al. showed that CD4+CD25highFOXP3+ Tregs were reconstituted in levels com‐ parable to those of normal individuals after autologous HSCT in 15 SLE patients [152]. In addi‐ tion, a novel Tregs subset (CD8+LAPhighCD103high) was induced and capable of maintaining remission through TGF‐β mediated suppression. On the contrary, Szodoray et al. did not find any significant differences in Tregs numbers (pre‐ and post‐transplant) in 12 patients with vari‐ ous systemic autoimmune diseases; only three lupus patients were enrolled in that study [153].

Concerning MSCT, a report on nine patients with refractory SLE showed good safety profile after 6 years; unfortunately, Tregs were not assessed in this study [154]. Limited case reports demonstrated a significant increase of peripheral Tregs in three lupus patients; however, clin‐ ical remission was not achieved [155, 156]. Of note, mesenchymal stem cells were shown to increase Tregs in 30 active lupus patients, in a dose‐dependent fashion, even after 1 week after transplantation, and this was sustained for 1 and 3 months after transplant [157]. In the same study, Th17 cells were accordingly reduced after 3 months.

#### **5.3. IL‐2‐based approaches**

Extensive research on IL‐2 and IL‐2 receptor (IL‐2R) biology has shed light on its critical impor‐ tance for the maintenance of immune tolerance by influencing Tregs number and function [132]. Administration of low doses of IL‐2 led to remission and decreased glucocorticoid dose in lupus patients [158], while it was shown that Tregs expansion (CD4+CD25highCD127low) and a decrease in T effectors/Tregs ratio were the primary mechanism [159]. The same results were observed in other diseases, such as GVHD and HCV‐related vasculitis [160]. Interestingly, IL‐2/anti‐IL‐2 immunocomplexes were capable of reducing the severity of renal inflammation in NZB/W F1 mice by inducing CD4+CD25+FOXP3+ Tregs. With regard to proteinuria, this approach was superior to the combination of glucocorticoids and mycophenolate mofetil, the current standard of care for LN [161].

#### **5.4. All‐trans retinoic acid (atRA)**

[147]. All the aforementioned studies reported a purity of approximately 90%, demonstrat‐ ing that this approach is feasible; on the other hand, survival of Tregs *in vivo* (after infusion) was limited with a dramatic decline after 14 days from infusion. There are several currently ongoing clinical trials based on adoptive Treg transfer mainly in solid organ transplantation [147]. Such therapeutic approaches have not been published yet in lupus patients; one phase I

Hematopoietic and mesenchymal stem cell transplantation (HSCT and MSCT, respectively) aim at immune reconstitution after intensive chemotherapy and have been implemented in

In the context of SLE, HSCT has been shown to induce long‐term remission for approximately 5 years in half patients, whereas relapse was usually mild [149, 150]. On the other hand, MSCT exerts potent immunosuppressive capacity since mesenchymal stem cells do not require MHC (major histocompatibility complex) restriction for their function [151]. The effects of these ther‐ apeutic approaches on Tregs numbers and function have a critical role with respect to their efficacy. Zhang et al. showed that CD4+CD25highFOXP3+ Tregs were reconstituted in levels com‐ parable to those of normal individuals after autologous HSCT in 15 SLE patients [152]. In addi‐ tion, a novel Tregs subset (CD8+LAPhighCD103high) was induced and capable of maintaining remission through TGF‐β mediated suppression. On the contrary, Szodoray et al. did not find any significant differences in Tregs numbers (pre‐ and post‐transplant) in 12 patients with vari‐ ous systemic autoimmune diseases; only three lupus patients were enrolled in that study [153]. Concerning MSCT, a report on nine patients with refractory SLE showed good safety profile after 6 years; unfortunately, Tregs were not assessed in this study [154]. Limited case reports demonstrated a significant increase of peripheral Tregs in three lupus patients; however, clin‐ ical remission was not achieved [155, 156]. Of note, mesenchymal stem cells were shown to increase Tregs in 30 active lupus patients, in a dose‐dependent fashion, even after 1 week after transplantation, and this was sustained for 1 and 3 months after transplant [157]. In the same

Extensive research on IL‐2 and IL‐2 receptor (IL‐2R) biology has shed light on its critical impor‐ tance for the maintenance of immune tolerance by influencing Tregs number and function [132]. Administration of low doses of IL‐2 led to remission and decreased glucocorticoid dose in lupus patients [158], while it was shown that Tregs expansion (CD4+CD25highCD127low) and a decrease in T effectors/Tregs ratio were the primary mechanism [159]. The same results were observed in other diseases, such as GVHD and HCV‐related vasculitis [160]. Interestingly, IL‐2/anti‐IL‐2 immunocomplexes were capable of reducing the severity of renal inflammation in NZB/W F1 mice by inducing CD4+CD25+FOXP3+ Tregs. With regard to proteinuria, this approach was superior to the combination of glucocorticoids and mycophenolate mofetil, the

clinical trial aiming to assess Treg efficacy in cutaneous lupus started in 2015 [148].

**5.2. Hematopoietic and mesenchymal stem cell transplantation**

study, Th17 cells were accordingly reduced after 3 months.

**5.3. IL‐2‐based approaches**

current standard of care for LN [161].

cases with refractory autoimmune diseases.

136 Lupus

This approach has been used in various autoimmune diseases with inconsistent and contra‐ dictory results, possibly due to the small sample sizes [162]. Limited data in lupus patients showed that Tregs could be induced by atRA [163]; however, these results were not confirmed [164]. In a more recent study, retinoic acid increased Treg numbers (and decreased Th17 cells) in lupus patients with low levels of vitamin A [165].

#### **5.5. Tolerogenic peptides**

The rationale behind the use of tolerogenic peptides in SLE therapeutics is that a dysregulated immune system can be modified by inducing tolerance against a specific antigen. This is a cru‐ cial component of this approach since non‐specific tolerance may lead to generalized immune suppression and secondary immunodeficiency. In this context, such different molecules (hCDR1, pCons, P140, etc.) have been administered in lupus prone mice with subsequent expansion of Tregs and suppression of effector cells and pro‐inflammatory cytokines [166, 167]. These encouraging results led to the first peptide‐based randomized controlled trial in SLE with 149 patients [168]. Although the effect on Tregs was not assessed, approximately 62% of the peptide‐treated patients achieved the primary clinical end‐point as compared to 38.6% of the placebo arm (all patients received standard of care therapy).

#### **5.6. Effect of other medications on Tregs**

Apart from the aforementioned approaches that implicate Tregs in their mechanism of action, medications commonly used in SLE patients have been demonstrated to increase their num‐ bers and/or restore their function. Several studies have demonstrated a significant Tregs expansion after treatment with glucocorticoids [121, 122, 169–171]. Moreover, intravenous methylprednisolone pulses led to a dramatic and sustained increase in CD4+CD25highFOXP3+ Tregs numbers, regardless of the initial clinical indication [121]; this was noted even from the first few days after the pulses [172]. The mechanism by which these medications lead to Treg proliferation is yet unknown; however, a steroid‐mediated up‐regulation of FOXP3 has been described [171].

Immunosuppressive drugs have also been shown to affect Tregs in active SLE. Cyclophosphamide pulse therapy led to a significant increase in Tregs numbers after the 4th month of administra‐ tion, which reflected clinical remission [121], although the effect of concomitant glucocorticoid treatment may have a role. Similar results were obtained with azathioprine and hydroxychlo‐ roquine [121]. Of note, polyclonal intravenous immunoglobulins (IVIGs) also led to Tregs increase, possibly through up‐regulation of FOX3 and intracellular IL‐10 and TGF‐β [173]. Rituximab was demonstrated to enhance the Tregs numbers and function in lupus patients whereas the increased and sustained FOXP3 mRNA expression was associated with favour‐ able outcome [174]. In general, *in vivo* expansion of Tregs after treatment might be the result of a change of Th1/Th17 to Th2 balance, which could lead to disease remission and not a direct drug‐specific reaction [121].

Other medications that are increasingly used in lupus patients and may affect Tregs include statins. These drugs display multiple beneficial effects in atherosclerosis through different mechanisms among which immune modulation is critical [175]. Several experiments in ani‐ mal models showed that statins increase the numbers and suppressive capacity of Tregs as well as their accumulation in the atherosclerotic plaque [176]. Atorvastatin, in particular, exerted similar results in human Tregs [177].

All the pre‐mentioned therapeutic interventions are summarized in **Table 1**.

#### **5.7. Barriers in Tregs‐based therapeutic approaches**

Although the above‐mentioned data are encouraging for SLE patients, several challenges still exist. The multiple phenotypes that have been used to characterize Tregs in the different stud‐ ies have demonstrated that all Tregs are not functionally capable of suppressing autoimmune responses [160]. In the chronic inflammatory environment of SLE, it cannot be predicted which regulatory cells are likely to function more beneficially; furthermore, effector cells are more capable of escaping regulatory mechanisms under these circumstances [106]. Furthermore, tissue distribution of Tregs, after infusion, is unknown, while their survival and maintenance of regulatory capacity have not been precisely defined in the context of SLE. Other consider‐ ations include technical aspects, such as the purity and cost‐effectiveness of these approaches.


**Table 1.** Therapeutic approaches targeting Tregs in SLE.

#### **6. Conclusion**

Other medications that are increasingly used in lupus patients and may affect Tregs include statins. These drugs display multiple beneficial effects in atherosclerosis through different mechanisms among which immune modulation is critical [175]. Several experiments in ani‐ mal models showed that statins increase the numbers and suppressive capacity of Tregs as well as their accumulation in the atherosclerotic plaque [176]. Atorvastatin, in particular,

Although the above‐mentioned data are encouraging for SLE patients, several challenges still exist. The multiple phenotypes that have been used to characterize Tregs in the different stud‐ ies have demonstrated that all Tregs are not functionally capable of suppressing autoimmune responses [160]. In the chronic inflammatory environment of SLE, it cannot be predicted which regulatory cells are likely to function more beneficially; furthermore, effector cells are more capable of escaping regulatory mechanisms under these circumstances [106]. Furthermore, tissue distribution of Tregs, after infusion, is unknown, while their survival and maintenance of regulatory capacity have not been precisely defined in the context of SLE. Other consider‐ ations include technical aspects, such as the purity and cost‐effectiveness of these approaches.

> clinical trials in other immune‐mediated

Moderate High purification rates,

results

Moderate Ongoing phase III clinical trials

Good Regardless of the

Good Rapid induction of Tregs

Good Accumulation of Tregs

plaques

Limited clinical trials Moderate Inconsistent clinical

Limited clinical trials Moderate IL‐2/anti‐IL‐2 complexes

low Tregs survival

provided favourable results in LN

agent used, probably an epiphenomenon to disease remission

in the atherosclerotic

low vitamin A

All the pre‐mentioned therapeutic interventions are summarized in **Table 1**.

**Therapy Mechanism Approach Efficacy Notes**

diseases

Retinoids Induction of Tregs Limited clinical trials Inconsistent Mainly in patients with

and one RCT

trials

trials

Induction of Tregs Limited observational trials

Limited observational

Experimental and limited observational

Increase of Tregs pool Experimental and

exerted similar results in human Tregs [177].

Adoptive transfer of *ex vivo* expanded

HSCT/MSCT Immune system

Glucocorticoids Up‐regulation of

Statins Enhanced numbers

Immunomodulating

agents

FOXP3

**Table 1.** Therapeutic approaches targeting Tregs in SLE.

IL‐2 Enhanced survival and

reconstitution

function of Tregs

Tolerogenic peptides Induction of Tregs Experimental studies

and function of Tregs

Tregs

138 Lupus

**5.7. Barriers in Tregs‐based therapeutic approaches**

Most well‐designed studies have concluded that Tregs are significantly depleted from the periphery of active lupus SLE patients and this reduction is in accordance with disease activ‐ ity. Moreover, Tregs follow alterations in disease activity (with inverse changes) quite reli‐ ably; numeric increase is not drug specific but characterizes disease remission. Their value as an activity biomarker has been demonstrated and may be helpful in assessing disease status in controversial circumstances. Their potential to be used for therapeutic purposes, either by direct adoptive transfer or by approaches aiming to increase their numbers, is quite promis‐ ing in the field of SLE.

#### **Author details**

Konstantinos Tselios<sup>1</sup> , Alexandros Sarantopoulos2 , Ioannis Gkougkourelas2 and Panagiota Boura2 \*

\*Address all correspondence to: boura@med.auth.gr

1 Centre for Prognosis Studies in the Rheumatic Diseases, Toronto Western Hospital, University of Toronto Lupus Clinic, Toronto, ON, Canada

2 Clinical Immunology Unit, 2nd Department of Internal Medicine, Hippokration General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece

#### **References**


[20] Marson A, Kretschmer K, Frampton GM, Jacobsen ES, Polansky JK, MacIsaac KD, et al. Foxp3 occupancy and regulation of key target genes during T‐cell stimulation. Nature. 2007;**445**:931. DOI: 10.1038/nature05478

[7] Kapp JA. Special regulatory T‐cell review: Suppressors regulated but unsuppressed.

[8] Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self‐tolerance main‐ tained by activated T cells expressing IL‐2 receptor alpha‐chain (CD25). Breakdown of a single mechanism of self‐tolerance causes various autoimmune diseases. Journal of

[9] Sakaguchi S, Wing K, Miyara M. Regulatory T cells‐ a brief history and perspective. European Journal of Immunology. 2007;**37**:S116–123. DOI: 10.1002/eji.200737593

[10] Thornton A, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin‐2 production. Journal of Experimental

[11] Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. Journal of Experimental Medicine. 2001;**193**:1285–1294.

[12] Liston A, Rudensky AY. Thymic development and peripheral homeostasis of regulatory T cells. Current Opinion in Immunology. 2007;**19**:176–185. DOI: 10.1016/j.coi.2007.02.005

[13] Shevach EM. From vanilla to 28 flavours: Multiple varieties of T regulatory cells.

[14] Miyara M, Amoura Z, Parizot C, Badoual C, Dorgham K, Trad S, et al. Global natural reg‐ ulatory T cell depletion in active systemic lupus erythematosus. Journal of Immunology.

[15] Brunkow ME, Jeffery EW, Hjierrild KA, Paeper B, Clark LB, Yasayko SA, et al. Disruption of a new forkhead/winged‐helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nature Genetics. 2001;**27**:68–73. DOI: 10.1038/83784

[16] Chatila TA, Blaeser F, Ho N, Lederman HM, Voulgaropoulos C, Helms C, et al. JM2, encoding a fork head‐related protein, is mutated in X‐linked autoimmunity‐allergic dysregulation syndrome. The Journal of Clinical Investigation. 2000;**106**:R75–R81. DOI:

[17] Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, et al. X‐linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent

[18] Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X‐linked syndrome (IPEX) is caused by mutations of Foxp3. Nature Genetics. 2001;**27**:20–21. DOI: 10.1038/83713

[19] Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the tran‐ scription factor FoxP3. Science. 2003;**299**:1057–1061. DOI: 10.1126/science.1079490

Immunology. 2008;**123**:28–32. DOI: 10.1111/j.1365‐2567.2007.02773.x

Medicine. 1998;**188**:287–296. DOI: 10.1084/jem.188.2.287

Immunity. 2006;**25**:195–201. DOI: 10.1016/j.immuni.2006.08.003

2005;**175**:8392–8400. DOI: https://doi.org/10.4049/jimmunol.175.12.8392

of mouse scurfy. Nature Genetics. 2001;**27**:18–20. DOI: 10.1038/83707

Immunology. 1995;**155**:1151–1164

140 Lupus

DOI: 10.1084/jem.193.11.1285

10.1172/JCI11679


[47] Tang Q, Boden EK, Henriksen KJ, Bour‐Jordan H, Bi M, Bluestone JA. Distinct roles of CTLA‐4 and TGF‐β in CD4+CD25+ regulatory T cell function. European Journal of Immunology. 2004;**34**:2996–3005. DOI: 10.1002/eji.200425143

[34] Workman CJ, Vignali DA. Negative regulation of T cell homeostasis by lymphocyte activation gene‐3 (CD223). Journal of Immunology. 2005;**174**:688–695. DOI: https://doi.

[35] Yamaguchi T, Hirota K, Nagahama K, Ohkawa K, Takahashi T, Nomura T, et al. Control of immune responses by antigen‐specific regulatory T cells expressing the folate recep‐

[36] Tran DQ, Glass DD, Uzel G, Darnell DA, Spalding C, Holland SM, et al. Analysis of adhesion molecules, target cells and role of IL‐2 in human FOXP3+ regulatory T cell suppressor function. Journal of Immunology. 2008;**182**:2929–2938. DOI: 10.4049/

[37] Zabransky DJ, Nirschl CJ, Durham NM, Park BV, Ceccato CM, Bruno TC, et al. Phenotypic and functional properties of Helios+regulatory T cells. PLoS One. 2012;7:e34547. DOI:

[38] Shu Y, Hu Q, Long H, Chang C, Lu Q, Xiao R. Epigenetic variability of CD4+CD25+ Tregs contributes to the pathogenesis of autoimmune diseases. Clinical Reviews in Allergy & Immunology. 29 September 2016 [Epub ahead of print]. DOI: 10.1007/s12016‐016‐8590‐3

[39] Ohl K, Tenbrock K. Regulatory T cells in systemic lupus erythematosus. European

[40] Chatenoud L, Bach JF. Adaptive human regulatory T cells: Myth or reality? The Journal

[41] Barrat FJ, Cua DJ, Boonstra A, Richards DF, Crain C, Savelkoul HF, et al. In vitro genera‐ tion of interleukin 10‐producing regulatory CD4 (+) T cells is induced by immunosup‐ pressive drugs and inhibited by T helper type 1 (Th1) ‐ and Th2‐inducing cytokines.

Journal of Experimental Medicine. 2002;**195**:603–616. DOI: 10.1084/jem.20011629

[42] Kemper C, Chan AC, Green JM, Brett KA, Murphy KM, Atkinson JP. Activation of human CD4+ cells with CD3 and CD46 induces a T‐regulatory cell 1 phenotype. Nature.

[43] Levings M, Gregori S, Tresoldi E, Cazzaniga S, Bonini C, Roncarolo MG. Differentiation of Tr1 cells by immature dendritic cells requires IL‐10 but no CD25+CD4+ Tr cells. Blood.

[44] Vieira PL, Christensen JR, Minaee S, O'Neill EJ, Barrat FJ, Boonstra A, et al. IL‐10‐secret‐ ing regulatory T cells do not express Foxp3 but have comparable regulatory func‐ tion to naturally occurring CD4+CD25+ regulatory T cells. Journal of Immunology.

[45] Langier S, Sade K, Kivity S. Regulatory T cells in allergic asthma. The Israel Medical

[46] Cerwenka A, Swain SL. TGF‐β1: Immunosuppressant and viability factor for T lymphocytes. Micro‐ bes and Infection. 1999;**1**:1291–1296. DOI: http://dx.doi.org/10.1016/S1286‐4579(99)00255‐5

2004;**172**:5986–5993. DOI: https://doi.org/10.4049/jimmunol.172.10.5986.

Journal of Immunology. 2015;**45**:344–355. DOI: 10.1002/eji.201344280

of Clinical Investigation. 2006;**116**:2325–2327. DOI: 10.1172/JCI29748

2003;**421**:388–392. DOI: 10.1038/nature01315

Association Journal. 2012;**14**:180–183

2004;**105**:1162–1169. DOI: 10.1182/blood‐2004‐03‐1211

tor. Immunity. 2007;**27**:145–159. DOI: 10.1016/j.immuni.2007.04.017

org/10.4049/jimmunol.174.2.688

jimmunol.0803827

142 Lupus

10.1371/journal.pone.0034547


[72] Fife BT, Pauken KE, Eagar TN, Obu T, Wu J, Tang Q, et al. Interactions between pro‐ grammed death ligand‐1 promote tolerance by blocking the T cell receptor‐induced stop signal. Nature Immunology. 2009;**10**:1185–1192. DOI: 10.1038/ni.1790

[60] Tselios K, Boura P, Kountouras J. T regulatory cells in Helicobacter pylori‐associated

[61] Furtado GC, Curotto de Lafaille MA, Kutchukhidze N, Lafaille JJ. Interleukin 2 signal‐ ling is required for CD4 (+) regulatory T cell function. Journal of Experimental Medicine.

[62] De la Rosa M, Rutz S, Dorninger H, Scheffold A. Interleukin‐2 is essential for CD4+CD25+ regulatory T cell function. European Journal of Immunology. 2004;**34**:2480–2488. DOI:

[63] Pandiyan P, Zheng L, Ishihara S, Reed J, Lenardo MJ. CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation‐mediated apoptosis of effector CD4+ T cells. Nature

[64] Barthlott T, Moncrieffe H, Veldhoen M, Atkins CJ, Christensen J, O' Garra A, et al. CD25+CD4+ T cells compete with naive CD4+ T cells for IL‐2 and exploit it for the induc‐ tion of IL‐10. International Immunology. 2005;**17**:279–288. DOI: 10.1093/intimm/dxh207

[65] Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, et al. Immunologic self‐tolerance maintained by CD4+CD25+ regulatory T cells constitutively express‐ ing cytotoxic T lymphocyte‐associated antigen 4. Journal of Experimental Medicine.

[66] Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte‐associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intesti‐ nal inflammation. Journal of Experimental Medicine. 2000;**192**:295–302. DOI: 10.1084/

[67] Marie JC, Letterio JJ, Gavin M, Rudensky AY. TGF‐β1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. Journal of Experimental Medicine.

[68] Wan YY, Flavell RA. Regulatory T cells, transforming growth factor‐β, and immune sup‐ pression. Proceedings of the American Thoracic Society. 2007;**4**:271–276. DOI: 10.1513/

[69] Liang B, Workman C, Lee J, Chew C, Dale BM, Colonna L, et al. Regulatory T cells inhibit dendritic cells by LAG‐3 engagement of MHC class II. Journal of Immunology.

[70] Kanamaru F, Youngnak P, Hashiguchi M, Nishioka T, Takahashi T, Sakaguchi S, et al. Co‐ stimulation via glucocorticoid‐induced TNF receptor in both conventional and CD15+ regulatory CD4+ T cells. Journal of Immunology. 2004;**172**:7306–7314. DOI: https://doi.

[71] Shevach EM, Stephens GL. The GITR‐GITRL interaction: Co‐stimulation or contra‐sup‐ pression of regulatory activity? Nature Reviews Immunology. 2006;**6**:613–618. DOI:

2008;**180**:5916–5926. DOI: https://doi.org/10.4049/jimmunol.180.9.5916

diseases. Immunogastroenterology. 2013;**2**:38–46. DOI: 10.7178/ig.27

2002;**196**:851–857. DOI: 10.1084/jem.20020190

Immunology. 2007;**8**:1353–1362. DOI: 10.1038/ni1536

2000;**192**:303–310. DOI: 10.1084/jem.192.2.303

2005;**201**:1061–1067. DOI: 10.1084/jem.20042276

10.1002/eji.200425274

144 Lupus

jem.192.2.295

pats.200701‐020AW

10.1038/nri1867

org/10.4049/jimmunol.172.12.7306


[98] Suri‐Prayer E, Amar AZ, McHugh R, Natarajan K, Marqulies DH, Shevach EM. Post‐ thymectomy autoimmune gastritis, fine specificity and pathogenicity of anti‐H/K APTase‐reactive T cells. European Journal of Immunology. 1999;**29**:669–677. DOI: 10.1002/(SICI)1521‐4141(199902)29:02<669::AID‐IMMU669>3.0.CO;2‐J

[85] Zarek PE, Huang CT, Lutz ER, Kowalski J, Horton MR, Linden J, et al. A2A receptor sig‐ nalling promotes peripheral tolerance by inducing T‐cell anergy and the generation of adaptive regulatory T cells. Blood. 2008;**111**:251–259. DOI: 10.1182/blood‐2007‐03‐081646

[86] Ernst PB, Garrison JC, Thompson LF. Much ado about adenosine: Adenosine synthesis and function in regulatory T cell biology. Journal of Immunology. 2010;**185**:1993–1998.

[87] Tadokoro CE, Shakhar G, Shen S, Ding Y, Lino AC, Maraver A, et al. Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. Journal of

[88] Houot R, Perrot I, Garcia E, Durand I, Lebecque S. Human CD4+CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation. Journal of Immunology. 2006;**176**:5293–5298. DOI: https://doi.org/10.4049/jimmunol.176.9.5293

[89] Cederbom L, Hall H, Ivars F. CD4+CD25+ regulatory T cells down‐regulate co‐stimulatory molecules on antigen‐presenting cells. European Journal of Immunology. 2000;**30**:1538–

[90] Oderup C, Cederbom L, Makowska A, Cilio CM, Ivars F. Cytotoxic T lymphocyte anti‐ gen‐4‐dependent down‐modulation of costimulatory molecules on dendritic cells in CD4+CD25+ regulatory T‐cell‐mediated suppression. Immunology. 2006;**118**:240–249.

[91] Kornete M, Piccirillo CA. Functional crosstalk between dendritic cells and Foxp3(+) regulatory T cells in the maintenance of immune tolerance. Frontiers in Immunology.

[92] Liu X, Nurieva RI, Dong C. Transcriptional regulation of follicular T‐helper (Tfh) cells.

[93] Burchill MA, Yang J, Vogtenhuber C, Blazar BR, Farrar MA. IL‐2 receptor beta‐dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. Journal of

Immunology. 2007;**178**:280–290. DOI: https://doi.org/10.4049/jimmunol.178.1.280

[94] Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regula‐ tory T cells. Nature Reviews Immunology. 2011;**11**:119–130. DOI: 10.1038/nri2916

[95] Chauhdry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A, et al. CD4+ regulatory T cells control Th17 responses in a STAT3‐dependent manner. Science. 2009;**326**:986–991.

[96] Wan YY. Regulatory T cells: Immune suppression and beyond. Cellular & Molecular

[97] McClymont SA, Putnam AL, Lee MR, Esensten JH, Liu W, Hulme MA, et al. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. Journal

of Immunology. 2011;**186**:3918–3926. DOI: 10.4049/jimmunol.1003099

Immunological Reviews. 2013;**252**:139–145. DOI: 10.1111/imr.12040

Immunology. 2010;**7**:204–210. DOI: 10.1038/cmi.2010.20

1543. DOI: 10.1002/1521‐ 4141(200006)30:6<1538::AID‐IMMU1538>3.0.CO;2‐X

Experimental Medicine. 2006;**203**:505–511. DOI: 10.1084/jem.20050783

DOI: 10.4049/jimmunol.1000108

146 Lupus

DOI: 10.1111/j.1365‐ 2567.2006.02362.x

DOI: 10.1126/science.1172702

2012;**3**:165. DOI: 10.3389/fimmu.2012.00165


[122] Tselios K, Sarantopoulos A, Gkougkourelas I, Papagianni A, Boura P. Increase of peripheral T regulatory cells during remission induction with cyclophosphamide in active systemic lupus erythematosus. International Journal of Rheumatic Diseases. 2014;**17**:790–795. DOI:10.1111/1756‐185X.12500

[111] Lyssouk EY, Torgashina AV, Soloviev SK, Nassonov EL, Bykovskaia SN. Reduced num‐ ber and function of CD4+CD25highFOXP3+ regulatory T cells in patients with systemic lupus erythematosus. Advances in Experimental Medicine and Biology. 2007;**601**:113–

[112] Barath S, Aleksza M, Tarr T, Sinka S, Szegedi G, Kiss E. Measurement of natural (CD4+CD25high) and inducible (CD4+IL‐10+) regulatory T cells in patients with sys‐ temic lupus erythematosus. Lupus. 2007;**16**:488–496. DOI: 10.1177/0961203307080226

[113] Tselios K, Sarantopoulos A, Gkougkourelas I, Boura P. CD4+CD25highFOXP3+ T regu‐ latory cells as a biomarker of disease activity in systemic lupus erythematosus: A pro‐

[114] Yates J, Whittington A, Mitchell P, Lechler RI, Lightstone L, Lombardi G. Natural regu‐ latory T cells: Number and function are normal in the majority of patients with sys‐ temic lupus erythematosus. Clinical and Experimental Immunology. 2008;**153**:44–55.

[115] Alvarado‐Sanchez B, Hernandez‐Castro B, Portales‐Perez D, Baranda L, Layseca‐Espinosa E, Abud‐Mendoza C, et al. Regulatory T cells in patients with systemic lupus erythema‐

tosus. Journal of Autoimmunity. 2006;**27**:110–118. DOI: 10.1016/j.jaut.2006.06.005 [116] Lin SC, Chen KH, Lin CH, Kuo CC, Ling QD, Chan CH. The quantitative analysis of peripheral blood FOXP3‐expressing T cells in systemic lupus erythematosus and rheu‐ matoid arthritis patients. European Journal of Clinical Investigation. 2007;**37**:987–996.

[117] Venigalla RK, Tretter T, Krienke S, Max R, Eckstein V, Blank N, et al. Reduced CD4+CD25‐ T cell sensitivity to the suppressive function of CD4+CD25highCD127low regulatory T cells in patients with active systemic lupus erythematosus. Arthritis and

[118] Zhang B, Zhang X, Tang F, Zhu L, Liu Y. Reduction of forkhead box P3 levels in CD4+CD25high T cells in patients with new‐onset systemic lupus erythematosus. Clinical and Experimental Immunology. 2008;**153**:182–187. DOI: 10.1111/j.1365‐2249.

[119] Kuhn A, Beissert S, Krammer PH. CD4+CD25+ regulatory T cells in systemic lupus erythematosus. Archives of Dermatological Research. 2009;**301**:71–81. DOI: 10.1007/

[120] Yang HX, Zhang W, Zhao LD, Li Y, Zhang FC, Tang FL, et al. Are CD4+CD25‐FOXP3+ cells in untreated new‐onset lupus patients regulatory T cells? Arthritis Research &

[121] Tselios K, Sarantopoulos A, Gkougkourelas I, Boura P. The influence of therapy on CD4+CD25highFOXP3+ regulatory T cells in systemic lupus erythematosus patients: A prospective study. Scandinavian Journal of Rheumatology. 2015;**44**:29–35. DOI:

spective study. Clinical and Experimental Rheumatology. 2014;**32**:630–639

119. DOI: 10.1007/978‐0‐387‐72005‐0\_12

148 Lupus

DOI: 10.1111/j.1365‐2249.2008.03665.x

DOI: 10.1111/j.1365‐2362.2007.01882.x

Therapy. 2009;**11**:R153. DOI: 10.1186/ar2829

10.3109/03009742.2014.922214

2008.03686.x

s00403‐008‐0891‐9

Rheumatism. 2008;**58**:2120–2130. DOI: 10.1002/art.23556


T cells prolongs survival of pancreatic islets‐ results of one year follow‐up. Clinical Immunology. 2014;**153**:23–30. DOI: 10.1016/j.clim.2014.03.016

[146] Theil A, Tuve S, Oelschlagel U, Maiwald A, Dohler D, Obmann D, et al. Adoptive trans‐ fer of allogeneic regulatory T cells into patients with chronic graft‐versus‐host disease. Cytotherapy. 2015;**17**:473–486. DOI: 10.1016/j.jcyt.2014.11.005

[134] Qin A, Wen Z, Zhou Y, Li Y, Li Y, Luo J, et al. MicroRNA‐126 regulates the induction and function of CD4+FOXP3+ regulatory T cells through PI3K/AKT pathway. Journal

of Cellular and Molecular Medicine. 2013;**17**:252–264. DOI: 10.1111/jcmm.12003 [135] Zhao M, Liang GP, Tang MN, Luo SY, Zhang J, Cheng WJ, et al. Total glucosides of paeony induces regulatory CD4+CD25+ T cells by increasing FOXP3 demethyl‐ ation in lupus CD4+ T cells. Clinical Immunology. 2012;**143**:180–187. DOI: 10.1016/j.

[136] Regna NL, Chafin CB, Hammond SE, Puthiyaveetil AG, Caudell DL, Reilly CM. Class I and II histone deacetylase inhibition by ITF2357 reduces SLE pathogenesis in vivo.

[137] Koga T, Mizui M, Yoshida N, Otomo K, Lieberman LA, Crispin JC, et al. KN‐93, an inhibitor of calcium/calmodulin‐dependent protein kinase IV promotes generation and function of FOXP3+ regulatory T cells in MRL/lpr MICE. Autoimmunity. 2014;**47**:445–

[138] Stohl W. Future prospects in biologic therapy for systemic lupus erythematosus. Nature

[139] Zheng SG, Wang JH, Koss MN, Quismorio Jr. F, Gray JD, Horwitz DA. CD4+ and CD8+ regulatory T cells generated ex vivo with IL‐2 and TGF‐beta suppress a stimu‐ latory graft‐versus‐host disease with a lupus‐like syndrome. Journal of Immunology

[140] Scalapino KJ, Tang Q, Bluestone JA, Bonyhadi ML, Daikh DI. Suppression of disease in New Zealand black/New Zealand white lupus‐prone mice by adoptive transfer of ex vivo expanded regulatory T cells. Journal of Immunology. 2006;**177**:1451–1459. DOI:

[141] Lan Q, Zhou X, Fan H, Chen M, Wang J, Ryffel B, et al. Polyclonal CD4+Foxp3+ Treg cells induce TGFβ‐dependent tolerogenic dendritic cells that suppress the murine lupus‐like syndrome. The Journal of Molecular Cell Biology. 2012;**4**:409–419. DOI:

[142] Cao T, Wenzel SE, Faubion WA, Harriman G, Li L. Enhanced suppressive function of regulatory T cells from patients with immune‐mediated diseases following successful ex vivo expansion. Clinical Immunology. 2010;**136**:329–337. DOI: 10.1016/j.clim.2010.04.014

[143] Hahn BH, Anderson M, Le E, La Cava A. Anti‐DNA Ig peptides promote Treg cell activ‐ ity in systemic lupus erythematosus patients. Arthritis and Rheumatism. 2008;**58**:2488–

[144] Bluestone JA, Buckner JH, Fitch M, Gitelman SE, Gupta S, Hellerstein MK, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Science Translational

[145] Marek‐Trzonkowska N, Mysliwiec M, Dobyszuk A, Grabowska M, Derkowska I, Juscinska J, et al. Therapy of type 1 diabetes with CD4+CD25highCD127‐ regulatory

Medicine. 2015;**7**:315ra189. DOI: 10.1126/scitranslmed.aad4134

Reviews Rheumatology. 2013;**9**:705–720. DOI: 10.1038/nrrheum.2013.136

2004;**172**:1531–1539. DOI: https://doi.org/10.4049/jimmunol.172.3.1531

Clinical Immunology. 2014;**151**:29–42. DOI: 10.1016/j.clim.2014.01.002

clim.2012.02.002

150 Lupus

450. DOI: 10.3109/08916934.2014.915954

https://doi.org/10.4049/jimmunol.177.3.1451

10.1093/jmcb/mjs040

2497. DOI: 10.1002/art.23609


[169] Suarez A, Lopez P, Gomez J, Gutierrez C. Enrichment of CD4+CD25high T cell pop‐ ulation in patients with systemic lupus erythematosus treated with glucocorticoids. Annals of the Rheumatic Diseases. 2006;**65**:1512–1517. DOI: 10.1136/ard.2005.049924

[157] Wang D, Huang S, Yuan X, Liang J, Xu R, Yao G, et al. The regulation of the Treg/ Th17 balance by mesenchymal stem cells in human systemic lupus erythematosus. Cellular & Molecular Immunology 5 October 2015 [Epub ahead of print]. DOI: 10.1038/

[158] He J, Zhang X, Wei Y, Sun X, Chen Y, Deng J, et al. Low‐dose interleukin‐2 treatment selectively modulated CD4+ T cell subsets in patients with systemic lupus erythemato‐

[159] Von Spee‐Mayer C, Siegert E, Abdirama D, Rose A, Klaus A, Alexander T, et al. Low‐ dose interleukin‐2 selectively corrects regulatory T cell defects in patients with sys‐ temic lupus erythematosus. Annals of the Rheumatic Diseases. 2016;**75**:1407–1415. DOI:

[160] Giang S, La Cava A. Regulatory T cells in SLE: Biology and use in treatment. Current

[161] Yan JJ, Lee JG, Jang JY, Koo TY, Ahn C, Yang J. IL‐2/anti‐IL‐2 complexes ameliorates lupus nephritis by expansion of CD4+CD25+FOXP3+ regulatory T cells. Kidney International. 30 November 2016 [Epub ahead of print]. DOI: 10.1016/j.kint.2016.09.022

[162] Miyabe Y, Miyabe C, Nanki T. Could retinoids be a potential treatment for rheumatic diseases? Rheumatology International. 2015;**35**:35–41. DOI: 10.1007/s00296‐014‐3067‐2

[163] Lu L, Ma J, Li Z, Lan Q, Chen M, Liu Y, et al. All‐trans retinoic acid promotes TGF‐β‐ induced Tregs via histone modification but not DNA demethylation on Foxp3 gene

[164] Sobel ES, Brusko TM, Butfiloski EJ, Hou W, Li S, Cuda CM, et al. Defective response of CD4+ T cells to retinoic acid and TGFβ in systemic lupus erythematosus. Arthritis

[165] Handono K, Firdausi SN, Pratama MZ, Endharti AT, Kalim H. Vitamin A improves Th17 and Treg regulation in systemic lupus erythematosus. Clinical Rheumatology.

[166] Sharabi A, Mozes E. The suppression of murine lupus by a tolerogenic peptide involves Foxp3‐expressing CD8+ cells that are required for the optimal induction and function of Foxp3‐expressing CD4 cells. Journal of Immunology. 2008;**181**:3243–3251. DOI: https://

[167] Singh RP, La Cava A, Hahn B. pConsensus peptide induces tolerogenic CD8+ T cells in lupus‐prone (NXBxNZW) F1 mice by differentially regulating Foxp3 and PD1 mol‐ ecules. Journal of Immunology. 2008;**180**:2069–2080. DOI: https://doi.org/10.4049/

[168] Zimmer R, Scherbarth HR, Rillo OL, Gomez‐Reino JJ, Muller S. Lupuzor/P140 peptide in patients with systemic lupus erythematosus: A randomized, double‐blind, placebo‐ controlled phase IIb clinical trial. Annals of the Rheumatic Diseases. 2013;**72**:1830–1835.

sus. Nature Medicine. 2016;**22**:991–993. DOI: 10.1038/nm.4148

Rheumatology Reports. 2016;**18**:67. DOI: 10.1007/s11926‐016‐0616‐6

locus. PLoS One. 2011;**6**:e24590. DOI: 10.1371/journal.pone.0024590

Research & Therapy. 2011;**13**:R106. DOI: 10.1186/ar3387

2016;**35**:631–638. DOI: 10.1007/s10067‐016‐3197‐x

doi.org/10.4049/jimmunol.181.5.3243

DOI: 10.1136/annrheumdis‐2012‐202460

jimmunol.180.4.2069

cmi.2015.89

152 Lupus

10.1136/annrheumdis‐2015‐207776


**Additional Complications in Lupus**

## **Accelerated Atherosclerosis in Patients with Systemic Lupus Erythematosus and the Role of Selected Adipocytokines in This Process**

Eugeniusz Hrycek, Iwona Banasiewicz‐Szkróbka, Aleksander Żurakowski, Paweł Buszman and Antoni Hrycek

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/68016

#### **Abstract**

Systemic lupus erythematosus (SLE) can affect various systems and organs. The most severe forms of the disease affect the kidneys, the central nervous system, and the heart. Cardiac and cardiovascular system diseases are inter alia caused by atherosclerosis, vasculitis, and thromboembolic events. Patients with SLE are at a higher risk of developing accelerated atherosclerosis. This process in SLE patients cannot be explained solely based on classical risk factors. Recently, some adipocytokines/adipokines have been indicated in the development of atherosclerosis, inflammation, and immune processes. It has also been postulated that adipokines might regulate the immune response and hence the atherogenic process. In this work, the factors contributing to accelerated atherosclerosis in SLE patients with special respect to vasculitis/vascular injury are presented, and selected adipocytokines, that is leptin, resistin, and adiponectin, with their relation to atherosclerosis and SLE, are under discussion.

**Keywords:** systemic lupus erythematosus, pathogenesis, atherosclerosis, adipocytokines, associations

#### **1. Introduction**

Systemic lupus erythematosus (SLE) is an organ‐nonspecific autoimmune disease, more prevalent in young women than in men. It is characterized by periods of varying activity, sometimes even spontaneous remission. However, there can also be life‐threatening disease flares, especially in those patients who undergo incorrect treatment.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The pathogenesis of SLE involves different factors and generally speaking, complex gene‐ environment interactions [1, 2]. More specifically, abnormal lymphocyte count (T‐helper/T‐ suppressor cell quotient) and defects in T‐ and B‐lymphocyte functions should be emphasized. Ineffective clearance of apoptotic cells [3, 4] and of immune complexes containing host autoantigens and autoantibodies may also play a role in the development of SLE. Host lipids may contribute to the formation of immune complexes leading to the production of anticardiolipin antibodies [5]. Extracellular DNA molecules generated from apoptotic cells may also contribute to SLE as they promote the origin of anti‐DNA autoantibodies, which are characteristic of the disease [6–8]. It should be noted though that the cause of SLE is not fully known.

As already mentioned, SLE is an organ‐non‐specific autoimmune disease that can affect almost any organ or system; the most severe forms affect the kidneys, the central nervous system, and the heart. Cardiac and cardiovascular system involvement may result from atherosclerosis, vasculitis, and thromboembolic lesions that are known to be interrelated processes [9, 10].

Although SLE patients constitute a small proportion of the population dying from cardiovascular events, they tend to suffer from cardiovascular complications at a young age [11]. Previous studies on SLE‐related mortality revealed that early deaths were associated with disease activity and infections, whereas late deaths frequently resulted from atherosclerotic disease [12, 13]. It is noteworthy that these patients often suffer from accelerated atherosclerosis, which is associated inter alia with lipid disturbances, vasculitis, and vascular injury. The latter result from the activity of autoantibodies and immune complexes, which, via the activation of the complement system, lead to autoimmune inflammation of the vascular wall. Long‐term side effects of lupus medications may also contribute to the development of cardiovascular disease (CVD) [4, 7, 11, 14, 15].

### **2. Factors contributing to accelerated atherosclerosis in patients with SLE with special respect to vasculitis/vascular injury**

Patients in early stages of SLE rarely exhibit cardiac manifestations. Nevertheless, in over 50% of severe cases, the heart is affected, and the patients suffer from pericarditis, myocarditis, Libman‐Sacks endocarditis, pulmonary hypertension, and coronary artery disease (CAD) the development of which is related to autoimmune processes characteristic of SLE [16].

Among CVD diagnosed in SLE patients, particular attention should be paid to angiopathy, which is due to a chronic inflammatory process within the vascular wall and underlies premature atherosclerosis [17–20]. Atherosclerosis is a progressive disease resulting from a multitude of factors, including altered composition of the extracellular matrix and activation of vascular smooth muscle cells in the arterial walls, which leads to atherosclerotic plaque formation.

Due to modern immunosuppressive therapy, prognosis in SLE patients has markedly improved. However, arterial disease (including CAD) and strokes still account for a large proportion of SLE‐related morbidity and mortality, while their pathogenesis has not been fully elucidated [21]. It has been estimated that the incidence of acute coronary syndromes is 50‐fold higher in patients with SLE compared to the control [19, 22–25], while the CAD mortality rate ranges from 3.5 to 15.7% [26]. It should be noted that diagnostic imaging revealed subclinical atherosclerosis in 30–52% of SLE patients [24, 25, 27, 28].

The pathogenesis of SLE involves different factors and generally speaking, complex gene‐ environment interactions [1, 2]. More specifically, abnormal lymphocyte count (T‐helper/T‐ suppressor cell quotient) and defects in T‐ and B‐lymphocyte functions should be emphasized. Ineffective clearance of apoptotic cells [3, 4] and of immune complexes containing host autoantigens and autoantibodies may also play a role in the development of SLE. Host lipids may contribute to the formation of immune complexes leading to the production of anticardiolipin antibodies [5]. Extracellular DNA molecules generated from apoptotic cells may also contribute to SLE as they promote the origin of anti‐DNA autoantibodies, which are characteristic of

the disease [6–8]. It should be noted though that the cause of SLE is not fully known.

diovascular disease (CVD) [4, 7, 11, 14, 15].

158 Lupus

**with special respect to vasculitis/vascular injury**

As already mentioned, SLE is an organ‐non‐specific autoimmune disease that can affect almost any organ or system; the most severe forms affect the kidneys, the central nervous system, and the heart. Cardiac and cardiovascular system involvement may result from atherosclerosis, vasculitis, and thromboembolic lesions that are known to be interrelated processes [9, 10].

Although SLE patients constitute a small proportion of the population dying from cardiovascular events, they tend to suffer from cardiovascular complications at a young age [11]. Previous studies on SLE‐related mortality revealed that early deaths were associated with disease activity and infections, whereas late deaths frequently resulted from atherosclerotic disease [12, 13]. It is noteworthy that these patients often suffer from accelerated atherosclerosis, which is associated inter alia with lipid disturbances, vasculitis, and vascular injury. The latter result from the activity of autoantibodies and immune complexes, which, via the activation of the complement system, lead to autoimmune inflammation of the vascular wall. Long‐term side effects of lupus medications may also contribute to the development of car-

**2. Factors contributing to accelerated atherosclerosis in patients with SLE** 

Patients in early stages of SLE rarely exhibit cardiac manifestations. Nevertheless, in over 50% of severe cases, the heart is affected, and the patients suffer from pericarditis, myocarditis, Libman‐Sacks endocarditis, pulmonary hypertension, and coronary artery disease (CAD) the development of which is related to autoimmune processes characteristic of SLE [16].

Among CVD diagnosed in SLE patients, particular attention should be paid to angiopathy, which is due to a chronic inflammatory process within the vascular wall and underlies premature atherosclerosis [17–20]. Atherosclerosis is a progressive disease resulting from a multitude of factors, including altered composition of the extracellular matrix and activation of vascular smooth muscle cells in the arterial walls, which leads to atherosclerotic plaque formation.

Due to modern immunosuppressive therapy, prognosis in SLE patients has markedly improved. However, arterial disease (including CAD) and strokes still account for a large proportion of SLE‐related morbidity and mortality, while their pathogenesis has not been fully elucidated [21]. It has been estimated that the incidence of acute coronary syndromes is 50‐fold higher The mechanisms of accelerated atherosclerosis in SLE patients are not fully understood and remain controversial. Its development cannot be accounted for based on traditional risk factors such as age, male sex, arterial hypertension, abnormalities in serum lipids, smoking, diabetes mellitus, obesity, and abnormal results of laboratory tests including high levels of C‐reactive protein (CPR), fibrinogen, and homocysteine [4, 11, 29–34]. Other causative factors that might promote accelerated atherosclerosis should also be considered [35].

It has been suggested that atherosclerosis could be caused by an immune reaction against autoantigens at the endothelial level, which include oxidized low‐density lipoprotein (LDL) and heat shock proteins (HSP) 60/65. Endothelial dysfunction plays a key role. It has also been speculated that immune mechanisms might be responsible for conversion of stable to instable plaque with resultant rupture [36].

Thus, it is not surprising that several autoimmune diseases, for example, SLE and antiphospholipid syndrome, are considered to raise the risk of CAD [37]; nevertheless, the precise mechanism is yet to be defined [38]. Multiple researchers believe that atherosclerosis is associated with immune responses [37, 39, 40]; it should be emphasized though that considering atherosclerosis as a solely autoimmune condition would be an oversimplification since metabolic disorders and hemodynamic factors are also involved in its development [41].

Although there is a lot of evidence that inflammation plays a central role in atherosclerosis, its pathogenesis is also associated with other risk factors often connected with SLE, that is arterial hypertension (especially renal hypertension), prolonged exposure to high doses of glucocorticoids (which, apart from having a beneficial anti‐inflammatory action, also influence blood pressure and glucose metabolism), lupus‐associated antiphospholipid syndrome, diabetes mellitus, and hypercholesterolemia [23, 25]. It should be noted that a regimen of ≤ 10 mg prednisone daily is considered safe in this respect [25]; however, the problem of metabolic disorders seen in SLE patients and its relation to glucocorticoid doses has not been satisfactorily elucidated. Contrary to steroids, antimalarial drugs used in SLE patients have a beneficial effect on their lipid profile.

Patients with SLE also exhibit other metabolic disturbances that may promote accelerated atherosclerosis and accelerated CAD. These include hypertriglyceridemia, high homocysteine levels, and early menopause [25].

The multifactorial etiology of atherosclerosis makes it difficult to unambiguously determine why SLE patients develop atherosclerotic lesions earlier in life and more frequently than the general population. Researchers are often confronted with inconsistent results [23, 42]; hence, it has been suggested SLE might be considered an independent risk factor for atherosclerosis including CAD [24, 43–45] and CVD [23].

The factors underlying the atherosclerotic process undoubtedly comprise pro‐inflammatory cytokines, antiphospholipid antibodies, antiendothelial cell antibodies (AECAs), and antineutrophil cytoplasmic antibodies (ANCAs), all acting directly on blood vessels or forming deposits of immune complexes. Monocyte chemotactic protein‐1 (MCP‐1) [7] is also involved; its higher concentrations were revealed in the blood of our study participants with mild‐to‐moderate SLE [46]. Other researchers investigated the relationships between atherosclerotic plaques in the carotid arteries, antiphospholipid antibodies, and peripheral blood leukocyte count, an established indicator of inflammation [25]. There is also a spectrum of vascular abnormalities in SLE, resulting from adverse effects of several drugs or induced by infections, etc. [15].

It is important to note that vascular injury may develop not only due to an inflammatory condition but also as a result of non‐inflammatory factors including environmental influences (toxicity, medication, or micro‐organisms), neoplastic process, etc. [14]. Vascular disease in SLE patients can occur due to a combination of different pathological processes, for example, atherosclerosis, clotting disorders, and systemic vasculitis associated with vascular wall injury (especially endothelial dysfunction), caused by an autoimmune process [24, 28].

SLE patients most typically exhibit cutaneous vasculitis; systemic vasculitis develops in 10–18% of these patients and, as a life‐threatening condition, may require aggressive therapy [14, 15].

Recently, it has been suggested that adipokines might play a causative role in the development of atherosclerosis, inflammatory, and immune processes [47]. These factors, secreted by the white adipose tissue, have autocrine‐like actions, locally affecting adipocyte biology. They also act as endocrine factors that regulate systemic processes, for example, food intake, insulin sensitivity, bone growth and energy homeostasis, and affect development of obesity and metabolic syndrome [40, 48]. Adipokines have been indicated in the link between immune response and atherosclerotic process [49].

Since patients with SLE develop metabolic syndrome, insulin resistance, dyslipidemia, or hypertension more frequently than the general population [50–54], the interest in the adipose tissue is justified in the group of rheumatic diseases [55, 56]. Identification of mechanisms common to inflammation and CVD might be of considerable interest especially in the context SLE, which is, potentially, a model disease for gaining a deeper insight into such mechanisms [13]. Generally, the relationship of adipokines to inflammation and coronary atherosclerosis in patients with SLE has not been fully elucidated [57]; for example, it has not been determined whether adiponectin concentrations in SLE result from metabolic disorders or inflammatory processes and nor has it been determined whether adipokine abnormalities associated with connective tissue diseases contribute to disease development or are caused by inflammation induced by other pro‐inflammatory factors [58].

#### **3. Adipocytokines and their relation to atherosclerosis and systemic lupus erythematosus**

White adipose tissue is a loose connective tissue composed of adipocytes and also containing adipocyte precursors, immune system cells fibroblasts, and other cell types [59]. Previously, this tissue had been considered an energy store (triglycerides) but now it is known to produce a number of biologically active substances that act at autocrine, paracrine, and endocrine levels. They regulate homeostasis through regulation of food intake, energy balance, lipid, and carbohydrate metabolism. They also modulate the insulin effects, angiogenesis, and vascular remodeling, regulate arterial pressure, affect inflammatory processes as well as associated immune response, and have metabolic effects including an impact on the development of atherosclerosis [47, 50, 57, 59–63]. Since the structure of these substances resembles that of the cytokine family, they have been referred to as adipocytokines or adipokines [60]. Their multifunctionality underlies the relationship between white adipose tissue, metabolic disorders, and autoimmune diseases [59]. Actions of selected adipokines are summarized in **Table 1**.

immune complexes. Monocyte chemotactic protein‐1 (MCP‐1) [7] is also involved; its higher concentrations were revealed in the blood of our study participants with mild‐to‐moderate SLE [46]. Other researchers investigated the relationships between atherosclerotic plaques in the carotid arteries, antiphospholipid antibodies, and peripheral blood leukocyte count, an established indicator of inflammation [25]. There is also a spectrum of vascular abnormalities in SLE, resulting

It is important to note that vascular injury may develop not only due to an inflammatory condition but also as a result of non‐inflammatory factors including environmental influences (toxicity, medication, or micro‐organisms), neoplastic process, etc. [14]. Vascular disease in SLE patients can occur due to a combination of different pathological processes, for example, atherosclerosis, clotting disorders, and systemic vasculitis associated with vascular wall injury (especially endothelial dysfunction), caused by an autoimmune process [24, 28].

SLE patients most typically exhibit cutaneous vasculitis; systemic vasculitis develops in 10–18% of these patients and, as a life‐threatening condition, may require aggressive therapy [14, 15]. Recently, it has been suggested that adipokines might play a causative role in the development of atherosclerosis, inflammatory, and immune processes [47]. These factors, secreted by the white adipose tissue, have autocrine‐like actions, locally affecting adipocyte biology. They also act as endocrine factors that regulate systemic processes, for example, food intake, insulin sensitivity, bone growth and energy homeostasis, and affect development of obesity and metabolic syndrome [40, 48]. Adipokines have been indicated in the link between immune

Since patients with SLE develop metabolic syndrome, insulin resistance, dyslipidemia, or hypertension more frequently than the general population [50–54], the interest in the adipose tissue is justified in the group of rheumatic diseases [55, 56]. Identification of mechanisms common to inflammation and CVD might be of considerable interest especially in the context SLE, which is, potentially, a model disease for gaining a deeper insight into such mechanisms [13]. Generally, the relationship of adipokines to inflammation and coronary atherosclerosis in patients with SLE has not been fully elucidated [57]; for example, it has not been determined whether adiponectin concentrations in SLE result from metabolic disorders or inflammatory processes and nor has it been determined whether adipokine abnormalities associated with connective tissue diseases contribute to disease development or are caused by inflamma-

**3. Adipocytokines and their relation to atherosclerosis and systemic** 

White adipose tissue is a loose connective tissue composed of adipocytes and also containing adipocyte precursors, immune system cells fibroblasts, and other cell types [59]. Previously, this tissue had been considered an energy store (triglycerides) but now it is known to produce a number of biologically active substances that act at autocrine, paracrine, and endocrine levels. They regulate homeostasis through regulation of food intake, energy balance, lipid,

from adverse effects of several drugs or induced by infections, etc. [15].

response and atherosclerotic process [49].

160 Lupus

tion induced by other pro‐inflammatory factors [58].

**lupus erythematosus**

The role of adipokines in atherosclerosis deserves particular attention as they modulate inflammatory processes and initiate its development [60]. Adipokines may constitute a link between impaired insulin sensitivity, obesity, chronic inflammation, and atherosclerosis in patients with SLE [57, 64]. It has been speculated that altered serum/plasma levels of adipokines in SLE might be related to coronary atherosclerosis, insulin resistance, and the inflammatory process [57]; however, these correlations need to be further explored and documented [65]. A deep insight into the mechanisms of adipokine actions would help develop new therapies—also for autoimmune disorders [58, 66].

It is assumed that resistin or leptin have pro‐inflammatory and proatherosclerotic effects; they have also been implicated in insulin resistance [57, 67]. Conversely, adiponectin has an inverse association with inflammatory states, atherosclerosis, and insulin resistance [68]; hence, independent of traditional risk factors, low level of adiponectin might also contribute to the development of the abovementioned diseases [57, 69]. However, other studies did not confirm these causative associations [69–71] with respect to total adiponectin levels but only to some adiponectin isoforms determined in the serum [69]. The reported research results are therefore inconsistent.

Resistin plays an important role in the inflammatory process, but its amount in adipocytes is quite small. Greater resistin concentrations have been found in adipose tissue monocytes and macrophages and peripheral blood monocytes [72, 73]. It is also present in neutrophils and is capable of inducing the production of IL‐6 and TNFα [42, 74–76]. These facts may indicate pro‐inflammatory properties of resistin [73]. Patients with severe inflammatory disease exhibit significant increases in plasma resistin [77]. It is also noteworthy that endothelial cells exhibit sensitivity to resistin.


**Table 1.** Relationships between selected adipokines and disease processes in humans.

Although the role of resistin in SLE has not been fully determined [57, 58], its concentrations in the peripheral blood of SLE patients are elevated and have been found to correlate with inflammatory markers, glomerular filtration rate, and glucocorticoid therapy [75, 78]. However, other studies did not reveal significant differences in serum resistin between patients with SLE and control participants [78]. Hence, reports on resistin levels in SLE patients are not consistent and its role remains to be elucidated.

It has been argued that resistin concentrations might be predictive of coronary atherosclerosis, acute coronary events, and associated mortality [74, 78, 79]. It has been hypothesized that resistin secreted from macrophages in atheromas could affect vascular cell function and promote atherosclerosis [80]. Furthermore, it has been suggested that the levels of serum resistin might help determine the severity of myocardial ischemia [81], and its reduction could possibly reduce the risk for CVD [47]. It has also been speculated that this adipokine is more related to the inflammatory process and atherosclerosis than to obesity and insulin resistance [47].

Plasma leptin is known to be proportional to the total amount of adipose tissue, and therefore it is directly related to obesity and associated CVDs including atherosclerosis. Leptin exerts its atherogenic effects via induction of endothelial dysfunction, stimulation of inflammatory response, oxidative stress, platelet aggregation, migration, hypertrophy, and proliferation of vascular smooth muscle cells [82]. It regulates blood pressure and this is probably independent of body adiposity [83]. Plasma leptin concentration correlates with markers of subclinical atherosclerosis such as extracranial carotid intima‐media thickness and coronary artery calcification. Beltowski [82] speculates that inhibition of leptin activity might slow down the progression of atherosclerosis in obese individuals with hyperleptinemia.

There are also data on the involvement of leptin in the immune response [77] and its modulatory effect on monocytes/macrophages, neutrophils, basophils, eosinophils, natural killer cells (NK), and dendritic cells [63, 84]. It has also been indicated in lymphocyte reactivity [85]. As already mentioned, leptin is considered a pro‐inflammatory adipokine; therefore, changes in its plasma levels observed in SLE patients are not surprising. Leptin modulates the cardiovascular risk in these patients [86], and several authors have suggested that the adipokine might act as an independent risk factor for CVD [68].

Patients with SLE had higher plasma leptin compared to the control [49, 57, 87, 88], but clinical relevance of leptin level changes in autoimmune disorders remains unclear [87]. Several researchers believe that leptin is involved in the pathogenesis thereof [88, 89]. The evaluation of leptin concentrations in SLE patients with correction for BMI (body mass index*)* also revealed higher levels in the study group [42]. However, other authors concluded that leptin levels in SLE patients were lower or comparable to those found in healthy controls [90, 91].

Adiponectin, the major product of adipocytes, functions as an autocrine/paracrine factor within the adipose tissue and exerts endocrine effects on distant tissues thus influencing whole‐body metabolism [92].

The role of adiponectin in SLE remains controversial. Several researchers observed an increase in adiponectin concentration in SLE patients [57, 91], while others did not find differences compared to the control [50, 58]. However, plasma adiponectin levels tend to be higher in patients with renal SLE in comparison to healthy controls and patients with non‐ renal SLE [93].

Although the role of adiponectin in SLE pathogenesis has not been fully elucidated [86], higher local and/or systemic concentrations of this adipokine have been noted in chronic inflammatory conditions including SLE [94]. Nevertheless, processes leading to adiponectin levels elevation in chronic inflammatory/autoimmune diseases are still to be clarified [94].

The significance of adiponectin in atherosclerosis also needs clarification [72]. It was postulated that the anti‐inflammatory effects of adiponectin were associated with the inhibition of pro‐inflammatory cytokines, decreased leukocyte adhesion, and enhanced production of anti‐inflammatory cytokines [95]. Adiponectin mediates inhibition of macrophage phagocytosis and decreases the production of IL‐6 and TNF. It strongly inhibits B‐lymphopoiesis, reduces T‐lymphocyte response, and induces the production of anti‐inflammatory agents (e.g., IL‐10) in human monocytes, macrophages, and dendritic cells [62, 63]. Adiponectin also suppresses monocyte adhesion to endothelial cells as well as migration and proliferation of smooth muscle cells [30]. Hence, it may exert a beneficial effect in the metabolic syndrome and coronary heart disease. Low adiponectin concentrations have been found to enhance insulin resistance and the risk for coronary heart disease. It is noteworthy, though, that, contrary to its protective role with respect to obesity and vascular disease, adiponectin seems to have pro‐inflammatory effects in joint diseases [58, 62, 63].

Summing up, it should be noted that metabolic disorders frequently seen in patients with SLE might result from the disease itself or genetic influences/long‐term treatment. Patients with SLE also tend to more frequently develop a classic metabolic disease, that is obesity, which is associated with chronic, although not severe, inflammatory conditions. The latter has an impact on insulin resistance‐related type 2 diabetes as well as on atherosclerosis and ischemic heart disease.

### **Author details**

Although the role of resistin in SLE has not been fully determined [57, 58], its concentrations in the peripheral blood of SLE patients are elevated and have been found to correlate with inflammatory markers, glomerular filtration rate, and glucocorticoid therapy [75, 78]. However, other studies did not reveal significant differences in serum resistin between patients with SLE and control participants [78]. Hence, reports on resistin levels in SLE

It has been argued that resistin concentrations might be predictive of coronary atherosclerosis, acute coronary events, and associated mortality [74, 78, 79]. It has been hypothesized that resistin secreted from macrophages in atheromas could affect vascular cell function and promote atherosclerosis [80]. Furthermore, it has been suggested that the levels of serum resistin might help determine the severity of myocardial ischemia [81], and its reduction could possibly reduce the risk for CVD [47]. It has also been speculated that this adipokine is more related to the inflammatory process and atherosclerosis than to obesity and insulin

Plasma leptin is known to be proportional to the total amount of adipose tissue, and therefore it is directly related to obesity and associated CVDs including atherosclerosis. Leptin exerts its atherogenic effects via induction of endothelial dysfunction, stimulation of inflammatory response, oxidative stress, platelet aggregation, migration, hypertrophy, and proliferation of vascular smooth muscle cells [82]. It regulates blood pressure and this is probably independent of body adiposity [83]. Plasma leptin concentration correlates with markers of subclinical atherosclerosis such as extracranial carotid intima‐media thickness and coronary artery calcification. Beltowski [82] speculates that inhibition of leptin activity might slow down the progression of atherosclerosis in obese individuals

There are also data on the involvement of leptin in the immune response [77] and its modulatory effect on monocytes/macrophages, neutrophils, basophils, eosinophils, natural killer cells (NK), and dendritic cells [63, 84]. It has also been indicated in lymphocyte reactivity [85]. As already mentioned, leptin is considered a pro‐inflammatory adipokine; therefore, changes in its plasma levels observed in SLE patients are not surprising. Leptin modulates the cardiovascular risk in these patients [86], and several authors have suggested that the adipokine

Patients with SLE had higher plasma leptin compared to the control [49, 57, 87, 88], but clinical relevance of leptin level changes in autoimmune disorders remains unclear [87]. Several researchers believe that leptin is involved in the pathogenesis thereof [88, 89]. The evaluation of leptin concentrations in SLE patients with correction for BMI (body mass index*)* also revealed higher levels in the study group [42]. However, other authors concluded that leptin levels in SLE patients were lower or comparable to those found in healthy controls [90, 91].

Adiponectin, the major product of adipocytes, functions as an autocrine/paracrine factor within the adipose tissue and exerts endocrine effects on distant tissues thus influencing

patients are not consistent and its role remains to be elucidated.

resistance [47].

162 Lupus

with hyperleptinemia.

whole‐body metabolism [92].

might act as an independent risk factor for CVD [68].

Eugeniusz Hrycek<sup>1</sup> \*, Iwona Banasiewicz‐Szkróbka<sup>1</sup> , Aleksander Żurakowski<sup>1</sup> , Paweł Buszman1,2 and Antoni Hrycek<sup>2</sup>

\*Address all correspondence to: ehrycek@gmail.com

1 American Heart of Poland, Chrzanów, Poland

2 Department of Internal, Autoimmune and Metabolic Diseases, Medical University of Silesia, Katowice, Poland

#### **References**


[15] Radic M, Kaliterna DM, Radic J. Vascular manifestations of systemic lupus erythematosus. Neth J Med 2013: 71; 10–16.

**References**

164 Lupus

2005: 26; 1–6.

wego. Dermatol Klin 2005: 7; 97–100.

erythematosus. PloS One 2013: 8; e55639.

Pol Przegl Kardiol 2011: 13; 171–176.

matosus. Pol Arch Med Wewn 2008: 118; 57–63.

Pol Merk Lek 2012: 32; 111–115.

49; 419–425.

687–690.

Immunol 2009: 70; 175–183.

[1] Hrycek A, Siekiera U, Cieślik P, Szkróbka W. HLA‐DRB1 and DQB1 alleles and gene polymorphisms of selected cytokines in systemic lupus erythematosus. Rheumatol Int

[2] Hrycek A, Olszanecka‐Glinianowicz M. Pylica płuc z towarzyszącym toczniem rumien-

[3] Jasiuk B, Reich A. Znaczenie apoptozy w patogenezie tocznia rumieniowatego układo‐

[4] Wade NS, Major AS. The problem of accelerated atherosclerosis in systemic lupus erythematosus: insights into a complex co‐morbidity. Thromb Haemost 2011: 106; 849–857.

[5] Jovanović V, Aziz NA, Lim YT, Poh ANA, Chan SJH, Pei EHX, Lew FCH, Shui G, Jenner AM, Bowen L, McKinney EF, Lyons PA, Kemeny MD, Smith KGC, Wenk MR, MacAry PA. Lipid anti‐lipid antibody responses correlate with disease activity in systemic lupus

[6] Su K‐Y, Pisetsky DS. The role of extracellular DNA in autoimmunity in SLE. Scand J

[7] Narshi CB, Giles IP, Rahman A. The endothelium: an interface between autoimmunity

[8] Hrycek A, Cieślik P. Annexin A5 and anti‐annexin antibodies in patients with systemic

[9] Irzyk K, Ciurzyński M. Zmiany naczyniowe w chorobach układowych tkanki łącznej.

[10] Gęsikowska K, Kandera‐Anasz Z, Mielczarek‐Palacz A, Sikora J, Machaj I, Smycz M. Toczeń rumieniowaty układowy – ciągle aktualny problem kliniczny i diagnostyczny.

[11] Swacha M, Więsik‐Szewczyk E, Olesińska M. Ocena ryzyka sercowo‐naczyniowego u chorych na toczeń rumieniowaty układowy – aspekty praktyczne. Reumatologia 2011:

[12] Urowitz MB, Bookman AA, Koehler BE, Gordon DA, Smythe HA, Ogryzlo MA. The bimodal mortality pattern of systemic lupus erythematosus. Am J Med 1976: 60; 221–225.

[13] Bruce IN. Atherogenesis and autoimmune disease: the model of lupus. Lupus 2005: 14;

[14] Cieślik P, Hrycek A, Kłuciński P. Vasculopathy and vasculitis in systemic lupus erythe-

and atherosclerosis in systemic lupus erythematosus? Lupus 2011: 20; 5–13.

lupus erythematosus. Rheumatol Int 2012: 32; 1335–1342.

iowatym układowym – opis przypadku. Pol Merk Lek 2008: 24; 18–19.


[41] Sherer Y, Zinger H, Shoenfeld Y. Atherosclerosis in systemic lupus erythematosus. Autoimmunity 2010: 43; 98–102.

[27] Doria A, Shoenfeld Y, Wu R, Gambari PF, Puato M, Ghirardello A, Gilburd B, Carbanese S, Patniak M, Zampieri S, Peter JB, Favaretto E, Laccarino L, Sherer Y, Todesco S, Pauletto P. Risk factors for subclinical atherosclerosis in a prospective cohort of patients with

[28] Croca SA, Rahman A. Imaging assessment of cardiovascular disease in systemic lupus

[29] De Leeuw K, Freire B, Smit AJ, Bootsoma H, Kallenberg CG, Bijl M. Traditional and non‐traditional risk factors contribute to the development of accelerated atherosclerosis

[30] Kahlenberg JM, Kaplan MJ. The interplay of inflammation and cardiovascular disease in

[31] Funakubo Asanuma Y. Accelerated atherosclerosis and inflammation in systemic lupus

[32] Skaggs BJ, Hahn BH, McMahon M. The role of the immune system in atherosclerosis: molecules, mechanisms an implications for management of cardiovascular risk and dis-

[33] Vadacca M, Zardi EM, Margiotta D, Rigon A, Cacciapaglia F, Arcarese L, Buzzulini F, Amoroso A, Afeltra A. Leptin, adiponectin and vascular stiffness parameters in women

[34] Parker B, Urowitz MB, Gladman DD, Lunt M, Bae S‐CH et al. Clinical associations of the metabolic syndrome in systemic lupus erythematosus: data from an international incep-

[35] McMahon M, Hahn BH, Skaggs BJ. Systemic lupus erythematosus and cardiovascular disease: prediction and potential for therapeutic intervention. Expert Rev Clin Immunol

[37] Shoenfeld Y, Gerli R, Doria A, Matsuura E, Cerinic MM, Ronda N, Jara LJ, Abu‐Shakra M, Meroni PL, Sherer Y. Accelerated atherosclerosis in autoimmune rheumatic diseases.

[38] Haque S, Gordon C, Isenberg D, Rahman A, Lanyon P et al. Risk fctors for clinical coronary heart disease in systemic lupus erythematosus: the lupus and atherosclerosis evalu-

[39] Shoenfeld Y, Sherer Y, Harats D. Atherosclerosis as an infectious, inflammatory and

[40] Gonzalez‐Gay MA, Vazquez‐Rodriguez TR, Garcia‐Unzueta MT, Berja A, Miranda‐Filloy JA, de Matias JM, Gonzalez‐Juanatey C, Liorca J. Visfatin is not associated with inflammation or metabolic syndrome in patients with severe rheumatoid arthritis undergoing

[36] Blasi C. The autoimmune origin of atherosclerosis. Atherosclerosis 2008: 201; 17–32

ease in patients with rheumatic diseases. Nat Rev Rheumatol 2012: 8; 214–223.

systemic lupus erythematosus. Ann Rheum Dis 2003: 62; 1071–1077.

erythematosus. Clin Develop Immunol 2012, Article ID 694143, 7 p.

systemic lupus erythematosus. Arth Res Ther 2011: 13; 203–213.

in patients with systemic lupus erythematosus. Lupus 2006: 15; 675–682.

erythematosus. Nihon Rhinsho Meneki Gakkai Kaishi 2012: 35; 470–480.

with systemic lupus erythematosus. Intern Emerg Med 2013: 8; 705–712.

tion cohort. Ann Rheum Dis 2013: 72; 1308–1314.

ation of risk [LASER] study. J Rheumatol 2010: 37; 322–329.

autoimmune disease. Trends Immunol 2001: 22; 293–295.

anti‐TNF‐α therapy. Clin Exp Rheumatol 2010: 28; 56–62.

2011: 7; 227–241.

166 Lupus

Circulation 2005: 112; 3337–3347.


[68] Wu Z, Zhao S. Adipocyte: a potential target for the treatment of atherosclerosis. Med Hypoth 2006: 67; 82–86.

[53] Tellels R, Lanna C, Ferreira G, Ribeiro A. Metabolic syndrome in patients with systemic lupus erythematosus: association with traditional risk factors for coronary heart disease

[54] dos Santos FMM, Borges MC, Telles RW, Correia MITD, Lanna CCD. Excess weight and associated risk factors in patients with systemic lupus erythematosus. Rheumatol Int

[55] Gómez R, Conde J, Scotece M, Gómez‐Reino J, Lago F, Gualillo O. What's new in our understanding of the role of adipokines in rheumatic diseases? Nat Rev Rheumatol

[56] Barbosa VS, Rȇgo J, da Silva NA. Possible role of adipokines in systemic lupus erythema-

[57] Chung CP, Long AG, Solus JF, Rho YH, Oeser A, Raggi P, Stein M. Adipocytokines in systemic lupus erythematosus: relationships to inflammation, insulin resistance and

[58] Krysiak R, Handzlik‐Orlik G, Okopień B. The role of adipokines in connective tissue

[59] Olewicz‐Gawlik A, Dańczak‐Pazdrowska A, Klama K, Mackiewicz S, Silny W, Hrycaj P. Rola adipokin w patogenezie twardziny układowej – badania własne i przegląd liter-

[60] Szadkowska A. Adipokiny w Miażdżyca u Dzieci i Młodzieży. Redakcja Mirosława

[61] Juge‐Aubry CHE, Henrichot E, Meier CHA. Adipose tissue: a regulator of inflammation.

[62] Lago F, Dieguez C, Gómez‐Reino J, Gualillo O. The emerging role of adipokines as mediators of inflammation and immune responses. Cytokine Growth Factor Rev 2007: 18;

[63] Lago F, Dieguez C, Gómez‐Reino J, Gualillo O. Adipokines as emerging mediators of immune response and inflammation. Nature Publishing Group. Nat Clin Pract

[64] Niedzwiedzka‐Rystwej P, Deptuła W. Tkanka tłuszczowa a układ odpornościowy.

[65] Al M, Ng L, Tyrrell P, Bargman J, Bradley T, Silverman E. Adipokines as novel biomarkers to pediatric systemic lupus erythematosus. Rheumatology 2009: 48; 497–501.

[66] Tilg H, Moschen AR. Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nature Publishing Group. Nat Rev Immunol 2006: 6; 773–783.

[67] Guzik TJ, Mangalat D, Korbut R. Adipokines – novel link between inflammation and

tosus and rheumatoid arthritis. Rev Bras Rheumatol 2012: 52; 271–287.

and lupus characteristics. Lupus 2010: 19; 803–809.

coronary atherosclerosis. Lupus 2009: 18; 799–806.

Best Pract Res Clin Endocrinol Metab 2005: 19; 547–566.

vascular function? J Physiol Pharmacol 2006: 57; 505–528.

diseases. Eur J Nutr 2012: 51; 513–528.

atury. Reumatologia 2009: 47; 329–331.

Urban. Wydawca Cornetis 2007r.

Rheumatol 2007: 3; 716–724.

Allergia Astma Immunol 2009: 15; 101–105.

313–325.

2013: 33; 681–688.

168 Lupus

2011: 7; 528–536.


## **Idiopathic Osteonecrosis and Atypical Femoral Fracture in Systemic Lupus Erythematosus**

Takeshi Kuroda and Hiroe Sato

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68143

#### **Abstract**

[81] Zheng H, Xu H, Xie N, Huang J, Fang H, Luo M. Association of serum resistin with

[83] Beltowski J. Role of leptin in blood pressure regulation and arterial hypertension. J

[84] Matarese G, Maschos S, Mantzoros CS. Leptin in immunology. J Immunol 2005: 174;

[85] Lord GM, Matarese G, Howard JK, Baker RJ, Blooms SR, Lechler RI. Leptin modulates the T‐cell immune response and reverses starvation‐induced immunosuppression.

[86] Scotece M, Conde J, Gómez R, López V, Lago F, Gómez‐Reino J, Gualillo O. Beyond fat mass: exploring the role of adipokines in rheumatic diseases. Sci World J 2011: 11;

[87] Garcia‐Gonzalez A, Gonzalez‐Lopez L,Valera‐Gonzalez IC, Gardona‐Muñoz EG, Salazar‐ Parmo M, González‐Ortiz M, Martinez‐Abundis E, Gamez‐Nawa JI. Serum leptin levels

[88] Xu W‐D, Zhang M, Zhang Y‐J, Liu S‐S, Pan H‐F, Ye DQ. Association between leptin and

[89] Otero M, Lago R, Gomez R, Dieguez C, Lago F, Gómez‐Reino J, Gualillo O. Towards a pro‐inflammatory and immunomodulatory emergin role of leptin. Rheumatology 2006:

[90] De Sanctis JB, Zabaleta M, Bianco NE, Garmendia JV, Rivas L. Serum adipokine levels in patients with systemic lupus erythematosus. Autoimmunity 2009: 42; 272–274.

[91] Wisłowska M, Rok M, Stępień K, Kuklo‐Kowalska A. Serum leptin in systemic lupus

[92] Lara‐Castro C, Fu Y, Chung BH, Garvey WT. Adiponectin and metabolic syndrome: mechanisms mediating risk for metabolic and cardiovascular diseases. Curr Opin

[93] Rovin BH, Song H, Herbert LA, Nadasdy T, Nadasdy G, Birmingham DJ, Yu CY, Nagaraja HN. Plasma, urine and renal expression of adiponectin in human systemic

[94] Fantuzzi G. Adiponectin and inflammation: consensus and controversy. J Allergy Clin

[95] Song H, Chan J, Rovin BH. Induction of chemokine expression by adiponectin in vitro is

in women with systemic lupus erythematosus. Rheumatol Int 2002: 22; 138–141.

systemic lupus erythematosus. Rheumatol Int 2014: 34; 559–563.

erythematosus. Rheumatol Int 2008: 28; 467–473.

lupus erythematosus. Kidney Intern 2005: 68; 1825–1833.

isoform dependent. Transl Res 2009: 54; 18–26.

peripheral arterial disease. Pol Arch Med Wewn 2013: 123; 680–685. [82] Beltowski J. Leptin and atherosclerosis. Atherosclerosis 2006: 189; 47–60.

Hypertens 2006: 24; 789–801.

Nature 1998: 394; 897–901.

3137–3142.

170 Lupus

1932–1947.

45; 944–950.

Lipidol 2007: 18; 263–270.

Immunol 2008: 121; 326–330.

Osteonecrosis and osteoporosis are frequent adverse effects of glucocorticoid therapy of systemic lupus erythematosus (SLE). Idiopathic osteonecrosis (ION) of the femoral head occurs in 3–40% of patients receiving glucocorticoid, and can also develop in other bones. Higher doses of glucocorticoid and steroid pulse therapy are considered to be risk factors for ION of the femoral head. To analyze these risk factors, it seems important to detect early changes in the femoral head by magnetic resonance imaging and to monitor early clinical events attributable to steroid therapy. Prophylaxis with statins and warfarin remains debatable. The use of glucocorticoid is increase the risk of bone fractures. Bisphosphonate (BP) is used for its prevention and treatment of osteoporosis. Atypical femoral fracture (AFF) has been recently recognized as a complication associated with BP use. AFF is considered to be a form of stress fracture; localized periosteal thickening of the lateral cortex is often present at the fracture site. The thickening has been recently recognized as a complication associated with the use of antiresorptive agents such as BP and denosumab. As long-term BP/glucocorticoid use is a risk factor for beaking in patients with SLE , temporary withdrawal of BP administration should be considered.

**Keywords:** systemic lupus erythematosus, glucocorticoid, osteonecrosis, bisphosphonate, atypical femoral fracture

#### **1. Introduction**

Systemic lupus erythematosus (SLE) is a chronic, inflammatory, systemic autoimmune disease of unknown etiology characterized by production of antinuclear autoantibodies. It mainly affects young women and shows a broad spectrum of manifestations such as general fatigue, skin rash, fever, and arthritis and disorders involving the kidney, heart, and central nervous system. These organ involvements occur in patients with severer disease status and indicate

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

a poor prognosis. Glucocorticoid has been used as a first-line therapy for SLE. Glucocorticoid exerts strong anti-inflammatory effects and is widely used for the treatment of uncontrolled disease activity in patients with SLE, such as central nervous system lupus (CNS), severe lupus nephritis, and other life-threatening conditions [1]. Glucocorticoid therapy is successful in most cases when high doses are employed, and as a result the prognosis of the SLE has improved remarkably. On the other hand, as glucocorticoid has adverse side effects on many organ systems, only the minimum effective dose is used for treatment. For example, skin thinning and purpura are commonly observed, and the risk of both cataracts and glaucoma is increased. Glucocorticoid use is associated with an increased risk of ischemic heart disease and heart failure, and also an increased risk of gastritis, gastric ulcer, and gastrointestinal bleeding. In the musculoskeletal system, osteoporosis is one of the more serious adverse effects of glucocorticoid [2], and osteonecrosis is also a significant problem [3]. Bisphosphonate (BP) is a key drug used for prevention and treatment of osteoporosis. The risk of osteonecrosis caused by glucocorticoid is higher in patients with SLE. Glucocorticoid causes a dose-dependent, mild increase in the fasting glucose level and a greater increase in postprandial hyperglycemia in patients without preexisting diabetes mellitus (DM), whereas it worsens control of the blood glucose level in patients with DM. The adverse effect of glucocorticoid on atherosclerotic vascular disease is thought to be mediated in part by elevated levels of nonfunctional lipoprotein. In patients with SLE, the adverse effects of glucocorticoid on lipid profiles are dosedependent, occurring only at prednisone doses exceeding 10 mg/day. Systemic glucocorticoid also has many effects on both innate and acquired immunity, resulting in a dose-dependent increase in the risk of infection, especially with common bacterial, viral, and fungal pathogens [4]. Conventional immunosuppressive agents such as mycophenolate mofetil, azathioprine, and cyclophosphamide are also widely used in the management of SLE, and current treatment regimens optimize the use of these agents while minimizing their potential toxicity [5]. Tacrolimus may be particularly useful as adjunctive therapy in patients with persistent proteinuria despite other therapies, and in the management of lupus nephritis in pregnancy. The advent of biological agents has advanced the treatment of SLE, particularly in patients with refractory disease. The CD20 monoclonal antibody rituximab and the anti-BLyS agent belimumab are widely used in clinical practice. The prognosis of SLE has improved markedly, and long-term survival has increased. Prior to 1955, fewer than 50% of patients survived 5 years after diagnosis whereas now, 10-year survival exceeds 90% [6 ]. Our recent data have also confirmed that the 5-year survival rates of patients diagnosed as having SLE before 1970, between 1970 and 1979, between 1980 and 1989, between 1990 and 1999, and after 2000 were 71.4, 83.1, 94.5, 93.4, and 96.4%, respectively. Previously, the causes of death in patients with SLE were mainly infection and renal disease, but recently atherosclerotic cardiovascular disease emerging in the long term has become a focus of concern. Musculoskeletal conditions that impair the quality of life have also become problematic, including osteonecrosis and atypical femoral fracture (AFF). Here we discuss osteonecrosis and AFF in patients with SLE.

#### **2. Incidence, etiology, and pathogenesis**

Idiopathic osteonecrosis (ION) of the femoral head occurs frequently (3–40%) in patients receiving glucocorticoid for underlying conditions such as nephrotic syndrome and renal transplantation [7–10]. It is also known to occur as one of the serious complications of glucocorticoid treatment *of* SLE. Among several factors *related to* ION, glucocorticoid therapy is considered to be one of *essential* importance [3]. There have been *a lot of* reports of ION onset in SLE patients to date, but the *exact* incidence of ION in this group is unknown. The etiology or pathogenesis of this disorder has not been fully clarified, and no prophylaxis has been established to date. The risk of ION in SLE patients is considered to be due to both the SLE itself and the concomitant use of glucocorticoid because, in occasional cases, ION has been noted in the absence of glucocorticoid therapy [3]. In addition, the risk of developing ION has been linked to numerous factors such as glucocorticoid use, alcohol consumption, cigarette smoking, and several rheumatic diseases including SLE. Although the pathogenesis remains unclear, involvement of lipid metabolism abnormality [11], hypercoagulability [12], oxidative stress [13], and vascular endothelial dysfunction [14] has been suggested from basic and clinical research. Several studies investigating the association of ION with steroid treatment have yielded conflicting results with regard to the cumulative dosage, maximum dose, route of administration, and duration of treatment. Glucocorticoid dose and duration seem to be important factors related to ION, but there is considerable controversy about this issue [3, 15–20]. ION may develop in patients who have received high-dose, short-term, or long-term steroid. However, in the early phase, the relationship between steroid and ION has yet been not fully investigated. In our present study, the patients were treated with steroid for the first time, and our observation period was short. Additionally, the initial dose of prednisolone (PSL) for treatment of SLE has sometimes been determined according to the patient's weight or body surface area (BSA) [21]. Therefore, we investigated the relationship of body mass index (BMI) [22] and BSA with the initial dose of PSL. We found that the initial dose of PSL, steroid pulse therapy, BMI, and BSA were not correlated with asymptomatic ION, similar to the results obtained by Sekiya et al. [23]. Also, we failed to identify any relationship between BMI, BSA, the initial dose of PSL per unit BW, the initial dose of PSL relative to BMI, or the initial dose of PSL relative to BSA. None of the factors evaluated were associated with asymptomatic ION. Recent meta-analysis data have shown that the likelihood of ION developing in patients receiving glucocorticoid at more than 20 mg/day is significantly higher than in patients receiving less than 20 mg/day. In the early phase, corticosteroid at over 20 mg/day may trigger ION. In addition, it has been revealed that increasing the steroid dose at the time of SLE recurrence is a risk factor for development of new ION [24].

a poor prognosis. Glucocorticoid has been used as a first-line therapy for SLE. Glucocorticoid exerts strong anti-inflammatory effects and is widely used for the treatment of uncontrolled disease activity in patients with SLE, such as central nervous system lupus (CNS), severe lupus nephritis, and other life-threatening conditions [1]. Glucocorticoid therapy is successful in most cases when high doses are employed, and as a result the prognosis of the SLE has improved remarkably. On the other hand, as glucocorticoid has adverse side effects on many organ systems, only the minimum effective dose is used for treatment. For example, skin thinning and purpura are commonly observed, and the risk of both cataracts and glaucoma is increased. Glucocorticoid use is associated with an increased risk of ischemic heart disease and heart failure, and also an increased risk of gastritis, gastric ulcer, and gastrointestinal bleeding. In the musculoskeletal system, osteoporosis is one of the more serious adverse effects of glucocorticoid [2], and osteonecrosis is also a significant problem [3]. Bisphosphonate (BP) is a key drug used for prevention and treatment of osteoporosis. The risk of osteonecrosis caused by glucocorticoid is higher in patients with SLE. Glucocorticoid causes a dose-dependent, mild increase in the fasting glucose level and a greater increase in postprandial hyperglycemia in patients without preexisting diabetes mellitus (DM), whereas it worsens control of the blood glucose level in patients with DM. The adverse effect of glucocorticoid on atherosclerotic vascular disease is thought to be mediated in part by elevated levels of nonfunctional lipoprotein. In patients with SLE, the adverse effects of glucocorticoid on lipid profiles are dosedependent, occurring only at prednisone doses exceeding 10 mg/day. Systemic glucocorticoid also has many effects on both innate and acquired immunity, resulting in a dose-dependent increase in the risk of infection, especially with common bacterial, viral, and fungal pathogens [4]. Conventional immunosuppressive agents such as mycophenolate mofetil, azathioprine, and cyclophosphamide are also widely used in the management of SLE, and current treatment regimens optimize the use of these agents while minimizing their potential toxicity [5]. Tacrolimus may be particularly useful as adjunctive therapy in patients with persistent proteinuria despite other therapies, and in the management of lupus nephritis in pregnancy. The advent of biological agents has advanced the treatment of SLE, particularly in patients with refractory disease. The CD20 monoclonal antibody rituximab and the anti-BLyS agent belimumab are widely used in clinical practice. The prognosis of SLE has improved markedly, and long-term survival has increased. Prior to 1955, fewer than 50% of patients survived 5 years after diagnosis whereas now, 10-year survival exceeds 90% [6 ]. Our recent data have also confirmed that the 5-year survival rates of patients diagnosed as having SLE before 1970, between 1970 and 1979, between 1980 and 1989, between 1990 and 1999, and after 2000 were 71.4, 83.1, 94.5, 93.4, and 96.4%, respectively. Previously, the causes of death in patients with SLE were mainly infection and renal disease, but recently atherosclerotic cardiovascular disease emerging in the long term has become a focus of concern. Musculoskeletal conditions that impair the quality of life have also become problematic, including osteonecrosis and atypical femoral

172 Lupus

fracture (AFF). Here we discuss osteonecrosis and AFF in patients with SLE.

Idiopathic osteonecrosis (ION) of the femoral head occurs frequently (3–40%) in patients receiving glucocorticoid for underlying conditions such as nephrotic syndrome and renal

**2. Incidence, etiology, and pathogenesis**

#### **3. Timing of osteonecrosis-related ischemia in patients with SLE**

Radiographically, at the earliest stage of ION, plain radiographs show normal features, whereas axial and coronal T1- and T2-weighted MR images show low-density signals in the femoral head (**Figure 1**).

From this viewpoint, osteonecrosis associated with renal transplantation can provide important information. The band patterns on MRI correspond to repair tissue located between necrotic and intact areas [25, 26]. Thus, there is a time lag between the occurrence of ischemia and the appearance of the band pattern. It has been reported that 1 month after internal fixation of a femoral neck fracture, MRI can reveal band patterns in the femoral head away

**Figure 1.** A T1-weighted image demonstrates a ring-like subchondral area of osteonecrosis (white arrow) present in the femoral head.

from the fracture line [27]. In patients who develop ION after renal transplantation, it is presumed that intraosseous ischemia occurs earlier than 6–12 weeks postoperatively [28, 29] when band patterns are observed on T1-weighted MRI. In experimental animal models of osteonecrosis, it has been shown that intraosseous ischemia occurs quite soon after administration of large doses of steroid, that is, on the fifth and third day, respectively. The total dose of steroid administered in the first 2 weeks after renal transplantation is related to ION development [30]. This suggests the occurrence of an event in the bone that may lead to the development of ION at a very early stage after steroid administration. The widespread use of MRI now makes it possible to detect osteonecrotic change in SLE patients soon after administration of glucocorticoid, thus facilitating early diagnosis. Nagasawa et al. reported that 33% of patients developed ION within 3 months after the start of glucocorticoid treatment and that symptomatic ION became apparent at 2 years and beyond [31]. Radiographically, a subchondral radiolucency known as the crescent sign appeared at a late stage in ION, indicating subchondral fracture. However, that study was a multicenter one, and several strategies were selected for treatment of SLE according to the clinical conditions of the patients, resulting in slight differences among the participating hospitals. Several strategies have been selected for treatment of SLE *in conformity with* the clinical conditions of the affected patients, and there are *many* differences among hospitals, such as the indications for immunosuppressants. However, for any study performed at a single institution, the strategy *of treatment*, the steroid *selection*, the initial dose of steroid, and concomitant drugs would be more uniform. In addition, the speed of steroid tapering would also be quite uniform. This would allow better clarification of the background factors associated with ION. On the basis of this concept, we investigated the early development of ION in a cohort of strictly selected SLE patients using MRI and the early changes in laboratory parameters associated with steroid therapy [32].

#### **4. Classification of osteonecrosis**

Diagnosis of ION of the femoral head relies on the combination of clinical symptoms and radiographs and/or magnetic resonance imaging (MRI) *changes*. To evaluate the evolution of ION of the femoral head, the Ficat (**Table 1**) [33] and the Association Research Circulation Osseous (ARCO) classification (**Table 2**) [34] are *generally* used to *evaluate* both imaging modalities. For comparative purposes, these classifications need to be reliable and *uniform definition* to provide sufficient therapy options for the patient.


**Table 1.** Scheme of Ficat classification (1985).

from the fracture line [27]. In patients who develop ION after renal transplantation, it is presumed that intraosseous ischemia occurs earlier than 6–12 weeks postoperatively [28, 29] when band patterns are observed on T1-weighted MRI. In experimental animal models of osteonecrosis, it has been shown that intraosseous ischemia occurs quite soon after administration of large doses of steroid, that is, on the fifth and third day, respectively. The total dose of steroid administered in the first 2 weeks after renal transplantation is related to ION development [30]. This suggests the occurrence of an event in the bone that may lead to the development of ION at a very early stage after steroid administration. The widespread use of MRI now makes it possible to detect osteonecrotic change in SLE patients soon after administration of glucocorticoid, thus facilitating early diagnosis. Nagasawa et al. reported that 33% of patients developed ION within 3 months after the start of glucocorticoid treatment and that symptomatic ION became apparent at 2 years and beyond [31]. Radiographically, a subchondral radiolucency known as the crescent sign appeared at a late stage in ION, indicating subchondral fracture. However, that study was a multicenter one, and several strategies were selected for treatment of SLE according to the clinical conditions of the patients, resulting in slight differences among the participating hospitals. Several strategies have been selected for treatment of SLE *in conformity with* the clinical conditions of the affected patients, and there are *many* differences among hospitals, such as the indications for immunosuppressants. However, for any study performed at a single institution, the strategy *of treatment*, the steroid *selection*, the initial dose of steroid, and concomitant drugs would be more uniform. In addition, the speed of steroid tapering would also be quite uniform. This would allow better clarification of the background factors associated with ION. On the basis of this concept, we investigated the early development of ION in a cohort of strictly selected SLE patients using MRI and the early changes in laboratory parameters associated with steroid therapy

**Figure 1.** A T1-weighted image demonstrates a ring-like subchondral area of osteonecrosis (white arrow) present in the

Diagnosis of ION of the femoral head relies on the combination of clinical symptoms and radiographs and/or magnetic resonance imaging (MRI) *changes*. To evaluate the evolution of ION of the femoral head, the Ficat (**Table 1**) [33] and the Association Research Circulation Osseous (ARCO) classification (**Table 2**) [34] are *generally* used to *evaluate* both imaging modalities. For comparative purposes, these classifications need to be reliable and *uniform* 

[32].

femoral head.

174 Lupus

**4. Classification of osteonecrosis**

*definition* to provide sufficient therapy options for the patient.


*Note*: (a) Location of femoral head necrosis: (1) medial third, (2) median third, and (3) lateral third. Size of femoral head necrosis: (A) <15%, (B) 15–30%, (C) >30%.

(b) Intrusion degree of femoral head contour: (A) <2 mm, (B) 2–4 mm, and (C) >4 mm.

**Table 2.** Scheme of ARCO classification system (1992).

#### **5. Early changes in MRI features and laboratory parameters of SLE patients**

In previous multicenter studies, several strategies were selected for treatment of SLE according to the clinical conditions of the patients, resulting in slight differences among the participating hospitals. This allowed us to investigate the very early development of ION at 3 months after the start of steroid therapy using MRI imaging to clarify the background factors associated with ION. We found that the prevalence of asymptomatic ION among our patients was 26.9%, similar to that described previously [23]. We found no differences in the clinical characteristics of the patients, such as sex, age, height, and body weight and clinical features. The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) *is shown to be a valid and reliable measure of disease activity of SLE patients* [35]. The SLEDAI score *is assessed using a combination of clinical history, physical examination, organ-specific function test and serological test*. Almost all of our patients showed high or very high disease activity at the time of steroid initiation. However, the SLEDAI score was not correlated with asymptomatic ION [32]. In SLE, as is the case for antiphospholipid syndrome (APS), about 30–40% of patients have detectable antiphospholipid antibodies [36] and a positive lupus anticoagulant test and anticardiolipin antibody are detected in approximately 10–30% and 20–40% of patients, respectively. The prevalence of a so-called clinically significant anticardiolipin profile is considered to be about 30–40% [37]. APS antibodies as a prothrombotic factor might predispose to ION by causing microvascular thrombosis. However, the link between APS antibodies and ION is controversial [38–41]. In our study, there was no significant association between APS antibody and ION [32]. A Japanese nationwide study revealed that cigarette smoking was an independent risk factor for ION [42]. However, in our present study, cigarette smoking was not correlated with ION in SLE patients [32].

#### **6. Serological parameters and ION**

Serological activity of SLE was determined on the basis of decreased CH50 and increased anti-DNA antibodies. We also investigated serological parameters such as C3, C4, CH50, and anti-ds DNA antibody, as well as renal function parameters such as the serum creatinine level, estimated glomerular filtration rate, and *proteinuria*. However, these factors were *not* correlated with ION. Thus, both *initial* serological *disease* activity and *initial* renal function, as the most common forms of organ involvement, did not appear to be correlated with ION [32]. Similar results were obtained in a previous single-center study [23].

#### **7. Lipid levels, statins, and ION**

Several studies of ION have indicated the association of lipid. Nagasawa et al. [31] investigated the rate of increase in serum total cholesterol (TC) levels 1 month after glucocorticoid administration in patients with new-onset SLE, and found that they were significantly high in the ION group. It was also found that lipid levels, and the rates of increase in almost all the TC and triglyceride (TG) parameters, were higher in the group that developed ION. TC levels tended to be higher than that in the non-ION group, and the maximum levels and *increasing rates* were significantly higher, suggesting that a rapid rise in serum lipids soon after an increase in glucocorticoid dose might affect the onset of ION [23]. Our data *suggested* that the level of TG both before and after the *initiation* of steroid *therapy* was higher in patients with ION [32]. *The TG level before PSL therapy was associated future risk of asymptomatic ION*. As the *association* between TG before PSL therapy and the initial dose of PSL was not significant, the effect of *TG* before PSL therapy on asymptomatic ION would not have been modified by the initial dose of subsequent PSL. Several studies have *shown* that a high TG level is a strong risk factor for *stroke and ischemic heart disease* [43–45]. ION is caused by partial or total *interruption* of blood flow to the femoral head, and SLE patients *thought to* develop asymptomatic ION through a similar mechanism. *Additionally*, it is well known that steroid *therapy* induces iatrogenic metabolic syndrome, and *from this point of view*, a high TG level is considered to be an important risk factor for asymptomatic ION. Furthermore, *the TC level* after steroid *therapy* tended to be higher in patients with asymptomatic ION. *However*, the levels of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were not correlated with asymptomatic ION. As described previously, the TC level after 1 month of steroid treatment was significantly higher in patients with asymptomatic ION. Our data are similar to those reported previously [46]. In the early phase, lipids—especially TG—play an important role in the development of ION in patients with SLE. HMG-CoA reductase inhibitors (statins) have been widely used for the treatment of dyslipidemia as well as for prevention of *ischemic heart* disease. *According to* a chicken model, Wang et al. suggested that lovastatin prevented steroid-induced ION [47], and a study by Nishida et al. using a rabbit model also *revealed* that pitavastatin had a similar effect [48]. In humans, it has been reported that the incidence of osteonecrosis was decreased by 1% by administration of statins in a study of 284 patients with various disorders (excluding SLE) who received glucocorticoid treatment [49]. We used pravastatin, pitavastatin, lovastatin, and atorvastatin for prevention of ION, but no such preventive effect was observed [32]. Until now, no randomized controlled trial has been reported to successfully prevent steroid-induced ION. In patients with SLE, treatment with statins alone is insufficient for prevention of ION.

#### **8. Prevention of osteonecrosis**

the start of steroid therapy using MRI imaging to clarify the background factors associated with ION. We found that the prevalence of asymptomatic ION among our patients was 26.9%, similar to that described previously [23]. We found no differences in the clinical characteristics of the patients, such as sex, age, height, and body weight and clinical features. The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) *is shown to be a valid and reliable measure of disease activity of SLE patients* [35]. The SLEDAI score *is assessed using a combination of clinical history, physical examination, organ-specific function test and serological test*. Almost all of our patients showed high or very high disease activity at the time of steroid initiation. However, the SLEDAI score was not correlated with asymptomatic ION [32]. In SLE, as is the case for antiphospholipid syndrome (APS), about 30–40% of patients have detectable antiphospholipid antibodies [36] and a positive lupus anticoagulant test and anticardiolipin antibody are detected in approximately 10–30% and 20–40% of patients, respectively. The prevalence of a so-called clinically significant anticardiolipin profile is considered to be about 30–40% [37]. APS antibodies as a prothrombotic factor might predispose to ION by causing microvascular thrombosis. However, the link between APS antibodies and ION is controversial [38–41]. In our study, there was no significant association between APS antibody and ION [32]. A Japanese nationwide study revealed that cigarette smoking was an independent risk factor for ION [42]. However, in

our present study, cigarette smoking was not correlated with ION in SLE patients [32].

Similar results were obtained in a previous single-center study [23].

Serological activity of SLE was determined on the basis of decreased CH50 and increased anti-DNA antibodies. We also investigated serological parameters such as C3, C4, CH50, and anti-ds DNA antibody, as well as renal function parameters such as the serum creatinine level, estimated glomerular filtration rate, and *proteinuria*. However, these factors were *not* correlated with ION. Thus, both *initial* serological *disease* activity and *initial* renal function, as the most common forms of organ involvement, did not appear to be correlated with ION [32].

Several studies of ION have indicated the association of lipid. Nagasawa et al. [31] investigated the rate of increase in serum total cholesterol (TC) levels 1 month after glucocorticoid administration in patients with new-onset SLE, and found that they were significantly high in the ION group. It was also found that lipid levels, and the rates of increase in almost all the TC and triglyceride (TG) parameters, were higher in the group that developed ION. TC levels tended to be higher than that in the non-ION group, and the maximum levels and *increasing rates* were significantly higher, suggesting that a rapid rise in serum lipids soon after an increase in glucocorticoid dose might affect the onset of ION [23]. Our data *suggested* that the level of TG both before and after the *initiation* of steroid *therapy* was higher in patients with ION [32]. *The TG level before PSL therapy was associated future risk of asymptomatic ION*. As the *association* between TG before PSL therapy and the initial dose of PSL was not significant, the effect of *TG* before PSL

**6. Serological parameters and ION**

176 Lupus

**7. Lipid levels, statins, and ION**

In patients at risk of osteonecrosis, several factors are controllable, and thus prevention is the best approach. Hyperlipidemia and DM should be managed appropriately and alcohol consumption minimized [42]. Smoking should also be avoided, if possible [50]. The dosage of glucocorticoid should be minimized as far as possible, as described previously. Statins may help to protect against osteonecrosis. One database review found that only 1% of 284 patients developed ION after treatment with statins before glucocorticoid use. In renal transplant recipients, among 338 patients who were treated with statins, 15 (4.4%) developed ION and among 2543 patients who were not treated with statins, 180 (7%) patients developed ION [49]. Antioxidant agents have been shown to inhibit osteonecrosis in animal models [51]. Further accumulation of similar studies is needed to clarify the preventive effect of statins against SLE-associated ION.

#### **9. Treatment of ION**

The management of ION is usually determined by the degree of femoral head involvement. If the subchondral shell remains intact, there is still a possibility of healing, but if collapse occurs, healing is impossible. If the necrotic area is small and there are no symptoms, ION should be followed up because some cases may spontaneously progress over time. If femoral head ION is diagnosed early, core depression is a commonly used form of prophylactic surgery to prevent the development of arthritis. One study has demonstrated long-term spontaneous repair of osteonecrotic lesions during low-dose glucocorticoid therapy [52]. Conservative treatment of ION involves limiting the degree of weight-bearing on the hip joint in conjunction with analgesia. In general, simple observation may be considered for asymptomatic lesions. Symptomatic lesions will likely progress to collapse, and if observation is chosen, the next joint-preserving procedure should be considered. Various forms of medication have been tried. BP is regularly used for prevention of insufficiency fractures, and several studies have shown that alendronate can reduce pain and slow the progression of collapse [53–55]. Treatment with BP is effective before subchondral collapse, but after subchondral collapse, the effects of treatment for inhibiting destruction of the femoral head are limited. Lipid-lowering agents, especially statins, are hypothesized to have a protective effect against ON. The prevalence of a clinically significant antiphospholipid profile is approximately 20% in SLE patients, and these antiphospholipid antibodies are believed to contribute to ION though hypercoagulation. Accordingly, anticoagulation therapy has been tried for prevention of ION in SLE patients, and warfarin has been modestly beneficial in this respect. One study of 60 SLE patients receiving prednisolone at more than 40 mg/day found that treatment with warfarin significantly reduced the onset of ION [31]. Among various physical modalities, extracorporeal shockwave therapy, hyperbaric oxygen, and pulsed electromagnetic therapy have yielded encouraging results, but further large prospective studies will be necessary to confirm these effects. Surgical treatments to prevent joint destruction include hip arthroplasty, core decompression, osteotomy, and vascularized bone grafting. Core decompression is commonly performed as prophylactic surgery for pre-collapse osteonecrosis of the femoral head. This decreases intramedullary pressure within the femoral head and neck, and has been postulated to improve blood circulation to the femoral head. Core decompression is often combined with bone grafting to help regenerate healthy bone and support cartilage at the hip joint. Bone grafting involves transplantation of healthy bone tissue to an area of the body where it is needed. Another option that has had some degree of success is harvesting and *in vitro* culture of autologous mesenchymal stem cells and their re-implantation into the core decompression site [56–58]. Long-term studies to confirm the success of this approach are still underway. Hip arthroplasty is the most commonly performed procedure for postcollapse lesions of the femoral head. Recent mid- and long-term studies have yielded satisfactory results [59]. Transtrochanteric anterior rotational osteotomy moves the symptomatic portion of the antero-superior femoral head out of the weight-bearing dome, enabling the normal posterior aspect of the head to bear weight, thus helping to preserve the joint [60]. These procedures have yielded favorable success rates, but are associated with a moderate risk of nonunion. Vascularized fibular grafting is a more complex procedure in which a segment of bone is taken from the fibula of the patient, along with the arterial and venous blood supply. This is then transplanted into a hole created in the femoral neck and head. Vascularized fibular grafting has yielded successful outcomes in patients with precollapse lesions and moderate success in those with postcollapse lesions [61]. Additionally, use of autogenic or allogeneic cortical bone grafts and cancellous bone grafts has yielded good results for the treatment of precollapse and/or early precollapse osteonecrotic lesions of the femoral head.

### **10. Atypical femoral fracture**

should be followed up because some cases may spontaneously progress over time. If femoral head ION is diagnosed early, core depression is a commonly used form of prophylactic surgery to prevent the development of arthritis. One study has demonstrated long-term spontaneous repair of osteonecrotic lesions during low-dose glucocorticoid therapy [52]. Conservative treatment of ION involves limiting the degree of weight-bearing on the hip joint in conjunction with analgesia. In general, simple observation may be considered for asymptomatic lesions. Symptomatic lesions will likely progress to collapse, and if observation is chosen, the next joint-preserving procedure should be considered. Various forms of medication have been tried. BP is regularly used for prevention of insufficiency fractures, and several studies have shown that alendronate can reduce pain and slow the progression of collapse [53–55]. Treatment with BP is effective before subchondral collapse, but after subchondral collapse, the effects of treatment for inhibiting destruction of the femoral head are limited. Lipid-lowering agents, especially statins, are hypothesized to have a protective effect against ON. The prevalence of a clinically significant antiphospholipid profile is approximately 20% in SLE patients, and these antiphospholipid antibodies are believed to contribute to ION though hypercoagulation. Accordingly, anticoagulation therapy has been tried for prevention of ION in SLE patients, and warfarin has been modestly beneficial in this respect. One study of 60 SLE patients receiving prednisolone at more than 40 mg/day found that treatment with warfarin significantly reduced the onset of ION [31]. Among various physical modalities, extracorporeal shockwave therapy, hyperbaric oxygen, and pulsed electromagnetic therapy have yielded encouraging results, but further large prospective studies will be necessary to confirm these effects. Surgical treatments to prevent joint destruction include hip arthroplasty, core decompression, osteotomy, and vascularized bone grafting. Core decompression is commonly performed as prophylactic surgery for pre-collapse osteonecrosis of the femoral head. This decreases intramedullary pressure within the femoral head and neck, and has been postulated to improve blood circulation to the femoral head. Core decompression is often combined with bone grafting to help regenerate healthy bone and support cartilage at the hip joint. Bone grafting involves transplantation of healthy bone tissue to an area of the body where it is needed. Another option that has had some degree of success is harvesting and *in vitro* culture of autologous mesenchymal stem cells and their re-implantation into the core decompression site [56–58]. Long-term studies to confirm the success of this approach are still underway. Hip arthroplasty is the most commonly performed procedure for postcollapse lesions of the femoral head. Recent mid- and long-term studies have yielded satisfactory results [59]. Transtrochanteric anterior rotational osteotomy moves the symptomatic portion of the antero-superior femoral head out of the weight-bearing dome, enabling the normal posterior aspect of the head to bear weight, thus helping to preserve the joint [60]. These procedures have yielded favorable success rates, but are associated with a moderate risk of nonunion. Vascularized fibular grafting is a more complex procedure in which a segment of bone is taken from the fibula of the patient, along with the arterial and venous blood supply. This is then transplanted into a hole created in the femoral neck and head. Vascularized fibular grafting has yielded successful outcomes in patients with precollapse lesions and moderate success in those with postcollapse lesions [61]. Additionally, use of autogenic or allogeneic cortical bone grafts and cancellous bone grafts has yielded good results for the treatment of

178 Lupus

precollapse and/or early precollapse osteonecrotic lesions of the femoral head.

Glucocorticoid-induced osteoporosis (GIO) is an important problem in patients with SLE. BP is a key drug used for prevention and treatment of GIO. Patients with SLE often need to continue glucocorticoid and BP therapy for a long time, even if they are young. AFF has recently been recognized as a complication of long-term BP use [62, 63]. AFF is defined as a fracture located along the femoral diaphysis from just distal to the lesser trochanter to just proximal to the supracondylar flare, as distinguished from typical femoral fracture which occurs at the femoral neck or intertrochanteric area and is related to osteoporosis [63]. Diagnosis of AFF requires the presence of four of five major features (**Table 3**).

Beaking is one of these features, and is defined as localized periosteal or endosteal thickening of the lateral cortex at the fracture site. Since cortical thickening at the fracture site characterizes stress fracture, the mechanism of AFF is considered to involve stress [63]. The agestandardized incidence rates of AFF have been reported to be 16 per 100,000 person-years for patients treated with BPs over 5 years and 133 for those treated with BPs over 10 years [62], and AFF occurs much less frequently than osteoporosis-related fractures. However, once it occurs, it takes much time to heal [64, 65], and the daily life activities of the patient are often impaired. Risk factors for AFF other than long-term BP use include glucocorticoid therapy [66, 67], complicating connective tissue disease [67], lateral bowing of the femur [68, 69], a low level of serum 25-hydroxyvitamin D [66], and female gender [70]. Glucocorticoid therapy has been reported to have an important impact on AFF [66, 67], although no significant

Major features

The fracture is associated with minimal or no trauma, as in a fall from a standing height or less

The fracture line originates at the lateral cortex and is substantially transverse in its orientation, although it may become oblique as it progresses medially across the femur

Complete fractures extend through both cortices and may be associated with a medial spike; incomplete fractures involve only the lateral cortex

The fracture is noncomminuted or minimally comminuted

Localized periosteal or endosteal thickening of the lateral cortex is present at the fracture site ("beaking" or "flaring")

Minor features

Generalized increase in cortical thickness of the femoral diaphyses

Unilateral or bilateral prodromal symptoms such as dull or aching pain in the groin or thigh

Bilateral incomplete or complete femoral diaphysis fractures

Delayed fracture healing

**Table 3.** ASBMR Task Force 2013 Revised Case Definition of AFFs [63].

To satisfy the case definition of AFF, the fracture must be located along the femoral diaphysis from just distal to the lesser trochanter to just proximal to the supracondylar flare.

In addition, at least four of the five major features must be present. None of the minor features is required but have sometimes been associated with these fractures.

association has yet been proved in several studies [71, 72]. Girgis et al. reviewed 152 femoral shaft fractures and classified 20 of them as AFF; they concluded that the use of glucocorticoid therapy for more than 6 months was significantly associated with AFF [66]. In a fracture location-, age-, and gender-matched case-control study, Saita et al. reviewed 2238 hip and femoral shaft fractures and diagnosed 10 of them as AFF, concluding that glucocorticoid therapy and complicating connective tissue disease were significant risk factors for AFF [67]. We recently evaluated the incidence of latent femoral beaking (**Figure 2**), which may precede AFF, in 125 patients with autoimmune diseases [65 (52%) with SLE] taking BP and glucocorticoid [73].

Our data revealed that the incidence of beaking was 8% and increased to 10% over 2 years. A case of complete AFF from the tip of the beaking occurred in one patient. The risk factors for beaking were BP therapy for a period of >4 years, age 40–60 years, and presence of diabetes mellitus [73]. Although few studies have investigated AFF in patients with SLE, the frequency of beaking in our study was thought to be higher than in conventional reports of AFF, possibly because all of the patients were taking BP (mean therapy duration 5.1 ± 2.7 years) and glucocorticoid (mean dose 10.0 ± 3.8 mg). Both BP and prolonged glucocorticoid therapy reduce bone remodeling [64, 74], thus, impairing the healing of microdamage occurring during normal daily life activities. BP also changes bone plasticity [75], and glucocorticoid therapy leads to a deterioration of bone quality [76]. Thus, a combination of BP and glucocorticoid therapies enhances microdamage accumulation, producing conditions in which beaking can easily occur. Generally, lateral bowing of the femur is considered to be a risk factor for AFF. Hyodo et al. indicated that

**Figure 2.** X-ray of the hip joint showing beaking (white arrow).

AFF located in the mid femur was significantly related to femoral bowing, whereas AFF in the proximal femur was related to glucocorticoid use [68]. In our previous study of patients with autoimmune diseases taking BP and glucocorticoid, the femoral beaking was mostly located at the subtrochanter, and was not related to lateral bowing of the femur [73]. Therefore, AFF and beaking in patients taking BP and glucocorticoid may generally occur irrespective of lateral femoral bowing. In order to properly benefit from BP and to minimize the risk of AFF, a BP "drug holiday" has been proposed. Postmenopausal women treated orally with BP for over 5 years can be considered for such a break in drug therapy if they have no osteoporotic fractures, their hip T score is >−2.5 and their fracture risk is not high [62]. For patients with GIO, few studies have investigated the safety and effectiveness of temporary drug withdrawal, and further studies are required. Because of the high frequency of beaking in patients taking BP and glucocorticoid [73], regular femoral X-ray screening for beaking is strongly recommended for AFF prevention, and once beaking is detected, a BP drug holiday should be considered.

#### **11. Conclusion**

association has yet been proved in several studies [71, 72]. Girgis et al. reviewed 152 femoral shaft fractures and classified 20 of them as AFF; they concluded that the use of glucocorticoid therapy for more than 6 months was significantly associated with AFF [66]. In a fracture location-, age-, and gender-matched case-control study, Saita et al. reviewed 2238 hip and femoral shaft fractures and diagnosed 10 of them as AFF, concluding that glucocorticoid therapy and complicating connective tissue disease were significant risk factors for AFF [67]. We recently evaluated the incidence of latent femoral beaking (**Figure 2**), which may precede AFF, in 125 patients with autoimmune diseases [65 (52%) with SLE] taking BP and glucocorticoid [73].

180 Lupus

Our data revealed that the incidence of beaking was 8% and increased to 10% over 2 years. A case of complete AFF from the tip of the beaking occurred in one patient. The risk factors for beaking were BP therapy for a period of >4 years, age 40–60 years, and presence of diabetes mellitus [73]. Although few studies have investigated AFF in patients with SLE, the frequency of beaking in our study was thought to be higher than in conventional reports of AFF, possibly because all of the patients were taking BP (mean therapy duration 5.1 ± 2.7 years) and glucocorticoid (mean dose 10.0 ± 3.8 mg). Both BP and prolonged glucocorticoid therapy reduce bone remodeling [64, 74], thus, impairing the healing of microdamage occurring during normal daily life activities. BP also changes bone plasticity [75], and glucocorticoid therapy leads to a deterioration of bone quality [76]. Thus, a combination of BP and glucocorticoid therapies enhances microdamage accumulation, producing conditions in which beaking can easily occur. Generally, lateral bowing of the femur is considered to be a risk factor for AFF. Hyodo et al. indicated that

**Figure 2.** X-ray of the hip joint showing beaking (white arrow).

SLE is a chronic inflammatory autoimmune disease mainly affecting young women; the mortality has recently been improved by treatments including glucocorticoid therapy. However, several adverse effects of glucocorticoid may decrease the quality of life. Even though some of these adverse effects have been overcome recently, AFF and ION are still persistent problems, and further work needs to be done to alleviate them.

#### **Acknowledgements**

This study was supported by a research grant from the Research Committee on Idiopathic Osteonecrosis of the Femoral Head of the Ministry of Health, Labour, and Welfare of Japan.

### **Author details**

Takeshi Kuroda\* and Hiroe Sato

\*Address all correspondence to: kurodat@med.niigata-u.ac.jp

Niigata University Health Administration Center, Nishi-ku, Niigata City, Japan

#### **References**

[1] Boumpas DT, Chrousos GP, Wilder RL, Cupps TR, Balow JE. (1993). Glucocorticoid therapy for immune-mediated diseases: basic and clinical correlates. Ann Intern Med. 15;119(12): 1198–1208.


[15] Abeles M, Urman JD, Rothfield NF. (1978). Aseptic necrosis of bone in systemic lupus erythematosus. Relationship to corticosteroid therapy. Arch Intern Med. 138(5): 750–754.

[2] Zonana-Nacach A, Barr SG, Magder LS, Petri M. (2000). Damage in systemic lupus erythematosus and its association with corticosteroids. Arthritis Rheum. 43(8): 1801–1808. [3] Calvo-Alén J, McGwin G, Toloza S, Fernández M, Roseman JM, Bastian HM, Cepeda EJ, González EB, Baethge BA, Fessler BJ, Vilá LM, Reveille JD, Alarcón GS; LUMINA Study Group. Systemic lupus erythematosus in a multiethnic US cohort (LUMINA): XXIV. (2006). Cytotoxic treatment is an additional risk factor for the development of symptomatic osteonecrosis in lupus patients: results of a nested matched case-control study. Ann

[4] Kirou KA, Boumpus DT. Dubois' lupus erytheamtosus and related syndrome. Wallace

[5] Kalunian KC, Kim M, Xie X, Baskaran A, Daly RP, Merrill JT. (2016). Impact of standard of care treatments and disease variables on outcomes in systemic lupus erythematosus trials: analysis from the Lupus Foundation of America Collective Data Analysis

[6] Moroni G, Quaglini S, Gallelli B, Banfi G, Messa P, Ponticelli C. (2007). The long-term outcome of 93 patients with proliferative lupus nephritis. Nephrol Dial Transplant.

[7] Abeles M, Urman JD, Rothfield NF. (1987). Aseptic necrosis of bone in systemic lupus erythematosus. Relationship to corticosteroid therapy. Arch Intern Med. 138(5):

[8] Koo KH, Kim R. (1995). Quantifying the extent of osteonecrosis of the femoral head. A

[9] Landmann J, Renner N, Gächter A, Thiel G, Harder F. (1978). Cyclosporin a and osteo-

[10] Mont MA, Hungerford DS. (1995). Non-traumatic avascular necrosis of the femoral

[11] Moskal JT, Topping RE, Franklin LL. (1997). Hypercholesterolemia: an association with

[12] Oinuma K, Harada Y, Nawata Y, Kobayashi K, Abe I, Kamikawa K, Moriya H. (2000). Sustained hemostatic abnormality in patients with steroid-induced osteonecrosis in the

[13] Ichiseki T, Matsumoto T, Nishino M, Kaneuji A, Katsuda S. (2004). Oxidative stress and vascular permeability in steroid-induced osteonecrosis model. J Orthop Sci. 9(5):

[14] Iuchi T, Akaike M, Mitsui T, Ohshima Y, Shintani Y, Azuma H, Matsumoto T. (2003). Glucocorticoid excess induces superoxide production in vascular endothelial cells and

early period after high dose corticosteroid therapy. J Orthop Sci. 5(4): 374–379.

DJ, Haln BH (Eds.) Saunders-Elsevier, Philadelphia. 2012. p. 597

new method using MRI. J Bone Joint Surg Br. 77(6): 875–880.

necrosis of the femoral head. J Bone Joint Surg Am. 69(8): 1226–1228.

osteonecrosis of the femoral head. Am J Orthop. 26(9): 609–612.

elicits vascular endothelial dysfunction. Circ Res. 92(1): 81–87.

Rheum Dis. 65(6): 785–790.

22(9): 2531–2539.

750–754.

182 Lupus

509–515.

Initiative. Eur J Rheumatol. 3(1): 13–19.

head. J Bone Joint Surg Am 77(3): 459–474.


systemic lupus erythematosus: a retrospective case-control study. Mod Rheumatol. 25(4): 590–594.

[41] Mok MY, Farewell VT, Isenberg DA. (2000). Risk factors for avascular necrosis of bone in patients with systemic lupus erythematosus: is there a role for antiphospholipid antibodies? Ann Rheum Dis. 59(6): 462–467.

[28] Kubo T, Yamazoe S, Sugano N, Fujioka M, Naruse S, Yoshimura N, Oka T, Hirasawa Y. (1997). Initial MRI findings of non-traumatic osteonecrosis of the femoral head in renal

[29] Fujioka M, Kubo T, Nakamura F, Shibatani M, Ueshima K, Hamaguchi H, Inoue S, Sugano N, Sakai T, Torii Y, Hasegawa Y, Hirasawa Y. (2001). Initial changes of non-traumatic osteonecrosis of femoral head in fat suppression images: bone marrow edema was not found before the appearance of band patterns. Magn Reson Imaging. 19 (7): 985–991.

[30] Saito M, Ueshima K, Fujioka M, Ishida M, Goto T, Arai Y, Ikoma K, Fujiwara H, Fukushima W, Kubo T. (2014). Corticosteroid administration within 2 weeks after renaltransplantation affects the incidence of femoral head osteonecrosis. Acta Orthop. 85(3):

[31] Nagasawa K, Tada Y, Koarada S, Tsukamoto H, Horiuchi T, Yoshizawa S, Murai K, Ueda A, Haruta Y, Ohta A. (2006). Prevention of steroid-induced osteonecrosis of femoral

[32] Kuroda T, Tanabe N, Wakamatsu A, Takai C, Sato H, Nakatsue T, Wada Y, Nakano M, Narita I. (2015). High triglyceride is a risk factor for silent osteonecrosis of the femoral

[33] Ficat RP. (1985). Idiopathic bone necrosis of the femoral head. Early diagnosis and treat-

[34] Gardeniers JW (1992). A new international classification of osteonecrosis of the ARCO Committee on terminology and classification. J Jpn Orthop Assoc 66(1): 18–20.

[35] Bombardier C, Gladman DD, Urowitz MB, Carton D, Chang CH. (1992). Derivation of the SLEDAI. A disease activity index for lupus patients. The committee on prognosis

[36] Galli M, Luciani D, Bertolini G, Barbui T. (2003). Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid

[37] Taraborelli M, Leuenberger L, Lazzaroni MG, Martinazzi N, Zhang W, Franceschini F, Salmon J, Tincani A, Erkan D. (2016). The contribution of antiphospholipid antibodies to

[38] Asherson RA, Lioté F, Page B, Meyer O, Buchanan N, Khamashta MA, Jungers P, Hughes GR. (1993). Avascular necrosis of bone and antiphospholipid antibodies in sys-

[39] Mont MA, Glueck CJ, Pacheco IH, Wang P, Hungerford DS, Petri M. (1997). Risk factors for osteonecrosis in systemic lupus erythematosus. J Rheumatol. 24(4): 654–662.

[40] Faezi ST, Hoseinian AS, Paragomi P, Akbarian M, Esfahanian F, Gharibdoost F, Akhlaghi M, Nadji A, Jamshidi AR, Shahram F, Nejadhosseinian M, Davatchi F. (2015). Non-corticosteroid risk factors of symptomatic avascular necrosis of bone in

syndrome:a systemtic review of the literature. Blood. 101(5): 1827–1832.

organ damage in systemic lupus erythematosus. Lupus. 25(12): 1365–1368.

head in systemic lupus erythematosus by anti-coagulant. Lupus. 15(6): 354–357.

head in systemic lupus erythematosus. Clin Rheumatol. 34(12): 2071–2077.

ment. J Bone Joint Surg Br. 67(1): 3–9.

studies in SLE. Arthritis Rheum. 35(6): 630–640.

temic lupus erythematosus. J Rheumatol. 20(2): 284–288.

allograft recipients. Magn Reson Imaging. 15(9): 1017–1023.

266–270.

184 Lupus


diaphyseal femoral fractures: second report of a task force of the American society for bone and mineral research. J Bone Miner Res. 29(1): 1–23.

[64] Odvina CV, Zerwekh JE, Rao DS, Maalouf N, Gottschalk FA, Pak CY. (2005). Severely suppressed bone turnover: a potential complication of alendronate therapy. J Clin Endocrinol Metab. 90(3): 1294–1301.

[52] Shigemura T, Nakamura J, Kishida S, Harada Y, Ohtori S, Kamikawa K, Ochiai N, Takahashi K. (2011). Incidence of osteonecrosis associated with corticosteroid therapy among different underlying diseases: prospective MRI study. Rheumatology (Oxford).

[53] Agarwala S, Shah S, Joshi VR. (2009). The use of alendronate in the treatment of avascular necrosis of the femoral head: follow-up to eight years. J Bone Joint Surg Br. 91(8):

[54] Lai KA1, Shen WJ, Yang CY, Shao CJ, Hsu JT, Lin RM. (2005). The use of alendronate to prevent early collapse of the femoral head in patients with nontraumatic osteonecrosis.

[55] Nishii T, Sugano N, Miki H, Hashimoto J, Yoshikawa H. (2006). Does alendronate prevent collapse in osteonecrosis of the femoral head? Clin Orthop Relat Res. 443:

[56] Gangji V, Toungouz M, Hauzeur JP. (2005). Stem cell therapy for osteonecrosis of the

[57] Hernigou P, Beaujean F. (2002). Treatment of osteonecrosis with autologous bone mar-

[58] Hauzeur JP, Gangji V. (2010). Phases 1–3 clinical trials using adult stem cells in osteone-

[59] Woo MS, Kang JS, Moon KH. (2014). Outcome of total hip arthroplasty for avascular necrosis of the femoral head in systemic lupus erythematosus. J Arthroplasty. 29(12):

[60] Atsumi T, Muraki M, Yoshihara S, Kajihara T. (1999). Posterior rotational osteotomy for the treatment of femoral head osteonecrosis. Arch Orthop Trauma Surg. 119(7–8):

[61] Malizos KN, Quarles LD, Seaber AV, Rizk WS, Urbaniak JR. (1993). An experimental canine model of osteonecrosis: characterization of the repair process. J Orthop Res. 11(3):

[62] Adler RA, El-Hajj Fuleihan G, Bauer DC, Camacho PM, Clarke BL, Clines GA, Compston JE, Drake MT, Edwards BJ, Favus MJ, Greenspan SL, McKinney R Jr., Pignolo RJ, Sellmeyer DE. (2016). Managing osteoporosis in patients on long-term bisphosphonate treatment: report of a task force of the American society for bone and mineral research.

[63] Shane E, Burr D, Abrahamsen B, Adler RA, Brown TD, Cheung AM, Cosman F, Curtis JR, Dell R, Dempster DW, Ebeling PR, Einhorn TA, Genant HK, Geusens P, Klaushofer K, Lane JM, McKiernan F, McKinney R, Ng A, Nieves J, O'Keefe R, Papapoulos S, Howe TS, van der Meulen MC, Weinstein RS, Whyte MP. (2014). Atypical subtrochanteric and

A randomized clinical study. J Bone Joint Surg Am. 87(10): 2155–2159.

femoral head. Expert Opin Biol Ther. 5(4): 437–442.

crosis and nonunion fractures. Stem Cells Int. 2010: 410170.

row grafting. Clin Orthop Relat Res. 405: 14–23.

50(11): 2023–2028.

1013–1018.

186 Lupus

273–279.

2267–2270.

388–393.

350–357.

J Bone Miner Res. 31(1): 16–35.


**Diet and Microbes in Lupus**

[74] Teitelbaum SL. (2012). Bone: the conundrum of glucocorticoid-induced osteoporosis.

[75] Tjhia CK, Odvina CV, Rao DS, Stover SM, Wang X, Fyhrie DP. (2011). Mechanical property and tissue mineral density differences among severely suppressed bone turnover (SSBT) patients, osteoporotic patients, and normal subjects. Bone. 49(6): 1279–1289. [76] Peel NF, Moore DJ, Barrington NA, Bax DE, Eastell R. (1995). Risk of vertebral fracture and relationship to bone mineral density in steroid treated rheumatoid arthritis. Ann

Nat Rev Endocrinol. 8(8): 451–452.

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Rheum Dis. 54(10): 801–806.

### **Diet and Microbes in the Pathogenesis of Lupus**

Xin M. Luo, Michael R. Edwards, Christopher M. Reilly, Qinghui Mu and S. Ansar Ahmed

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/68110

#### **Abstract**

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with no known cure. It is characterized by severe and persistent inflammation that damages multiple organs. To date, treatment and prevention of disease flares have relied on long-term use of anti-inflammatory drugs where side effects are of particular concern. There is a need for better understanding of the disease and for better approaches in SLE treatment and management. In this chapter, we delineate the roles of diet and microbes in the pathogenesis of SLE.

**Keywords:** diet, microbiota, lupus, hygiene hypothesis, epigenetics

#### **1. Introduction**

Systemic lupus erythematosus (SLE) is an autoimmune disorder with complex genetic and environmental etiology. It is characterized by severe and persistent inflammation that leads to tissue damage in multiple organs. The cause is unclear, and there is currently no cure. Current standard of care treatments for SLE are primarily nonselective immunosuppressants, and not all patients respond to these regimens. Although current therapies can treat acute symptoms and reduce the risk of renal failure associated with SLE, side effects are a major cause of concern. Patients taking long-term immunosuppressants are prone to higher incidence of and more severe infections. There is an imperative need to develop new treatment strategies against SLE, for which a better understanding of disease pathogenesis is required. In this chapter, we delineate the roles of diet and microbes in the pathogenesis of SLE.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. The hygiene hypothesis**

*Introduction*. Environmental factors are known to impact lupus progression in both human and mouse. The role of environmental factors in the etiology of SLE is evidenced by the dramatic difference in disease incidence between West Africans and African-Americans, both derived from the same ethnic group but exposed to different environments. In addition, two remarkable cases of disease amelioration have been reported in SLE patients who had experienced multiple microbial infections. In lupus-prone mice, infections have also been demonstrated to attenuate lupus-like disease. The mechanisms behind these observations are unclear, but improvement in hygiene and absence of certain microbes may have contributed to the higher incidence and faster progression of lupus disease. In this subsection, we will describe the hygiene hypothesis and its relationship with the pathogenesis of SLE.

*SLE and the hygiene hypothesis*. Proposed by Strachan [1], the hygiene hypothesis initially states that the increased incidence of allergies stems from "declining family size, improvements in household amenities, and higher standards of personal cleanliness." Since then, the scope of the hypothesis has expanded to cover several autoimmune diseases, including type 1 diabetes (T1D), inflammatory bowel disease and multiple sclerosis. Many infectious agents have been described to be protective against autoimmunity [2, 3], whereas mouse models of these autoimmune diseases, such as NOD mice for T1D, are known to develop more progressive clinical signs in cleaner environments. In the case for SLE, it has been long recognized that lupus-prone mice exhibit different disease courses in different animal facilities, suggesting an important role for environmental factors in the etiology of SLE. In addition, the incidence of SLE increased at least threefold in the second half of the twentieth century [4, 5], which is strongly correlated with the increase in hygiene and the decrease of infections, particularly those of the gastrointestinal tract [6]. We therefore have recently extended the hygiene hypothesis to SLE [7].

*Protective infectious agents against SLE*. The increase in hygiene could reduce infections that are either pathogenic or protective in SLE. Known triggers of lupus development include parvovirus B-19 [8], rubella virus [9], Epstein-Barr virus and cytomegalovirus (CMV) [7, 10–15]. Infectious agents that have been shown to be protective against SLE, on the other hand, include *Helicobacter pylori*, hepatitis B virus (HBV), *Toxoplasma gondii*, malaria parasites such as *Plasmodium berghei* and *Plasmodium chabaudi* and more recently described, helminths. In a large cohort of African-Americans, *H. pylori* seronegativity was found to be associated with an increased risk and earlier onset of SLE [16, 17]. For HBV, only 2.5% of SLE patients were found to be positive for an HBVspecific antibody, suggesting prior infection, whereas in the general population, >10% of people are found positive for the same antibody [9, 18]. Besides human studies, it has been shown that the infection of *T. gondii* protects New Zealand Black (NZB)/New Zealand White (NZW) F1 hybrid (NZB/W F1) lupus-prone mice from developing SLE-like disease, with significantly decreased mortality, ameliorated lupus nephritis and reduced autoantibodies in the serum [19, 20]. Infections with malaria-causing parasites also protected NZB/W F1 mice from developing clinical signs of murine lupus [21–24]. In another classical mouse model of SLE, MRL/Mp-Faslpr (MRL/lpr), helminthic infection and administration of helminth (worm)-derived molecules have been recently shown to induce the regulatory functions of the immune system and attenuate SLE-like disease such as glomerulonephritis and development of anti-nuclear antibodies [25–27]. Similar results have also been obtained in NZB/W F1 mice, although with a different worm-derived compound [28]. Interestingly, all these protective infectious agents are highly prevalent in Africa where the incidence of SLE is low. Due to vaccinations and the increase in hygiene such as higher quality of drinking water, people in North America and Europe are much less likely to be infected by these agents. Conversely, SLE is of high prevalence in Western developed countries, especially for people of African descent.

**2. The hygiene hypothesis**

192 Lupus

hygiene hypothesis to SLE [7].

*Introduction*. Environmental factors are known to impact lupus progression in both human and mouse. The role of environmental factors in the etiology of SLE is evidenced by the dramatic difference in disease incidence between West Africans and African-Americans, both derived from the same ethnic group but exposed to different environments. In addition, two remarkable cases of disease amelioration have been reported in SLE patients who had experienced multiple microbial infections. In lupus-prone mice, infections have also been demonstrated to attenuate lupus-like disease. The mechanisms behind these observations are unclear, but improvement in hygiene and absence of certain microbes may have contributed to the higher incidence and faster progression of lupus disease. In this subsection, we will

*SLE and the hygiene hypothesis*. Proposed by Strachan [1], the hygiene hypothesis initially states that the increased incidence of allergies stems from "declining family size, improvements in household amenities, and higher standards of personal cleanliness." Since then, the scope of the hypothesis has expanded to cover several autoimmune diseases, including type 1 diabetes (T1D), inflammatory bowel disease and multiple sclerosis. Many infectious agents have been described to be protective against autoimmunity [2, 3], whereas mouse models of these autoimmune diseases, such as NOD mice for T1D, are known to develop more progressive clinical signs in cleaner environments. In the case for SLE, it has been long recognized that lupus-prone mice exhibit different disease courses in different animal facilities, suggesting an important role for environmental factors in the etiology of SLE. In addition, the incidence of SLE increased at least threefold in the second half of the twentieth century [4, 5], which is strongly correlated with the increase in hygiene and the decrease of infections, particularly those of the gastrointestinal tract [6]. We therefore have recently extended the

*Protective infectious agents against SLE*. The increase in hygiene could reduce infections that are either pathogenic or protective in SLE. Known triggers of lupus development include parvovirus B-19 [8], rubella virus [9], Epstein-Barr virus and cytomegalovirus (CMV) [7, 10–15]. Infectious agents that have been shown to be protective against SLE, on the other hand, include *Helicobacter pylori*, hepatitis B virus (HBV), *Toxoplasma gondii*, malaria parasites such as *Plasmodium berghei* and *Plasmodium chabaudi* and more recently described, helminths. In a large cohort of African-Americans, *H. pylori* seronegativity was found to be associated with an increased risk and earlier onset of SLE [16, 17]. For HBV, only 2.5% of SLE patients were found to be positive for an HBVspecific antibody, suggesting prior infection, whereas in the general population, >10% of people are found positive for the same antibody [9, 18]. Besides human studies, it has been shown that the infection of *T. gondii* protects New Zealand Black (NZB)/New Zealand White (NZW) F1 hybrid (NZB/W F1) lupus-prone mice from developing SLE-like disease, with significantly decreased mortality, ameliorated lupus nephritis and reduced autoantibodies in the serum [19, 20]. Infections with malaria-causing parasites also protected NZB/W F1 mice from developing clinical signs of murine lupus [21–24]. In another classical mouse model of SLE, MRL/Mp-Faslpr (MRL/lpr), helminthic infection and administration of helminth (worm)-derived molecules

describe the hygiene hypothesis and its relationship with the pathogenesis of SLE.

It is most remarkable, however, that multiple infections led to complete amelioration of lupus disease in two female patients previously diagnosed with the most active form of SLE [20]. Both women were of childbearing age, where pregnancy was found to be a trigger of lupus flares. In addition, their disease was refractory to all available treatments. Due to drug-induced immunosuppression, they experienced multiple infections over several months that included CMV and *Staphylococcus aureus*, which unfortunately induced sepsis. One patient also experienced infections of *Pseudomonas aeruginosa* and *Pneumocystis carinii*, whereas the other experienced multiple *Escherichia coli* urinary tract infections. Both were treated with antivirals such as ganciclovir and antibiotics such as vancomycin and trimethoprim-sulfamethoxazole. Remarkably, the course of lupus disease changed dramatically in both women with improved autoantibody titers. They were reported to be symptom free after 3–5 years of follow-ups, and one patient even had a normal pregnancy afterwards. While the authors of the reports suggested that the infections had caused the amelioration of SLE symptoms, it is likely that the combination of infections and antiviral/antibacterial treatments had contributed to the change in disease course.

*Mechanisms of protection*. A couple of mechanisms could explain the protective effects of infectious agents against SLE. The first mechanism is competition. It is likely that the strong immune responses elicited by infectious agents can compete successfully for homeostatic signals (cytokines, growth factors, etc.) against the autoimmune response elicited by weaker autoantigens. The second mechanism is that regulatory cells stimulated by infectious agents can dampen autoimmune response. For example, type I interferons induced by some infectious agents can induce the generation of IL-10-producing regulatory T cells (Treg or Tr1 cells) [29, 30] and limit the production of IL-17 from T cells [31], both of which are mechanisms to suppress autoimmunity. Besides regulatory T cells, anti-inflammatory dendritic cells (DCs) that produce IL-10 and transforming growth factor β (TGF-β) and drive T helper (Th)-2 responses can be also induced by parasitic infections [3]. In addition, parasitic infections can promote IL-4 secretion from basophils and NKT cells, leading to a Th2-biased response to dampen Th1/Th17-induced autoimmunity [3]. Bacterial products, on the other hand, have been shown to directly induce IL-10-producing Treg cells [32]. Finally, toll-like receptors (TLR) appear to mediate the effects of protective infectious agents, which are TLR agonists, in preventing autoimmunity [6]. It is worth noting that these mechanistic studies were performed with regard to T1D.

Specifically for SLE, several studies have tried to pinpoint the mechanisms by which protective infectious agents prevent lupus-like disease in mice. In response to *T. gondii* infection, the levels of interferon (IFN)-γ and IL-10 decreased in NZB/W F1 mice, suggesting the repression of disease-facilitating Th1 and Th2 cytokines in the development of lupus-like nephritis [19]. In addition, alterations of the redox state in kidney and liver tissues may explain the protective effects of malarial infections against lupus nephritis [21]. A parasitederived compound tuftsin-phosphorylcholine, on the other hand, was able to enhance the expression of TGF-β and IL-10 as well as the expansion of Treg cells in NZB/W F1 mice, while at the same time inhibiting the expression of IFN-γ and IL-17 [28]. This suggests a shift of the balance between Th1/Th17 and Treg toward a Treg-biased phenotype that may in turn attenuate lupus-like disease. Moreover, infection of *Schistosoma mansoni* skewed the Th1:Th2 balance to a Th2 phenotype in MRL/lpr mice, which significantly changed the pathophysiology of glomerulonephritis from diffuse proliferative lupus nephritis (more severe) to membranous lupus nephritis (less severe) [25]. Furthermore, it has been found that a parasitic worm product ES-62 targets MyD88-dependent effector functions of B cells (promoting regulatory B cells and inhibiting plasmablast differentiation) to suppress autoantibody production and proteinuria in MRL/lpr mice [26, 27]. Altogether, these studies suggest that infectious agents can protect against the development of SLE by inducing the regulatory functions of immune cells.

#### **3. Diet and SLE**

*Introduction*. Environmental factors beyond microbial infections contribute to the pathogenesis and severity of disease progression in mouse models, as well as lupus patients. Nutritional components including vitamins, caloric excess or restriction, polyunsaturated fatty acid composition, excess sodium intake and exogenous hormone containing compounds all influence the clinical signs and immune system cellular responses. Nutritional compounds such as vitamin D, omega-3 fatty acids and conjugated linoleic acid appear to have beneficial effects on some parameters of lupus, while high levels of vitamin E, omega-6 fatty acids, a modern "western diet," and high levels of circulating adipokines may exacerbate disease flares. Various commonly consumed phytoestrogens exert complex and differential effects, leading to regulation or exacerbation of SLE clinical signs. Modulating the immune system through dietary intervention to promote regulation of the immune response may form an adjunctive treatment to reduce signs of SLE both for maintenance as well as during disease flare-ups, while decreasing necessary dosages of systemic medications. In this section, we will discuss the role of diet in the pathogenesis of SLE.

*Vitamin A*. The three main isoforms of the ubiquitously expressed nuclear retinoic acid receptor (RAR), α, β and γ, heterodimerize with the retinoic X receptor (RXR) isoforms. Retinoic acid binding to these heterodimers leads to promotion of retinoic acid-responsive genes through retinoic acid response elements (RAREs). The RXR receptors are also associated with multiple nuclear receptors, including peroxisome proliferator-activated receptor (PPAR) and vitamin D receptor (VDR). Retinoic acid and 1,25-dihydroxyvitamin D3 (D3), the active hormonal metabolite of vitamin D, have been described to potentially antagonize each other's effects due to the similar binding patterns of RAR and VDR to RXR [33].

Retinoic acid has many roles in the development of both innate and cell-mediated immunity. Exposure of antigen presenting cells (APCs) to retinoic acid is critical for cell development and establishment of normal cellular function, leading to an increase in co-stimulatory molecules and induction of the expression of CD11b, a critical adhesion marker, on murine splenocytes and human monocytes [34, 35]. Retinoic acid appears to have differential effects on the induction of immune responses. 9-cis retinoic acid decreases the production of IL-12 in macrophages. However, when combined with lipopolysaccharide (LPS), it augmented the production of nitric oxide, leading to promotion of the Th2 phenotype through regulation of cytokine production by APCs [34, 36, 37].

the levels of interferon (IFN)-γ and IL-10 decreased in NZB/W F1 mice, suggesting the repression of disease-facilitating Th1 and Th2 cytokines in the development of lupus-like nephritis [19]. In addition, alterations of the redox state in kidney and liver tissues may explain the protective effects of malarial infections against lupus nephritis [21]. A parasitederived compound tuftsin-phosphorylcholine, on the other hand, was able to enhance the expression of TGF-β and IL-10 as well as the expansion of Treg cells in NZB/W F1 mice, while at the same time inhibiting the expression of IFN-γ and IL-17 [28]. This suggests a shift of the balance between Th1/Th17 and Treg toward a Treg-biased phenotype that may in turn attenuate lupus-like disease. Moreover, infection of *Schistosoma mansoni* skewed the Th1:Th2 balance to a Th2 phenotype in MRL/lpr mice, which significantly changed the pathophysiology of glomerulonephritis from diffuse proliferative lupus nephritis (more severe) to membranous lupus nephritis (less severe) [25]. Furthermore, it has been found that a parasitic worm product ES-62 targets MyD88-dependent effector functions of B cells (promoting regulatory B cells and inhibiting plasmablast differentiation) to suppress autoantibody production and proteinuria in MRL/lpr mice [26, 27]. Altogether, these studies suggest that infectious agents can protect against the development of SLE by inducing the

*Introduction*. Environmental factors beyond microbial infections contribute to the pathogenesis and severity of disease progression in mouse models, as well as lupus patients. Nutritional components including vitamins, caloric excess or restriction, polyunsaturated fatty acid composition, excess sodium intake and exogenous hormone containing compounds all influence the clinical signs and immune system cellular responses. Nutritional compounds such as vitamin D, omega-3 fatty acids and conjugated linoleic acid appear to have beneficial effects on some parameters of lupus, while high levels of vitamin E, omega-6 fatty acids, a modern "western diet," and high levels of circulating adipokines may exacerbate disease flares. Various commonly consumed phytoestrogens exert complex and differential effects, leading to regulation or exacerbation of SLE clinical signs. Modulating the immune system through dietary intervention to promote regulation of the immune response may form an adjunctive treatment to reduce signs of SLE both for maintenance as well as during disease flare-ups, while decreasing necessary dosages of systemic medications. In this section, we will discuss

*Vitamin A*. The three main isoforms of the ubiquitously expressed nuclear retinoic acid receptor (RAR), α, β and γ, heterodimerize with the retinoic X receptor (RXR) isoforms. Retinoic acid binding to these heterodimers leads to promotion of retinoic acid-responsive genes through retinoic acid response elements (RAREs). The RXR receptors are also associated with multiple nuclear receptors, including peroxisome proliferator-activated receptor (PPAR) and vitamin D receptor (VDR). Retinoic acid and 1,25-dihydroxyvitamin D3 (D3), the active hormonal metabolite of vitamin D, have been described to potentially antagonize each other's

effects due to the similar binding patterns of RAR and VDR to RXR [33].

regulatory functions of immune cells.

the role of diet in the pathogenesis of SLE.

**3. Diet and SLE**

194 Lupus

The development and differentiation of B and T cells are also influenced by the exposure to retinoic acid. Administration of retinoic acid contributes to increased CD19<sup>+</sup> B cell numbers in the bone marrow and spleen, accelerated B cell maturation and increased expression of Pax-5, a transcription factor promoting early B cell development and expansion [33, 34]. Conversely, retinoic acid is reported by several groups to decrease mature B cell expansion by arresting B cells in the G0/G1 phase through increased expression of p27 (Kip1). This restricted proliferation may be necessary for the increased expression of CD38 leading to increased plasma cell numbers and production of IgG1 [34, 38–40]. Retinoic acid has also been reported to increase the proliferation of memory B cells when stimulated with the TLR9 ligand CpG and retinoic acid. Stimulation of B cells with both molecules led to a threefold increase in immunoglobulin secretion compared with cells stimulated with CpG alone [34].

Vitamin A deficiency has been associated with a decrease in CD4<sup>+</sup> T cells, a decrease in the CD4<sup>+</sup> to CD8<sup>+</sup> ratio, decreased splenic germinal center formation, decreased total splenocytes, as well as splenic and thymus organ mass [41–43]. Deficiency of vitamin A also contributed to an increase in a Th1-driven immune response, with increased IFN-γ and IL-12 and a decrease in IL-5 and IL-10 production [41]. Administration of retinoic acid contributed to an increase in CD4:CD8 ratio and the promotion of a Th2-driven immune response with an increase in IL-5 production, while repressing IFN-γ and the Th1 response [37, 44]. The administration of a single dose of all-*trans*-retinoic acid in a murine model of lupus nephritis reduced the disease severity and production of Th1-associated cytokines IL-2 and IFN-γ [45]. Another study in the same NZB/W F1 model of lupus reported prolonged survival with decreases in total IgG, IgG2a and anti-dsDNA autoantibodies in the serum [46]. A major contribution to the development of lupus nephritis is the deposition of immunoglobulins and complement proteins in the glomeruli. Multiple groups have reported the protective effect of retinoic acid on the development of glomerular disease, leading to decreased proteinuria and glomerular damage, while maintaining similar levels of IgG and C3 deposition within the glomeruli [47]. Another study reported that retinoic acid inhibits Th1-phenotype-related genes T-bet and IRF-1, leading to a decrease in Th1-related cytokines and altering the Th1/Th2 balance, skewing toward a type-2 response [48].

Retinoic acid administration to MRL/lpr mice contributes to the sustained function of natural T regulatory (Treg) cells and promotes the differentiation of inducible Treg cells in the peripheral tissues [36]. Enhanced differentiation of T cells to Treg cells was shown in the small intestine lamina propria, as well as upregulation of gut-homing receptors on Treg cells by the binding of retinoic acid [36, 49]. There is discrepancy as to whether DCs or macrophages are the predominant cell type to induce Treg cell differentiation in the small intestinal lamina propria, while DCs are known to contribute to Th17 cell differentiation. It has been shown that human SLE patients exhibit a defective induction of Treg cells through TGF-β induction and retinoic acid expansion [37]. This impaired induction led to increased numbers of Treg cells with defective regulatory function abilities, causing the inducible Treg cells to be ineffective at controlling the autoimmune inflammatory response. The studies described above clearly show that cells of both the innate and adaptive immune system are regulated by vitamin A/ retinoic acid. Therefore, it is conceivable that vitamin A and its metabolites may have an effect on patients suffering from SLE.

*Vitamin D*. Vitamin D3 and its metabolites have been extensively studied and have shown to influence multiple functions in the body, including the abilities to affect bone density and strength, and to modulate both the innate and adaptive immune branches. In this chapter, we will focus on the evidence of vitamin D's metabolites influence on the human immune system as it pertains to lupus. Vitamin D3 administration contributes to decreased proinflammatory cytokine production by human monocytes, along with decrease dendritic cell maturation, while increasing IL-10, impairing the differentiation of T cells into Th1 cells [50]. D3 is also able to decrease the expression of MHC class II, CD40, CD80 and CD86 co-stimulatory molecules, decreasing the ability of DCs to present antigens to T and B cells. The ability of D3 to activate human monocytes and macrophages in vitro led to increased cathelicidin and IL-1 production [48].

D3 decreases the expression of IL-2, IFN-γ and T cell proliferation in vitro, as well as decreasing CD8<sup>+</sup> cytotoxic activity. It is important in the regulation of many genes, including IL-2 and IFN-γ in T cells, through interaction with the vitamin D receptor-RXR heterodimer. Similar to retinoic acid, D3 inhibits the induction of the Th1-mediated immunity response in favor of the Th2 phenotype, through enhancing the production of IL-4 and decreasing IFNγ. The Th17 cell response is also mitigated by vitamin D through the inhibition of IL-6 and IL-23 production, leading to the promotion of the Treg cell phenotype through expansion of Foxp3 [51, 52].

It has not yet been determined if D3 acts directly on B cells to decrease B cell proliferation and induction of apoptosis. D3 is also associated with decreased plasma cell differentiation and a subsequent decrease in IgG secretion. In one study, the authors showed that D3 has the ability to upregulate p27 mRNA; however, p18 and p21 were not similarly upregulated in human purified B cells from patients with SLE. The p27 gene is associated with inhibition of the cell cycle, leading to decreased proliferation in activated human B cells, especially memory B cells [53].

Mouse models of SLE have shown decreased proteinuria and prolonged survival when supplemented with D3. A commonly used therapy for human SLE patients is the administration of glucocorticoids to reduce inflammation and to broadly dampen the immune response [54]. Glucocorticoids have been shown to decrease the expression of the vitamin D receptor in multiple cell types. Conversely, D3 interferes with both glucocorticoid receptor and androgen receptor responsiveness through reduced receptor expression and impaired translocation to the nucleus due to alterations in phosphorylation status [55]. When administered as a medication, D3 can lead to adverse effects, including hypercalcemia and bone resorption [48].

binding of retinoic acid [36, 49]. There is discrepancy as to whether DCs or macrophages are the predominant cell type to induce Treg cell differentiation in the small intestinal lamina propria, while DCs are known to contribute to Th17 cell differentiation. It has been shown that human SLE patients exhibit a defective induction of Treg cells through TGF-β induction and retinoic acid expansion [37]. This impaired induction led to increased numbers of Treg cells with defective regulatory function abilities, causing the inducible Treg cells to be ineffective at controlling the autoimmune inflammatory response. The studies described above clearly show that cells of both the innate and adaptive immune system are regulated by vitamin A/ retinoic acid. Therefore, it is conceivable that vitamin A and its metabolites may have an effect

*Vitamin D*. Vitamin D3 and its metabolites have been extensively studied and have shown to influence multiple functions in the body, including the abilities to affect bone density and strength, and to modulate both the innate and adaptive immune branches. In this chapter, we will focus on the evidence of vitamin D's metabolites influence on the human immune system as it pertains to lupus. Vitamin D3 administration contributes to decreased proinflammatory cytokine production by human monocytes, along with decrease dendritic cell maturation, while increasing IL-10, impairing the differentiation of T cells into Th1 cells [50]. D3 is also able to decrease the expression of MHC class II, CD40, CD80 and CD86 co-stimulatory molecules, decreasing the ability of DCs to present antigens to T and B cells. The ability of D3 to activate human monocytes and macrophages in vitro led to increased cathelicidin

D3 decreases the expression of IL-2, IFN-γ and T cell proliferation in vitro, as well as decreas-

and IFN-γ in T cells, through interaction with the vitamin D receptor-RXR heterodimer. Similar to retinoic acid, D3 inhibits the induction of the Th1-mediated immunity response in favor of the Th2 phenotype, through enhancing the production of IL-4 and decreasing IFNγ. The Th17 cell response is also mitigated by vitamin D through the inhibition of IL-6 and IL-23 production, leading to the promotion of the Treg cell phenotype through expansion of

It has not yet been determined if D3 acts directly on B cells to decrease B cell proliferation and induction of apoptosis. D3 is also associated with decreased plasma cell differentiation and a subsequent decrease in IgG secretion. In one study, the authors showed that D3 has the ability to upregulate p27 mRNA; however, p18 and p21 were not similarly upregulated in human purified B cells from patients with SLE. The p27 gene is associated with inhibition of the cell cycle, leading to decreased proliferation in activated human B cells, especially memory B cells

Mouse models of SLE have shown decreased proteinuria and prolonged survival when supplemented with D3. A commonly used therapy for human SLE patients is the administration of glucocorticoids to reduce inflammation and to broadly dampen the immune response [54]. Glucocorticoids have been shown to decrease the expression of the vitamin D receptor in multiple cell types. Conversely, D3 interferes with both glucocorticoid receptor and androgen receptor responsiveness through reduced receptor expression and impaired translocation to

cytotoxic activity. It is important in the regulation of many genes, including IL-2

on patients suffering from SLE.

and IL-1 production [48].

ing CD8<sup>+</sup>

196 Lupus

Foxp3 [51, 52].

[53].

Though in vitro evidence strongly supports an immune-regulatory role of vitamin D and its metabolites in the adaptive immune system, there are conflicting reports regarding vitamin D deficiency in SLE patients and the supportive role of vitamin D supplementation. The majority of studies describe decreased serum levels of D3 in patients with SLE compared to healthy patients, which correlated with increased disease activity. The current consensus is that vitamin D serum levels correlate inversely with disease activity, and vitamin D deficiency is associated with increased disease activity or severity [51, 56].

*Vitamin E*. Vitamin E is a potent antioxidant supporting the normal structure and function of cells by reducing damaging free radical reactive oxygen species (ROS). ROS have been implicated in tissue damage and an increase in pro-inflammatory cytokine production [57]. These factors support the development of autoimmune and degenerative disease severity. The preferentially absorbed form of vitamin E is α-tocopherol, which contributes to the normal function of the immune system in humans and mice. In one study, lowered antioxidant ability was found to precede the diagnosis of SLE, and oxidative damage was increased prior to diagnosis of SLE. This suggests that the decreased ability to control oxidative damage is a potential risk factor for development of SLE [58, 59].

Multiple studies in mouse models of SLE have shown that the dose of vitamin E had differential effects on the disease. Low-dose α-tocopherol increases the lifespan of NZB/W F1 mice while decreasing proteinuria, anti-dsDNA autoantibody IgG in the serum, IL-6 and IFN-γ production from splenocytes [60]. In the MRL/lpr mouse model, low-dose administration of vitamin E increases lifespan and IL-2 production. In contrast, high-dose vitamin E increases anti-DNA and cardiolipin IgM, as well as IL-4 and IL-10 from activated splenocytes. The authors conclude that the decreased survival in high-dose groups was due to the imbalance of Th1/Th2 cytokines, with Th2 cytokines leading to hyperactivation of the B cells in the MRL/ lpr model [61]. Vitamin E also increases the pro-inflammatory chemokine MIP-1α in MRL/ lpr mice, which activates granulocytes and promotes pro-inflammatory cytokine production [62]. A study comparing plasma antioxidant status between healthy patients and patients diagnosed with SLE showed that SLE patients have impaired plasma antioxidant status as well as a decreased antioxidant intake. The study was small and did not take into account dietary restrictions or food intolerance, limiting the ability to draw firm conclusions for a large set of SLE patients [58]. The above studies demonstrate that the dose of vitamin E is a critical determinant in amelioration or exacerbation of murine lupus. Response to vitamin E supplementation in human SLE patients needs to be thoroughly investigated to determine whether vitamin E supplementation is appropriate.

*Omega 3:6 PUFA*. Poly-unsaturated fatty acids (PUFAs) have been a popular topic of research in health due to their wide range of effects that can be exerted on the body. Omega-3 PUFAs generally have an anti-inflammatory role, lowering the severity of autoimmune disease and increasing survival in mouse models of SLE. In contrast, diets that are high in saturated fatty acids and omega-6 PUFAs lead to higher anti-dsDNA antibody, proteinuria and inflammatory cytokines in mice [63, 64]. Mouse survival rates were not measured in these studies. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the main bioactive constituents of omega-3 rich fish oil. SLE patients were shown to have decreased EPA in erythrocytes along with a decrease in the EPA to pro-inflammatory arachidonic acid ratio [65]. The evidence in human studies and clinical trials using omega-3 PUFAs in SLE patients is inconclusive at this time, with multiple studies showing no demonstrable effect on SLE disease activity index or other clinical scores and no effect of omega-3 fatty acids on glucocorticoid requirements when used as immunosuppressive medication.

In a mouse study using NZB/W F1 female mice, higher on concentrations of EPA and DHA led to increased lifespan, decreased glomerulonephritis, decreased anti-dsDNA antibodies, as well as a reduction in pro-inflammatory cytokines IL-1β, IL-6 and TNFα in splenocytes, relative to controls. The authors also demonstrated a reduction in nuclear factor-κB (NF-κB) and p65 nuclear translocation in mice fed higher concentrations of both EPA and DHA [66]. In studies using the MRL/lpr mouse model, fish oil altered pro-inflammatory chemokine production, leading to a decrease in RANTES and MCP-1 from splenocytes [62]. In NZB/W F1 mice, omega-6 PUFAs, conversely, led to an increase in IL-6 and TNFα production as well as prostaglandin E2 from macrophages, a decrease in TGF-β mRNA from splenocytes and lower anti-dsDNA IgG in the serum [67].

There is evidence that n-3 PUFAs remodel the lipid rafts in T cells, which can lead to a decrease in intracellular signaling through the T cell receptor, as well as binding to multiple PPARs. Omega-3 PUFAs bind to PPARα in T and B cells, whereas they bind to PPARγ in cells of myeloid lineage, leading to alterations in gene expression. The binding capacity to PPARs by n-3 and n-6 PUFAs is equal, suggesting that gene modulation between the different families is unlikely, though the possibility exists for differences in cellular metabolism of n-3 and n-6 PUFAs to allow for distinct PPAR activation [65]. In certain instances, the evidence is contradictory between ex vivo and in vivo studies, and cytokine levels have been shown to be opposite in mouse models compared to human data [65]. While there is inconclusive evidence for the anti-inflammatory effects of n-3 PUFAs in human clinical trials, it is still recommended by some that rheumatic patients supplement their diet with long-chain n-3 PUFA and gamma linoleic acid [65].

*Conjugated linoleic acid*. Conjugated linoleic acid (CLA) has been described to provide health benefits in humans and makes up a group of isomers of linoleic acid. These fatty acids occur naturally in dairy and beef. CLA is considered to have anti-carcinogenic, anti-atherosclerotic and immune-enhancing abilities, with respect to lymphocytic cytotoxic function, macrophage activation and lymphocyte proliferation [68]. The isomer of CLA that demonstrates the strongest anti-inflammatory effect is *cis*9, *trans*11-CLA, which accounts for up to 90% of the dietary CLAv [68].

Supplementation with CLA leads to a reduction in the loss of body mass during end-stage disease, along with an increase in survival after the onset of proteinuria in the NZB/W F1 mouse model [69, 70]. In a non-autoimmune mouse model and cell lines, CLA has a profound effect on macrophage functions. In BALB/c mice, CLA ameliorates LPS-induced body wasting and anorexia in vivo, decreases the production of nitric oxide from macrophages, and decreases the serum concentration of TNFα. Splenocytes from BALB/c mice fed the CLA supplement also have a lower level of IL-4 production and an increase in IL-2 production after stimulation with concanavalin A [71]. CLA also decreases macrophage adhesion as well as downregulating multiple atherogenic genes associated with leukocyte adhesion, while inducing the antagonist IL-1Rα in human umbilical cord vein endothelial cells and the RAW macrophage cell line [72].

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the main bioactive constituents of omega-3 rich fish oil. SLE patients were shown to have decreased EPA in erythrocytes along with a decrease in the EPA to pro-inflammatory arachidonic acid ratio [65]. The evidence in human studies and clinical trials using omega-3 PUFAs in SLE patients is inconclusive at this time, with multiple studies showing no demonstrable effect on SLE disease activity index or other clinical scores and no effect of omega-3 fatty acids on glucocorticoid

In a mouse study using NZB/W F1 female mice, higher on concentrations of EPA and DHA led to increased lifespan, decreased glomerulonephritis, decreased anti-dsDNA antibodies, as well as a reduction in pro-inflammatory cytokines IL-1β, IL-6 and TNFα in splenocytes, relative to controls. The authors also demonstrated a reduction in nuclear factor-κB (NF-κB) and p65 nuclear translocation in mice fed higher concentrations of both EPA and DHA [66]. In studies using the MRL/lpr mouse model, fish oil altered pro-inflammatory chemokine production, leading to a decrease in RANTES and MCP-1 from splenocytes [62]. In NZB/W F1 mice, omega-6 PUFAs, conversely, led to an increase in IL-6 and TNFα production as well as prostaglandin E2 from macrophages, a decrease in TGF-β mRNA from splenocytes and lower

There is evidence that n-3 PUFAs remodel the lipid rafts in T cells, which can lead to a decrease in intracellular signaling through the T cell receptor, as well as binding to multiple PPARs. Omega-3 PUFAs bind to PPARα in T and B cells, whereas they bind to PPARγ in cells of myeloid lineage, leading to alterations in gene expression. The binding capacity to PPARs by n-3 and n-6 PUFAs is equal, suggesting that gene modulation between the different families is unlikely, though the possibility exists for differences in cellular metabolism of n-3 and n-6 PUFAs to allow for distinct PPAR activation [65]. In certain instances, the evidence is contradictory between ex vivo and in vivo studies, and cytokine levels have been shown to be opposite in mouse models compared to human data [65]. While there is inconclusive evidence for the anti-inflammatory effects of n-3 PUFAs in human clinical trials, it is still recommended by some that rheumatic patients supplement their diet with long-chain n-3 PUFA and gamma

*Conjugated linoleic acid*. Conjugated linoleic acid (CLA) has been described to provide health benefits in humans and makes up a group of isomers of linoleic acid. These fatty acids occur naturally in dairy and beef. CLA is considered to have anti-carcinogenic, anti-atherosclerotic and immune-enhancing abilities, with respect to lymphocytic cytotoxic function, macrophage activation and lymphocyte proliferation [68]. The isomer of CLA that demonstrates the strongest anti-inflammatory effect is *cis*9, *trans*11-CLA, which accounts for up to 90% of the dietary

Supplementation with CLA leads to a reduction in the loss of body mass during end-stage disease, along with an increase in survival after the onset of proteinuria in the NZB/W F1 mouse model [69, 70]. In a non-autoimmune mouse model and cell lines, CLA has a profound effect on macrophage functions. In BALB/c mice, CLA ameliorates LPS-induced body wasting and anorexia in vivo, decreases the production of nitric oxide from macrophages, and decreases the serum concentration of TNFα. Splenocytes from BALB/c mice fed the CLA supplement

requirements when used as immunosuppressive medication.

anti-dsDNA IgG in the serum [67].

linoleic acid [65].

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CLAv [68].

CLA is able to exert immune-modulatory effects on dendritic cells and subsequent differentiation of T cells in mice. DCs produce multiple cytokines after activation and express MHC II on the cell surface to facilitate interactions with T cells and B cells. Exposure of DCs to CLA led to a decrease in IL-12 and an increase in IL-10 production, along with decreased migration to the lymph nodes [73]. CLA decreased the expression of MHC II and costimulatory molecules CD80 and CD86 on the surface of DCs, reducing the DC's ability to trigger T cell and B cells responses [74]. When exposed to LPS stimulation in mice fed a CLA rich diet, serum levels of IFN-γ, IL-1β and IL-12p40 were decreased [75]. CLA decreases the ability of DCs to induce the T helper cell differentiation into Th1 and Th17 cells, as well as directly suppressing the production of IL-17 and IL-2 by Th17 cells [74]. The mechanism for CLA influencing the DC response after activation with LPS was shown to be through suppression of both NF-κB and IRF3 downstream of TLR4 [75]. The immune-modulatory effects have been well described in mouse models, though data for the effects of CLA in human SLE patients are lacking at this time.

*Western diet and obesity*. Considerable attention has recently been given to the attributes of dietary components that make up the typical diet in the developed countries, termed the "western diet." These factors include high-fat, high-protein, high-sugar and high-salt, as well as consumption of a large portion of processed foods. These nutritional components promote obesity, metabolic syndrome and cardiovascular disease. In addition, a recent study has shown the role of the "western diet" in promotion of autoimmune disorders [76]. White adipose tissue is now considered to be a major endocrine organ, secreting more than 50 adipokines including leptin, adiponectin, resistin and visfatin, along with pro-inflammatory cytokines IL-6 and TNFα [76, 77]. Excess white adipose tissue drives a low-level steady state inflammation, which may contribute to various autoimmune and inflammatory-mediated diseases.

The connection between obesity and the development of SLE is complex, and contradictory information exists among studies. There was no correlation found in Danish women between obesity and the incidence of SLE after 11 years [77, 78]. This cohort study had multiple limitations, such as reliance on survey questionnaires regarding pre-pregnancy weights and diagnoses, potential socio-economic bias with women of lower socioeconomic status being underrepresented in the Danish National Birth Cohort, and the confounding factor of pregnancy and childbirth during the study, which do not allow for conclusions to be drawn regarding the link between obesity and SLE [78]. Childhood obesity rates correlated positively with incidence of childhood-onset SLE. The adipokine leptin has been extensively studied and exerts multiple effects on the body [79]. Leptin promotes satiety and stimulation of energy expenditure, by acting on the hypothalamic nuclei, as well as contributing to fertility, bone metabolism and having a profound effect on the immune system. Lack of leptin, due to starvation, testosterone or glucocorticoids, can lead to immunosuppression, while upregulation of leptin by inflammatory cytokines or female sex hormones, 17β-estradiol and progesterone, can lead to inflammation [79]. Leptin can induce the production of IL-6, IL-12 and TNFα pro-inflammatory cytokines from macrophages, while stimulating the proliferation of naïve T cells and promoting a Th1 immune response. Leptin inhibits the production of Th2 cytokines IL-4 and IL-10, along with inhibiting T regulatory cell proliferation [80]. The formation of a pro-inflammatory state through increased leptin levels may contribute to autoimmune disease development.

Leptin is commonly elevated in obese patients as well as SLE patients, which contributes to the survival of autoreactive T cells, and a decrease in the functional T regulatory cells, while promoting the proliferation of Th17 cells in a mouse model of SLE [81, 82]. Circulating serum leptin levels as well as leptin secretion have been shown to be higher in females compared to males, both in obese and non-obese subjects. The sex difference in leptin secretion may be a contributing factor in the development of female-predominant SLE. Leptin deficiency in murine models of SLE has led to decreased anti-dsDNA and a decrease in severity of SLE symptoms [83, 84]. The increased level of circulating leptin may also contribute to the exacerbation of cardiovascular damage in some SLE patients [85, 86]. Adiponectin deficiency was correlated with an increase in SLE disease severity [87, 88]; however, another study found increased levels of adiponectin in the serum and urine of human patient with lupus nephritis [89]. Little information is available regarding other adipokines and their involvement with SLE development.

Obesity is correlated with a predisposition to metabolic syndrome, with increased rates of hypertension, dyslipidemia and atherosclerosis, as well as subsequent cardiovascular disease. Atherosclerosis and cardiovascular disease are highly prevalent in patients with SLE, and a significant number of patient deaths occur due to cardiovascular disease, while obesity has been linked to worsening renal disease and cardiovascular disease in patients suffering from SLE [77]. High levels of inflammatory markers and elevated leptin concentrations found in SLE patients, and mice have been correlated with decreased cognitive function, increased renal damage and increased cardiovascular risks [90, 91].

Diets high in salt (sodium chloride) are found in multiple regions throughout the world, mainly in developed countries where processed foods and "fast food" are prevalent. Excess sodium intake has been attributed to cardiovascular disease and hypertension, as well as stroke, and is now being studied in the development of autoimmune diseases [76]. T cells in a hyperosmotic environment show an increase in p38/MAPK as well as transcription factor NFAT5, leading to an altered cellular response [92]. Sodium can be stored in the human body in various tissues, leading to hyper-osmotic environments in multiple tissues during high-dietary salt intake. Human and murine T cells were investigated under high salt conditions *in vivo* that led to the promotion of Th17 differentiation in vitro [93]. High salt intake has been associated with a decrease in glucocorticoid therapy response in a study of 260 Chinese SLE patients treated with prednisone and followed for 12 weeks [94]. Further studies need to be performed to determine the mechanism for hyperosmotic conditions to promote the highly pro-inflammatory Th17 cell differentiation.

*Phytoestrogens*. Endogenously produced estrogen contributes significantly to the development of SLE and disease progression. Estrogen is able to suppress IL-2, enhancing the effect of autoantigens. 17β-estradiol implants led to an increase in IFN-γ, nitric oxide and a range of cytokines and chemokines in murine splenocytes [95]. The estrogen receptors on cells comprise two subtypes, α and β. Estrogen receptor-α (ERα) mRNA was increased in the peripheral blood mononuclear cells of SLE patients, while estrogen receptor-β (ERβ) was decreased. ERα was shown to be predominantly pro-inflammatory in a mouse model of SLE, leading to increased proteinuria and decreased survival time when activated, while ERβ showed an immunosuppressive phenotype by decreased autoantibody anti-DNA IgG2 [96, 97].

Phytoestrogens are plant compounds that exert an estrogenic or an anti-estrogenic effect on the human body through interaction with both estrogen receptor α and β, with a preferential binding to ERβ [97]. Phytoestrogens can also block the binding of more potent estrogenic compounds to the ER and regulate target genes. Many types of phytoestrogens are found naturally, with soy-based phytoestrogens, isoflavones, present in many of the foods consumed in industrialized societies. Due to the ubiquity of phytoestrogens in normal diets, controlled human trials are lacking with regard to the specific effects of phytoestrogens on SLE disease development. Data from mouse experiments involving supplementation of isoflavones to MRL/lpr mice show a decrease in IFN-γ from splenocytes with a decrease in anti-dsDNA and cardiolipin levels in the serum, along with a reduction in proteinuria and renal damage through preferential binding of the ERβ [98]. Exclusion of dietary isoflavones through diets using casein as the protein source rather than soy led to amelioration of glomerulonephritis [99]. Alfalfa sprout extract supplementation to MRL/lpr mice produced similar results with decreased renal damage and increased survival, while decreasing IFN-γ and IL-4 production by splenocytes. Alfalfa sprout extract was also able to reduce TNFα, IL-6 and IL-1β pro-inflammatory cytokines in a mouse model of endotoxic shock [100]. Coumestrol, one phytoestrogen in alfalfa, was able to decrease anti-dsDNA IgG and decrease proteinuria in the NZB/W F1 mouse model [101]. However, these findings have not been observed in human SLE patients, where ingestion of alfalfa tablets, which contain all phytoestrogen components of the alfalfa plant, exacerbated disease severity [102].

Multiple phytoestrogens have been shown to upregulate VDR expression in various human cell lines. Genistein and glycitein upregulated VDR transcription and translation in colon cancer cells, while resveratrol and genistein also increased VDR expression in breast cancer cells [103, 104]. Along with receptor expression, genistein decreased CYP24, an enzyme responsible for metabolism of the vitamin D metabolite D3 [105]. Phytoestrogens are abundant in many diets throughout the world and are able to contribute to the modulation of the immune system through interaction with estrogen receptors, potentially ameliorating or exacerbating patients' clinical signs.

#### **4. The role of gut microbiota**

starvation, testosterone or glucocorticoids, can lead to immunosuppression, while upregulation of leptin by inflammatory cytokines or female sex hormones, 17β-estradiol and progesterone, can lead to inflammation [79]. Leptin can induce the production of IL-6, IL-12 and TNFα pro-inflammatory cytokines from macrophages, while stimulating the proliferation of naïve T cells and promoting a Th1 immune response. Leptin inhibits the production of Th2 cytokines IL-4 and IL-10, along with inhibiting T regulatory cell proliferation [80]. The formation of a pro-inflammatory state through increased leptin levels may contribute to auto-

Leptin is commonly elevated in obese patients as well as SLE patients, which contributes to the survival of autoreactive T cells, and a decrease in the functional T regulatory cells, while promoting the proliferation of Th17 cells in a mouse model of SLE [81, 82]. Circulating serum leptin levels as well as leptin secretion have been shown to be higher in females compared to males, both in obese and non-obese subjects. The sex difference in leptin secretion may be a contributing factor in the development of female-predominant SLE. Leptin deficiency in murine models of SLE has led to decreased anti-dsDNA and a decrease in severity of SLE symptoms [83, 84]. The increased level of circulating leptin may also contribute to the exacerbation of cardiovascular damage in some SLE patients [85, 86]. Adiponectin deficiency was correlated with an increase in SLE disease severity [87, 88]; however, another study found increased levels of adiponectin in the serum and urine of human patient with lupus nephritis [89]. Little information is available regarding other adipokines and their involvement with

Obesity is correlated with a predisposition to metabolic syndrome, with increased rates of hypertension, dyslipidemia and atherosclerosis, as well as subsequent cardiovascular disease. Atherosclerosis and cardiovascular disease are highly prevalent in patients with SLE, and a significant number of patient deaths occur due to cardiovascular disease, while obesity has been linked to worsening renal disease and cardiovascular disease in patients suffering from SLE [77]. High levels of inflammatory markers and elevated leptin concentrations found in SLE patients, and mice have been correlated with decreased cognitive function, increased

Diets high in salt (sodium chloride) are found in multiple regions throughout the world, mainly in developed countries where processed foods and "fast food" are prevalent. Excess sodium intake has been attributed to cardiovascular disease and hypertension, as well as stroke, and is now being studied in the development of autoimmune diseases [76]. T cells in a hyperosmotic environment show an increase in p38/MAPK as well as transcription factor NFAT5, leading to an altered cellular response [92]. Sodium can be stored in the human body in various tissues, leading to hyper-osmotic environments in multiple tissues during high-dietary salt intake. Human and murine T cells were investigated under high salt conditions *in vivo* that led to the promotion of Th17 differentiation in vitro [93]. High salt intake has been associated with a decrease in glucocorticoid therapy response in a study of 260 Chinese SLE patients treated with prednisone and followed for 12 weeks [94]. Further studies need to be performed to determine the mechanism for hyperosmotic conditions to promote the highly pro-inflammatory

renal damage and increased cardiovascular risks [90, 91].

immune disease development.

200 Lupus

SLE development.

Th17 cell differentiation.

*Introduction*. The mammalian gut harbors trillions of microorganisms known as the microbiota. Increasing evidence in recent years suggests that host microbiota and immune system interact to maintain tissue homeostasis in healthy individuals. The importance of microbiota on the host is highlighted by altered immune responses in the absence of commensal bacteria. Higher susceptibility to infectious pathogens and in some cases, attenuated symptoms in autoimmune disorders, has been observed in mice raised under germ-free conditions. Indeed, perturbation of the host microbiota, especially that in the gut, has been shown to be associated with many autoimmune diseases, including SLE. Changes of gut microbiota in nephritic lupus mice versus healthy controls have recently been described, where a decrease of *Lactobacillaceae* and an increase of *Lachnospiraceae* were observed in lupus-prone mice. A cross-sectional study has also shown that a lower *Firmicutes* to *Bacteroidetes* ratio was present in the fecal microbiota of SLE patients with inactive disease, which is consistent with observations in other autoimmune diseases. In addition, oral antibiotics are known to trigger lupus flares, again suggesting a role for commensal bacteria in SLE. In this section, we will describe the existing data and a proposed role of gut microbiota in the pathogenesis of SLE.

*Gut microbiota and SLE*. Evidence is rapidly growing that mutualistic bacteria contribute to the development of a healthy functioning immune system, as well as the development of aberrant immune responses. The past 10 years have seen a rapid growth in the understanding of how the commensal microbiota is able to contribute to health and disease with the exploration of 16S rRNA sequencing along with the use of gnotobiotic animals. These newer methods of DNA sequencing have allowed researchers to investigate the effects of specific species or strains of organisms and how they interact with the immune system. Gnotobiotic animals are born into a sterile environment and develop under sterile conditions so that a specific group of bacteria, viruses, eukaryotes or parasites can be introduced to the animal, and the resulting effects can be studied without the interference of other organisms [106]. Intestinal microbial dysbiosis can lead to immune system effects at distant sites of the body [49, 107–112]. The systemic immune system is influenced by microbiota in the intestines, suggesting that microbiota at other sites of the body can also have a systemic effect on the host's immune system.

The host's genetic make-up is the primary factor in determining the composition of one's adult microbiota. Other contributing factors include mother's health in utero, the method of delivery during birth, if one is either breast fed or fed formula, the use of antibiotics during early childhood and types of foods consumed prior to, and during, weaning. The adult microbiota is not a static entity, however, and can be modified through many different environmental factors, and the composition will also change with age. Many of the previously discussed contributions to SLE are associated with alterations in the microbiota. Obese patients have an increased *Firmicutes* to *Bacterioidetes* ratio, with members of *Firmicutes* leading to increased energy harvest from dietary nutrients [113]. The "western diet" is associated with alterations in the gut microbiota as well. Children from a rural village in the West African country of Burkina Faso had increased microbial richness and greater *Prevotella* with decreased *Bacteroides* levels compared to European children. The microbiota of the Burkina Faso children also produced more short chain fatty acids than the intestinal microbiota of the European children [114]. These findings of agrarian societies with increased *Prevotella* and decreased *Bacteroides* have been supported in studies comparing rural South Americans and people from Bangladesh to people living in industrialized regions [115]. The source of dietary fat can impact microbiota composition as well, as mice fed diets with a fat source from either milk-fat, lard, safflower oil or a low-fat diet, all resulted in distinct phylogenetic profiles [116, 117].

interact to maintain tissue homeostasis in healthy individuals. The importance of microbiota on the host is highlighted by altered immune responses in the absence of commensal bacteria. Higher susceptibility to infectious pathogens and in some cases, attenuated symptoms in autoimmune disorders, has been observed in mice raised under germ-free conditions. Indeed, perturbation of the host microbiota, especially that in the gut, has been shown to be associated with many autoimmune diseases, including SLE. Changes of gut microbiota in nephritic lupus mice versus healthy controls have recently been described, where a decrease of *Lactobacillaceae* and an increase of *Lachnospiraceae* were observed in lupus-prone mice. A cross-sectional study has also shown that a lower *Firmicutes* to *Bacteroidetes* ratio was present in the fecal microbiota of SLE patients with inactive disease, which is consistent with observations in other autoimmune diseases. In addition, oral antibiotics are known to trigger lupus flares, again suggesting a role for commensal bacteria in SLE. In this section, we will describe

202 Lupus

the existing data and a proposed role of gut microbiota in the pathogenesis of SLE.

at other sites of the body can also have a systemic effect on the host's immune system.

The host's genetic make-up is the primary factor in determining the composition of one's adult microbiota. Other contributing factors include mother's health in utero, the method of delivery during birth, if one is either breast fed or fed formula, the use of antibiotics during early childhood and types of foods consumed prior to, and during, weaning. The adult microbiota is not a static entity, however, and can be modified through many different environmental factors, and the composition will also change with age. Many of the previously discussed contributions to SLE are associated with alterations in the microbiota. Obese patients have an increased *Firmicutes* to *Bacterioidetes* ratio, with members of *Firmicutes* leading to increased energy harvest from dietary nutrients [113]. The "western diet" is associated with alterations in the gut microbiota as well. Children from a rural village in the West African country of Burkina Faso had increased microbial richness and greater *Prevotella* with decreased *Bacteroides* levels compared to European children. The microbiota of the Burkina Faso children also produced more short chain fatty acids than the intestinal microbiota of the European children [114]. These findings of agrarian societies with increased *Prevotella* and decreased *Bacteroides* have been supported in studies comparing rural South Americans and people from Bangladesh to people living in industrialized regions [115]. The source of dietary fat can impact microbiota composition as well, as mice fed diets with a fat source

*Gut microbiota and SLE*. Evidence is rapidly growing that mutualistic bacteria contribute to the development of a healthy functioning immune system, as well as the development of aberrant immune responses. The past 10 years have seen a rapid growth in the understanding of how the commensal microbiota is able to contribute to health and disease with the exploration of 16S rRNA sequencing along with the use of gnotobiotic animals. These newer methods of DNA sequencing have allowed researchers to investigate the effects of specific species or strains of organisms and how they interact with the immune system. Gnotobiotic animals are born into a sterile environment and develop under sterile conditions so that a specific group of bacteria, viruses, eukaryotes or parasites can be introduced to the animal, and the resulting effects can be studied without the interference of other organisms [106]. Intestinal microbial dysbiosis can lead to immune system effects at distant sites of the body [49, 107–112]. The systemic immune system is influenced by microbiota in the intestines, suggesting that microbiota Murine studies on how the microbiota affects the immune system have emerged recently. At the level of the intestinal lamina propria, bacteria compete with host defenses and establish homeostasis. T cells are kept in check between pro-inflammatory Th1, Th2 and Th17 cells as well as innate lymphoid cells, and the anti-inflammatory T regulatory cells [118–120]. Specific bacterial groups can elicit distinct immune responses, as evidenced by mice colonized with segmented filamentous bacteria developed elevated levels of Th17 cells in the lamina propria [121, 122]. Colonization of previously germ-free mice with clostridial strains from cluster IV and XIVa resulted in an expansion of the T regulatory cell population in the lamina propria and systemically [123]. Polysaccharide A derived from *Bacteroides fragilis* is able to induce IL-10 production in the lamina propria through binding to TLR2 on T regulatory cells, inhibiting the expansion of the local Th17 cell population [124]. Only a single study, reported in 1999, to date has explored the development of SLE in a germ-free mouse strain. That study used MRL/lpr mice and showed no difference in disease progression between conventionally raised mice and germ-free mice [125]. Due to the *Fas* mutation and the underlying cause of lupus disease in this strain of mice, the MRL/lpr strain may not be the best strain to understand the impact of gut microbiome in a germ-free setting on lupus. Few murine studies investigating the microbiota's role in SLE have been published to date. Regardless, gut microbiome in MRL/lpr mice has recently been shown to impact lupus. In the MRL/lpr mouse model of SLE, young female mice had lower numbers of *Lactobacilli* species and increased numbers of *Lachnospiraceae* density in the murine feces. The administration of retinoic acid restored the *Lactobacilli* density and correlated with reduced clinical signs and a reverse of multiple lupus-associated microbial functions. The increased density of *Lachnospiraceae* was also overrepresented in female lupus-prone mice along with butyrate producing *Clostridiaceae* species. There is potential that use of *Lactobacilli* containing probiotics and vitamin A may benefit lupus patients [49, 107].

There have been few human trials to determine the impact on intestinal microbiota specifically on SLE development. In 20 human SLE patients in remission compared to healthy controls, a decreased *Firmicutes* to *Bacterioidetes* ratio was found in fecal samples. This change in fecal microbiota is associated with an increase in oxidative phosphorylation and glycan utilization by the host microbiota [126, 127]. In contrast to the murine studies, *Lachnospiraceae* and *Clostridia* were associated with healthy patients, rather than lupus patients [126]. The intestinal microbiota is a complex conglomeration that can contribute to modulation of the local and systemic immune system. Current and future work will begin to target specific groups and mechanisms of microbial contribution to systemic autoimmune disease pathogenesis.

#### **5. Bacterial metabolites and mechanisms of action**

*Introduction*. Bacterial metabolites produced by the gut microbiota may have profound effects on immune function. Recently, several groups have found that short-chain fatty acids (SCFAs) produced by gut microbiota, especially butyrate produced by Clostridia, can promote the differentiation of regulatory T (Treg) cells in the colon, spleen and lymph nodes and suppress inflammation [128–131]. Treg deficiency leads to autoimmunity, while re-introduction of Treg cells can rescue animals from the disease. In human SLE, a pathogenic role of dysfunctional Treg cells has been suggested. In particular, the imbalance between Treg and Th17 cells and a bias toward IL 17-producing cells (both Th17 cells and double-negative T cells can produce IL-17 in SLE) are widely recognized for SLE. In the gut, the number and diversity of butyrate-producing bacteria are subject to factors related to age, disease and to diet. Butyrate and SCFAs are inhibitors of histone deacetylases (HDACs). HDAC inhibition can result in altering gene expression by making the chromatin more accessible to transcription factors by acetylating histone proteins at specific lysine residues and may also lead to post-translational modification of several transcription factors that reside both cytosolic and nuclear. Below, we have outlined the influence of SCFAs (predominately butyrate) on HDAC activity on the epithelia in the gut microbiota as it is likely to play an important role in regulation of the gut microbiota and lupus.

*Role of epigenetics in immunity*. Although genome-wide association studies have identified many genes that may play a role in the initiation or progression of SLE [132–134], these studies do not account for risk attributed to heritable factors [135] and have failed to identify a unifying switch. Epigenetics is the process in which alterations in gene expression and phenotype occur which are heritable but do not alter the DNA sequence [136]. There is increasing evidence that epigenetics plays a key role in SLE pathogenesis and epigenetically targeted therapies may be efficacious [137, 138]. In regard to epigenetics, interactions between DNA and core histone proteins are important in regulating the accessibility of transcription factors to bind promoter regions and thus regulate gene expression [139]. Histone acetyltransferases (HATs) and HDACs can alter the charge and subsequent binding affinity of core histone proteins to the chromatin through removal or addition of acetyl groups on lysine residues, respectively [140–142]. Recent investigations have revealed that HATs and HDACs are also capable of modifying lysine residues on numerous non-histone nuclear and cytosolic proteins [141, 143], which has driven some researchers to alternatively refer to the enzymes as lysine (K) acetyltransferases (KATs) and lysine deacetylases (KDACs).

*HDAC enzymes*. There are 18 mammalian HDACs, which remove acetyl groups from lysine residues in histones and other proteins to control multiple cellular functions including transcription, cell cycle kinetics, cell signaling and cellular transport processes [144]. HDACs are classified based on structure, homology to yeast HDACs and function into classes I–IV [145, 146]. Class I HDACs (HDAC-1, -2, -3 and -8) are nuclear exclusive enzymes found in a wide range of tissues and cells lines where they are known for histone modification and repression of transcription [147, 148]. Class II HDACs are further subdivided into class IIa (HDAC-4, -5, -7 and -9) and class IIb (HDAC-6 and -10) based on domain organization [149] and exhibit selective tissue expression, nucleocytoplasmic shuttling and function through recruitment of distinct cofactors [148]. Class III comprises the sirtuins, which act through a distinct NAD<sup>+</sup> dependent mechanism and are not considered "classical" HDACs [147]. HDAC11 is the sole member of class IV as phylogenetic analysis revealed very low similarity to HDACs in the other classes [150].

In addition to their initial relevance in cancer biology [151], HDAC enzymes are now increasingly being investigated as regulators of inflammation and immunity [147]. As reviewed by Shakespear et al., HDACs are documented to play a role in myeloid development, toll-like receptor (TLR) and interferon (IFN) signaling in innate immune cells, antigen presentation and development and function of B and T lymphocytes [147]. Subsequently, pharmacologic inhibition of HDACs has been evaluated as a possible treatment modality in a wide spectrum of diseases, including inflammatory and autoimmune diseases [152].

differentiation of regulatory T (Treg) cells in the colon, spleen and lymph nodes and suppress inflammation [128–131]. Treg deficiency leads to autoimmunity, while re-introduction of Treg cells can rescue animals from the disease. In human SLE, a pathogenic role of dysfunctional Treg cells has been suggested. In particular, the imbalance between Treg and Th17 cells and a bias toward IL 17-producing cells (both Th17 cells and double-negative T cells can produce IL-17 in SLE) are widely recognized for SLE. In the gut, the number and diversity of butyrate-producing bacteria are subject to factors related to age, disease and to diet. Butyrate and SCFAs are inhibitors of histone deacetylases (HDACs). HDAC inhibition can result in altering gene expression by making the chromatin more accessible to transcription factors by acetylating histone proteins at specific lysine residues and may also lead to post-translational modification of several transcription factors that reside both cytosolic and nuclear. Below, we have outlined the influence of SCFAs (predominately butyrate) on HDAC activity on the epithelia in the gut microbiota as it is likely to play an important role in regulation of the gut

*Role of epigenetics in immunity*. Although genome-wide association studies have identified many genes that may play a role in the initiation or progression of SLE [132–134], these studies do not account for risk attributed to heritable factors [135] and have failed to identify a unifying switch. Epigenetics is the process in which alterations in gene expression and phenotype occur which are heritable but do not alter the DNA sequence [136]. There is increasing evidence that epigenetics plays a key role in SLE pathogenesis and epigenetically targeted therapies may be efficacious [137, 138]. In regard to epigenetics, interactions between DNA and core histone proteins are important in regulating the accessibility of transcription factors to bind promoter regions and thus regulate gene expression [139]. Histone acetyltransferases (HATs) and HDACs can alter the charge and subsequent binding affinity of core histone proteins to the chromatin through removal or addition of acetyl groups on lysine residues, respectively [140–142]. Recent investigations have revealed that HATs and HDACs are also capable of modifying lysine residues on numerous non-histone nuclear and cytosolic proteins [141, 143], which has driven some researchers to alternatively refer to the enzymes as lysine (K) acetyltransferases (KATs) and lysine deacetylases

*HDAC enzymes*. There are 18 mammalian HDACs, which remove acetyl groups from lysine residues in histones and other proteins to control multiple cellular functions including transcription, cell cycle kinetics, cell signaling and cellular transport processes [144]. HDACs are classified based on structure, homology to yeast HDACs and function into classes I–IV [145, 146]. Class I HDACs (HDAC-1, -2, -3 and -8) are nuclear exclusive enzymes found in a wide range of tissues and cells lines where they are known for histone modification and repression of transcription [147, 148]. Class II HDACs are further subdivided into class IIa (HDAC-4, -5, -7 and -9) and class IIb (HDAC-6 and -10) based on domain organization [149] and exhibit selective tissue expression, nucleocytoplasmic shuttling and function through recruitment of distinct cofactors [148]. Class III comprises the sirtuins, which act through a distinct NAD<sup>+</sup>

dependent mechanism and are not considered "classical" HDACs [147]. HDAC11 is the sole member of class IV as phylogenetic analysis revealed very low similarity to HDACs in the


microbiota and lupus.

204 Lupus

(KDACs).

other classes [150].

*SCFAs*. Intestinal bacteria provide the hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community and has been reviewed by others [153]. Recent studies have shown that metabolism and immunology are intertwined and the field of immunometabolism has emerged which examines how gut metabolites influence immune cell function [154]. In the gut, carbohydrates resistant to breakdown in the stomach and small intestine are subject to colonic fermentation to result in the production of SCFAs, containing1–6 carbon atoms. Anaerobic bacteria generate the major SCFAs and include acetate, propionate and butyrate. The SCFA butyrate is produced by fermentation of dietary fiber by the intestinal microbiota and is the primary energy source of colonocytes [155]. Recently, Imhann and coworkers showed that patients with inflammatory bowel disease (IBD) had a decrease in the genus *Roseburia*. Furthermore, they noted that *Roseburia* spp. is acetate-to-butyrate converters suggesting that a lack of butyrate may contribute to IBD [156]. Furthermore, a reduction on butyrate producing *Roseburia* ssp. has been associated with chronic kidney disease progression [157]. On the other hand, butyrate has seen shown to aggravate dextran sulfate sodium induce colitis in an animal model [158]. In regard to HDAC inhibition, butyric acid has been reported to specifically inhibit the class I HDACs (1, 2, 3 and 8) [159].

*T cells in gut immunity*. Balanced mucosal immunity in the gut is critical for host homeostasis and defense. Naïve CD4<sup>+</sup> T cells when activated differentiate into T helper cell [Th1, Th2, Th17 or follicular helper (Tfh)] depending on cytokine exposure and B cell or antigen presenting cell influence. One specific subset of T cells (Treg cells) acts to suppress effect T cell function. The majority of Treg cells develop in the thymus (nTregs) and are selected for by strong or intermediate T cell receptor (TCR) signals while escaping negative selection [160]. Additionally, Treg cells can also be induced under certain circumstances from naïve T cells in the periphery (iTregs). This can happen systemically and has been shown to occur at interface with the environment in both whole animal and in in vitro assays [161]. Treg differentiation is strongly influenced by the presence of the anti-inflammatory cytokine transforming growth factor β (TGF-β). Furthermore, expression of the transcription factor Foxp3 is essential for Treg development and function and is regulated by genomic regulatory elements termed conserved noncoding DNA sequences (CNS). While CNS1 is unnecessary for nTreg differentiation, it has been reported to be crucial for iTreg generation in gut-associated lymphoid tissues (GALT) [162]. CNS2 is required for Foxp3 expression in the progeny of dividing Treg cells. CNS3 controls *de novo* Foxp3 expression and nTreg differentiation [163]. Studies have shown that Treg cells expressing transcription factor Foxp3 have a key role in limiting inflammatory responses in the intestine [164, 165].

*Treg-Th17 balance*. Th17 is a subset of T helper cells and serves to maintain the mucosal barrier and contribute to pathogen clearance at mucosal surfaces. However, they have also been implicated in autoimmune and inflammatory disorders as the loss of Th17 cells at mucosal surfaces has been shown to allow chronic inflammation and microbial translocation. In the gut, there exists a balance of Treg-Th17 cells as the signals that cause Th17s to differentiate actually inhibit Treg differentiation [166]. Since both Treg and Th17 cells are both pertinent to gut homeostasis and immune regulation, the balance of these T cells is critical for homeostasis [167]. Treg cells prevent systemic and tissue-specific autoimmunity and inflammatory lesions at mucosal interfaces [165]. Mice deficient in iTregs spontaneously developed pronounced Th2-type pathologies at mucosal sites including in the gastrointestinal tract and lungs and shows hallmarks of allergic inflammation and asthma [168]. Studies have shown that in the gut, iTreg cells are the prominent phenotype and are rapidly induced following naïve T cell activation which is dependent on Notch2-singling and may be somewhat independent of TGF-β [165]. This suggests that whereas nTreg cells generated in the thymus appear sufficient for control of systemic and tissue-specific autoimmunity, extrathymic differentiation of iTregs affects the commensal microbiota composition and serves a distinct, essential function in maintaining the inflammatory response at mucosal interfaces. Furthermore, when the animals were given the SCFA (butyrate), Treg cell differentiation in the gut increased and this was dependent on CNS1 expression [129]. In addition to butyrate, de novo iTreg generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of HDAC inhibition, but not acetate, which lacks this HDAC-inhibitory activity, suggesting that bacterial metabolites mediate communication between the commensal microbiota and the immune system. Other studies have also reported that a butyrate-mediated increase in the Treg cell subset in vivo was due to increased extrathymic generation of Treg cells and not due to their increased nTreg cells [169]. In addition to butyrate inducing a Treg phenotype, butyrate has also been shown to increases macrophage phagocytosis and killing of bacteria. When Treg cells were cultured with stimulated macrophages exposed to IL-4 and butyrate, less inflammatory cytokines were produced compared to macrophages treated with IL-4 indicating that microbial-derived butyrate decreases inflammatory mediator production in the gut [170]. In other studies using the antibiotic vancomycin, which targets Gram-positive bacteria, the level of iTregs was reduced. This could be due to the decrease in *Roseburia* spp. that is the butyrate-producing, Gram-positive anaerobic bacteria that inhabit the human colon. However, when specific pathogen-free mice were treated with a combination of vancomycin and SCFAs, the reduction in iTregs was completely restored suggesting that SCFAs play a role in iTreg homeostasis [128].

*HDAC and Treg-Th17 balance in lupus*. HDAC inhibition has been shown to decrease disease in lupus-prone MRL/lpr and NZB/W F1 mice [171–174]. Mechanisms by which HDAC inhibition decreases SLE disease have previously been reviewed by Reilly and others [142, 175]. HDAC inhibition may act in several ways including correction the hypoacetylation states of histones H3 and H4 [176], increased CD4<sup>+</sup> CD25<sup>+</sup> Foxp3+ Treg cells [172, 174], reduced Th1 and Th17-inducing cytokines (IL-12 and IL-23) as well as Th1-attracting chemokines [142], and inhibition of germline and post-switch immunoglobulin transcripts in splenic B cells [177]. More importantly in SLE, decreased renal disease (glomerulonephritis and proteinuria) has been consistently reported in studies investigating the use of HDAC inhibitors to treat lupus in various mouse models [171–174]. Pan and selective HDAC inhibitors are being evaluated in the clinic for inflammatory diseases with some mixed results and adverse effects such as fatigue, nausea, vomiting, diarrhea, thrombocytopenia, neutropenia and cardiac irregularities [178]. Investigations of specific functions for each HDAC isoform in knockout mice have revealed that elimination of class I and class IIa HDACs result in embryonic lethal phenotypes or fatal cardiac, vascular, musculoskeletal or neural crest defects, and specific HDAC isoform activity is required for normal cells development [178, 179]. The observed butyrate-mediated increase in the Treg cell subsets in vivo due to increased extrathymic generation of Treg cells and not due to their increased thymic output would support a role of HDACi in gut homeostasis [169]. In studies involving lupus patients, while Treg or Th17 cells alone were not correlated with SLE development, the ratio of Treg to Th17 cells in active SLE patients was significantly lower than that in inactive SLE patients and healthy controls. Moreover, corticosteroid treatment increased the ratio of Treg to Th17 cells in active SLE patients. Indeed, the Treg/Th17 cell ratio is inversely correlated with the severity of active SLE, indicating that in active SLE, there appears to exist an imbalance between Treg and Th17 cells [180]. Inducible Tregs cells are dependent on the expression of the transcription factor Foxp3 which may be transiently expressed allowing for plasticity of the Tregs/Th17 phenotype [181]. Interestingly, when Foxp3 is acetylated, the transcription factor becomes more stable and has greater propensity to bind DNA yielding a more stable and effective Treg population [182]. In addition to the regulation of Treg differentiation, butyrate has also been shown to increase the tri-methylation of lysine 27 on histone 3 (H3K27me3) in the promoter of nuclear factor-κB1 (NF-κB1) in colon tissue resulting in repression of inflammation [183]. In our lupus mouse studies, we found a marked depletion of lactobacilli in our lupus animals and increases in *Lachnospiraceae* compared to age-matched health controls. Interestingly, we also found that *Lachnospiraceae*, butyrate-producing genera, was more abundant in the gut of lupus-prone mice at specific time points during lupus progression [49]. Whether this was causative or in response to disease pathogenesis is an active area of investigation in our laboratory. Nonetheless, our results and others demonstrate the dynamics of gut microbiota as that bacterial production of SCFAs and butyrate may play a role in the initiation and progression of inflammation and autoimmunity.

#### **Author details**

*Treg-Th17 balance*. Th17 is a subset of T helper cells and serves to maintain the mucosal barrier and contribute to pathogen clearance at mucosal surfaces. However, they have also been implicated in autoimmune and inflammatory disorders as the loss of Th17 cells at mucosal surfaces has been shown to allow chronic inflammation and microbial translocation. In the gut, there exists a balance of Treg-Th17 cells as the signals that cause Th17s to differentiate actually inhibit Treg differentiation [166]. Since both Treg and Th17 cells are both pertinent to gut homeostasis and immune regulation, the balance of these T cells is critical for homeostasis [167]. Treg cells prevent systemic and tissue-specific autoimmunity and inflammatory lesions at mucosal interfaces [165]. Mice deficient in iTregs spontaneously developed pronounced Th2-type pathologies at mucosal sites including in the gastrointestinal tract and lungs and shows hallmarks of allergic inflammation and asthma [168]. Studies have shown that in the gut, iTreg cells are the prominent phenotype and are rapidly induced following naïve T cell activation which is dependent on Notch2-singling and may be somewhat independent of TGF-β [165]. This suggests that whereas nTreg cells generated in the thymus appear sufficient for control of systemic and tissue-specific autoimmunity, extrathymic differentiation of iTregs affects the commensal microbiota composition and serves a distinct, essential function in maintaining the inflammatory response at mucosal interfaces. Furthermore, when the animals were given the SCFA (butyrate), Treg cell differentiation in the gut increased and this was dependent on CNS1 expression [129]. In addition to butyrate, de novo iTreg generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of HDAC inhibition, but not acetate, which lacks this HDAC-inhibitory activity, suggesting that bacterial metabolites mediate communication between the commensal microbiota and the immune system. Other studies have also reported that a butyrate-mediated increase in the Treg cell subset in vivo was due to increased extrathymic generation of Treg cells and not due to their increased nTreg cells [169]. In addition to butyrate inducing a Treg phenotype, butyrate has also been shown to increases macrophage phagocytosis and killing of bacteria. When Treg cells were cultured with stimulated macrophages exposed to IL-4 and butyrate, less inflammatory cytokines were produced compared to macrophages treated with IL-4 indicating that microbial-derived butyrate decreases inflammatory mediator production in the gut [170]. In other studies using the antibiotic vancomycin, which targets Gram-positive bacteria, the level of iTregs was reduced. This could be due to the decrease in *Roseburia* spp. that is the butyrate-producing, Gram-positive anaerobic bacteria that inhabit the human colon. However, when specific pathogen-free mice were treated with a combination of vancomycin and SCFAs, the reduction in iTregs was completely restored suggesting that SCFAs play a role

*HDAC and Treg-Th17 balance in lupus*. HDAC inhibition has been shown to decrease disease in lupus-prone MRL/lpr and NZB/W F1 mice [171–174]. Mechanisms by which HDAC inhibition decreases SLE disease have previously been reviewed by Reilly and others [142, 175]. HDAC inhibition may act in several ways including correction the hypoacetylation states of

CD25<sup>+</sup>

and Th17-inducing cytokines (IL-12 and IL-23) as well as Th1-attracting chemokines [142], and inhibition of germline and post-switch immunoglobulin transcripts in splenic B cells [177]. More importantly in SLE, decreased renal disease (glomerulonephritis and proteinuria) has been consistently reported in studies investigating the use of HDAC inhibitors to

Foxp3+

Treg cells [172, 174], reduced Th1-

in iTreg homeostasis [128].

206 Lupus

histones H3 and H4 [176], increased CD4<sup>+</sup>

Xin M. Luo\*, Michael R. Edwards, Christopher M. Reilly, Qinghui Mu and S. Ansar Ahmed

\*Address all correspondence to: xinluo@vt.edu

Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

#### **References**

[1] Strachan DP. Hay fever, hygiene, and household size. BMJ. 1989;299(6710):1259–60.


[18] Ram M, Anaya JM, Barzilai O, Izhaky D, Porat Katz BS, Blank M, et al. The putative protective role of hepatitis B virus (HBV) infection from autoimmune disorders. Autoimmun Rev. 2008;7(8):621–5.

[2] Cooke A. Infection and autoimmunity. Blood Cells Mol Dis. 2009;42(2):105–7.

comparison of worldwide disease burden. Lupus. 2006;15(5):308–18.

2011;23(2):122–9.

208 Lupus

2001;30(6):360–2.

2015;44(2):143–9.

2004;31(8):1546–50.

Rheum. 1999;42(1):46–50.

and diet? Front Immunol. 2015;6:608.

or foes? Trends Immunol. 2009;30(8):409–14.

[3] Zaccone P, Cooke A. Infectious triggers protect from autoimmunity. Semin Immunol.

[4] Uramoto KM, Michet CJ, Jr., Thumboo J, Sunku J, O'Fallon WM, Gabriel SE. Trends in the incidence and mortality of systemic lupus erythematosus, 1950–1992. Arthritis

[5] Danchenko N, Satia JA, Anthony MS. Epidemiology of systemic lupus erythematosus: a

[7] Mu Q, Zhang H, Luo XM. SLE: another autoimmune disorder influenced by microbes

[8] Severin MC, Levy Y, Shoenfeld Y. Systemic lupus erythematosus and parvovirus B-19: casual coincidence or causative culprit? Clin Rev Allergy Immunol. 2003;25(1):41–8. [9] Kivity S, Agmon-Levin N, Blank M, Shoenfeld Y. Infections and autoimmunity—friends

[10] Hayashi T, Lee S, Ogasawara H, Sekigawa I, Iida N, Tomino Y, et al. Exacerbation of systemic lupus erythematosus related to cytomegalovirus infection. Lupus. 1998;7(8):561–4.

[11] James JA, Kaufman KM, Farris AD, Taylor-Albert E, Lehman TJ, Harley JB. An increased prevalence of Epstein-Barr virus infection in young patients suggests a possible etiology

[12] Nawata M, Seta N, Yamada M, Sekigawa I, Lida N, Hashimoto H. Possible triggering effect of cytomegalovirus infection on systemic lupus erythematosus. Scand J Rheumatol.

[13] Nelson P, Rylance P, Roden D, Trela M, Tugnet N. Viruses as potential pathogenic agents

[14] Rasmussen NS, Draborg AH, Nielsen CT, Jacobsen S, Houen G. Antibodies to early EBV, CMV, and HHV6 antigens in systemic lupus erythematosus patients. Scand J Rheumatol.

[15] Sundar K, Jacques S, Gottlieb P, Villars R, Benito ME, Taylor DK, et al. Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) in the mouse can elicit the production of

[16] Esposito S, Bosis S, Semino M, Rigante D. Infections and systemic lupus erythematosus.

[17] Sawalha AH, Schmid WR, Binder SR, Bacino DK, Harley JB. Association between systemic lupus erythematosus and Helicobacter pylori seronegativity. J Rheumatol.

anti-dsDNA and anti-Sm antibodies. J Autoimmun. 2004;23(2):127–40.

for systemic lupus erythematosus. J Clin Investig. 1997;100(12):3019–26.

in systemic lupus erythematosus. Lupus. 2014;23(6):596–605.

Eur J Clin Microbiol Infect Dis. 2014;33(9):1467–75.

[6] Bach JF. Infections and autoimmune diseases. J Autoimmun. 2005;25(Suppl):74–80.


[46] Nozaki Y, Yamagata T, Yoo BS, Sugiyama M, Ikoma S, Kinoshita K, et al. The beneficial effects of treatment with all-trans-retinoic acid plus corticosteroid on autoimmune nephritis in NZB/WF mice. Clin Exp Immunol. 2005;139(1):74–83.

[31] Henry T, Kirimanjeswara GS, Ruby T, Jones JW, Peng K, Perret M, et al. Type I IFN signaling constrains IL-17A/F secretion by gammadelta T cells during bacterial infections.

[32] Alyanakian MA, Grela F, Aumeunier A, Chiavaroli C, Gouarin C, Bardel E, et al. Transforming growth factor-beta and natural killer T-cells are involved in the protective effect

[33] Mora JR, Iwata M, von Andrian UH. Vitamin effects on the immune system: vitamins A

[34] Ross AC, Chen Q, Ma Y. Augmentation of antibody responses by retinoic acid and

[35] Ma Y, Ross AC. The anti-tetanus immune response of neonatal mice is augmented by retinoic acid combined with polyriboinosinic:polyribocytidylic acid. Proc Natl Acad Sci

[36] Zhou X, Kong N, Wang J, Fan H, Zou H, Horwitz D, et al. Cutting edge: all-trans retinoic acid sustains the stability and function of natural regulatory T cells in an inflammatory

[37] Sobel ES, Brusko TM, Butfiloski EJ, Hou W, Li S, Cuda CM, et al. Defective response of CD4(+) T cells to retinoic acid and TGFbeta in systemic lupus erythematosus. Arthritis

[38] Ross AC, Chen Q, Ma Y. Vitamin A and retinoic acid in the regulation of B-cell develop-

[39] Duriancik DM, Lackey DE, Hoag KA. Vitamin A as a regulator of antigen presenting

[40] Long KZ, Santos JI, Rosado JL, Estrada-Garcia T, Haas M, Al Mamun A, et al. Vitamin A supplementation modifies the association between mucosal innate and adaptive immune responses and resolution of enteric pathogen infections. Am J Clin Nutr. 2011;93(3):578–85.

[41] Hall JA, Cannons JL, Grainger JR, Dos Santos LM, Hand TW, Naik S, et al. Essential role for retinoic acid in the promotion of CD4(+) T cell effector responses via retinoic acid

[42] Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science. 2007;317(5835):256–60.

[43] Ross AC. Vitamin A and retinoic acid in T cell-related immunity. Am J Clin Nutr.

[44] Nozaki Y, Yamagata T, Sugiyama M, Ikoma S, Kinoshita K, Funauchi M. Anti-inflammatory effect of all-trans-retinoic acid in inflammatory arthritis. Clin Immunol. 2006;119(3):272–9.

[45] Kinoshita K, Yoo BS, Nozaki Y, Sugiyama M, Ikoma S, Ohno M, et al. Retinoic acid reduces autoimmune renal injury and increases survival in NZB/W F1 mice. J Immunol.

ment and antibody production. Vitamins Hormon. 2011;86:103–26.

of a bacterial extract on type 1 diabetes. Diabetes. 2006;55(1):179–85.

and D take centre stage. Nat Rev Immunol. 2008;8(9):685–98.

costimulatory molecules. Semin Immunol. 2009;21(1):42–50.

J Immunol. 2010;184(7):3755–67.

210 Lupus

U S A. 2005;102(38):13556–61.

Res Ther. 2011;13(3):R106.

cells. J Nutr. 2010;140(8):1395–9.

2012;96(5):1166S–72S.

2003;170(11):5793–8.

receptor alpha. Immunity. 2011;34(3):435–47.

milieu. J Immunol. 2010;185(5):2675–9.


[75] Yu Y CP, Vanden Heuvel JP. Conjugated linoleic acid decreases production of proinflammatory products in macrophages: evidence for a PPAR gamma-dependent mechanism. Biochim Biophys Acta. 2002;1581(3):89–99.

[61] Hsieh CC, Lin BF. Opposite effects of low and high dose supplementation of vitamin E

[62] Venkatraman J MK. Effects of dietary omega3 and omega6 lipids and vitamin E on chemokine levels in autoimmune-prone MRLMpJ-lprlpr mice. J Nutr Biochem. 2002;13(8):479.

[63] Fernandes G. Dietary lipids and risk of autoimmune disease. Clin Immunol

[64] Lin BF, Jeng SJ, Chiang BL, Huang CC. Dietary fat affects lipids and anti-cardiolipin antibody levels in autoimmune-prone NZB/W F1 mice. Br J Nutr. 1997;77(4):657–69.

[65] Fritsche K. Fatty acids as modulators of the immune response. Ann Rev Nutr. 2006;26:

[66] Reifen R BM, Afek A, Kopilowiz Y, Sklan D, Gershwin ME, German B, Yoshida S, Shoenfeld Y. Dietary polyunsaturated fatty acids decrease anti-dsDNA and anti-cardiolipin antibodies production in idiotype induced mouse model of systemic lupus erythe-

[67] Chandrasekar B TD, Venkatraman JT, Fernandes G. Dietary omega-3 lipids delay the onset and progression of autoimmune lupus nephritis by inhibiting transforming growth factor beta mRNA and protein expression. J Autoimmunol. 1995;8(3):381–93.

[68] Reynolds CM, Roche HM. Conjugated linoleic acid and inflammatory cell signalling.

[69] Halade GV, Williams PJ, Veigas JM, Barnes JL, Fernandes G. Concentrated fish oil (Lovaza(R)) extends lifespan and attenuates kidney disease in lupus-prone short-lived

[70] Yang M PM, Cook ME. Dietary conjugated linoleic acid protects against end stage disease of systemic lupus erythematosus in the NZB/W F1 mouse. Immunopharmacol

[71] Yang M CM. Dietary conjugated linoleic acid decreased cachexia, macrophage tumor necrosis factor-alpha production, and modifies splenocyte cytokines production. Exp

[72] Lee Y, Vanden Heuvel JP. Inhibition of macrophage adhesion activity by 9trans,11trans-

[73] Dowling JK, McCoy CE, Doyle SL, BenLarbi N, Canavan M, O'Neill LA, et al. Conjugated linoleic acid suppresses IRF3 activation via modulation of CD14. J Nutr Biochem.

[74] Draper E, DeCourcey J, Higgins SC, Canavan M, McEvoy F, Lynch M, et al. Conjugated linoleic acid suppresses dendritic cell activation and subsequent Th17 responses. J Nutr

Prostaglandins Leukot Essent Fatty Acids. 2010;82(4–6):199–204.

(NZB×NZW)F1 mice. Exp Biol Med. 2013;238(6):610–22.

conjugated linoleic acid. J Nutr Biochem. 2010;21(6):490–7.

on survival of MRL/lpr mice. Nutrition. 2005;21(9):940–8.

Immunopathol. 1994;72(2):193–7.

matosus. Lupus. 1998;7(3):192–7.

Immunotoxicol. 2000;22(3):433–49.

Biol Med. 2003;228(1):51–8.

Biochem. 2014;25(7):741–9.

2013;24(5):920–8.

45–73.

212 Lupus


[102] Akaogi J, Barker T, Kuroda Y, Nacionales DC, Yamasaki Y, Stevens BR, et al. Role of nonprotein amino acid L-canavanine in autoimmunity. Autoimmun Rev. 2006;5(6):429–35.

[88] Barbosa Vde S RJ, Antônio da Silva N. Possible role of adipokines in systemic lupus erythematosus and rheumatoid arthritis. Rev Bras Reumatol. 2012;52(2):278–87.

[89] Loghman M HA, Broumand B, Ataipour Y, Tohidi M, Marzbani C, Fakharran M. Association between urinary adiponectin level and renal involvement in systemic

[90] Gilbert EL, Ryan MJ. High dietary fat promotes visceral obesity and impaired endothelial function in female mice with systemic lupus erythematosus. Gend Med.

[91] Ryan MJ. The pathophysiology of hypertension in systemic lupus erythematosus. Am

[92] Kleinewietfeld M, Manzel A, Titze J, Kvakan H, Yosef N, Linker RA, et al. Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells. Nature.

[93] Wu C, Yosef N, Thalhamer T, Zhu C, Xiao S, Kishi Y, et al. Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1. Nature. 2013;496(7446):513–7.

[94] Zou YF, Xu JH, Tao JH, Xu SQ, Liu S, Chen SY, et al. Impact of environmental factors on efficacy of glucocorticoids in Chinese population with systemic lupus erythematosus.

[95] Khan D, Dai R, Karpuzoglu E, Ahmed SA. Estrogen increases, whereas IL-27 and IFNgamma decrease, splenocyte IL-17 production in WT mice. Eur J Immunol. 2010;40(9):

[96] Menon R WS, Thomas LN, Allred CD, Dabney A, Azcarate-Peril MA, Sturino JM. Diet complexity and estrogen receptor β status affect the composition of the murine intesti-

[97] Morito K AT, Hirose T, Kinjo J, Hasegawa J, Ogawa S, Inoue S, Muramatsu M, Masamune Y. Interaction of phytoestrogens with estrogen receptors alpha and beta (II). Biol Pharm

[98] Hong Y WT, Huang C, Cheng W, Lin B. Soy isoflavones supplementation alleviates disease severity in autoimmune-prone MRL-lpr/lpr mice. Lupus. 2008;17(9):814–21.

[99] Zhao JH, Sun SJ, Horiguchi H, Arao Y, Kanamori N, Kikuchi A, et al. A soy diet accelerates renal damage in autoimmune MRL/Mp-lpr/lpr mice. Int Immunopharmacol.

[100] Hong YH HC, Wang SC, Lin BF. The ethyl acetate extract of alfalfa sprout ameliorates disease severity of autoimmune-prone MRL-lpr/lpr mice. Lupus. 2009;18(3):206–15.

[101] Schoenroth LJ, Hart DA, Pollard KM, Fritzler MJ. The effect of the phytoestrogen coumestrol on the NZB/W F1 murine model of systemic lupus. J Autoimmun.

lupus erythematous. Int J Rheum Dis. 2016;19(7):678–84.

J Physiol Regul Integr Comp Physiol. 2009;296(4):R1258–67.

nal microbiota. Appl Environ Microbiol. 2013;79(18):5763–73.

2011;8(2):150–5.

214 Lupus

2013;496(7446):518–22.

Bull. 2002;25(1):48–52.

2005;5(11):1601–10.

2004;23(4):323–32.

2549–56.

Inflammation. 2013;36(6):1424–30.


[129] Arpaia N, Campbell C, Fan X, Dikiy S, van der Veeken J, deRoos P, et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature. 2013;504(7480):451–5.

[116] Huang EY, Leone VA, Devkota S, Wang Y, Brady MJ, Chang EB. Composition of dietary fat source shapes gut microbiota architecture and alters host inflammatory mediators

[117] Hildebrandt MA, Hoffmann C, Sherrill-Mix SA, Keilbaugh SA, Hamady M, Chen YY, et al. High-fat diet determines the composition of the murine gut microbiome indepen-

[118] Tlaskalova-Hogenova H, Stepankova R, Hudcovic T, Tuckova L, Cukrowska B, Lodinova-Zadnikova R, et al. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 2004;93(2–3):97–108.

[119] Owyang C, Wu GD. The gut microbiome in health and disease. Gastroenterology.

[120] Cerf-Bensussan N, Gaboriau-Routhiau V. The immune system and the gut microbiota:

[121] Ivanov, II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell. 2009;139(3):485–98.

[122] Wu HJ, Ivanov, II, Darce J, Hattori K, Shima T, Umesaki Y, et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity.

[123] Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota.

[124] Ochoa-Reparaz J, Mielcarz DW, Wang Y, Begum-Haque S, Dasgupta S, Kasper DL, et al. A polysaccharide from the human commensal *Bacteroides fragilis* protects against

[125] Maldonado MA KV, MacDonald GC, Chen F, Reap EA, Balish E, Farkas WR, Jennette JC, Madaio MP, Kotzin BL, Cohen PL, Eisenberg RA. The role of environmental antigens in the spontaneous development of autoimmunity in MRL-lpr mice. J Immunol.

[126] Hevia A, Milani C, Lopez P, Cuervo A, Arboleya S, Duranti S, et al. Intestinal dysbiosis associated with systemic lupus erythematosus. mBio. 2014;5(5):e01548–14.

[127] de Araujo Navas EA, Sato EI, Pereira DF, Back-Brito GN, Ishikawa JA, Jorge AO, et al. Oral microbial colonization in patients with systemic lupus erythematous: correlation

[128] Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly YM, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science.

CNS demyelinating disease. Mucosal Immunol. 2010;3(5):487–95.

with treatment and disease activity. Lupus. 2012;21(9):969–77.

in mouse adipose tissue. J Parenter Enter Nutr. 2013;37(6):746–54.

dently of obesity. Gastroenterology. 2009;137(5):1716–24 e1–2.

friends or foes? Nat Rev Immunol. 2010;10(10):735–44.

2014;146(6):1433–6.

216 Lupus

2010;32(6):815–27.

1999;162(11):6322–30.

2013;341(6145):569–73.

Nature. 2013;500(7461):232–6.


[159] Newman JC, Verdin E. Beta-hydroxybutyrate: much more than a metabolite. Diabetes Res Clin Pract. 2014;106(2):173–81.

[144] Li G, Jiang H, Chang M, Xie H, Hu L. HDAC6 alpha-tubulin deacetylase: a potential therapeutic target in neurodegenerative diseases. J Neurol Sci. 2011;304(1–2):1–8.

[145] Yang Y, Rao R, Shen J, Tang Y, Fiskus W, Nechtman J, et al. Role of acetylation and extracellular location of heat shock protein 90alpha in tumor cell invasion. Cancer Res. 2008;68(12):

[146] de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J. 2003;370(Pt

[147] Shakespear MR, Halili MA, Irvine KM, Fairlie DP, Sweet MJ. Histone deacetylases as regulators of inflammation and immunity. Trends Immunol. 2011;32(7):335–43.

[148] Verdin E, Dequiedt F, Kasler HG. Class II histone deacetylases: versatile regulators.

[149] Martin M, Kettmann R, Dequiedt F. Class IIa histone deacetylases: regulating the regu-

[150] Yang XJ, Seto E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast

[151] Marks PA, Rifkind RA, Richon VM, Breslow R. Inhibitors of histone deacetylase are

[152] Dinarello CA, Fossati G, Mascagni P. Histone deacetylase inhibitors for treating a spec-

[153] Nagpal R, Kumar M, Yadav AK, Hemalatha R, Yadav H, Marotta F, et al. Gut microbiota in health and disease: an overview focused on metabolic inflammation. Benef

[154] Rathmell JC. Metabolism and autophagy in the immune system: immunometabolism

[155] Scott KP, Gratz SW, Sheridan PO, Flint HJ, Duncan SH. The influence of diet on the gut

[156] Imhann F, Vich Vila A, Bonder MJ, Fu J, Gevers D, Visschedijk MC, et al. Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of

[157] Jiang S, Xie S, Lv D, Zhang Y, Deng J, Zeng L, et al. A reduction in the butyrate producing species *Roseburia* spp. and *Faecalibacterium prausnitzii* is associated with chronic

kidney disease progression. Antonie Van Leeuwenhoek. 2016;109(10):1389–96.

[158] Zhang Q, Wu Y, Wang J, Wu G, Long W, Xue Z, et al. Accelerated dysbiosis of gut microbiota during aggravation of DSS-induced colitis by a butyrate-producing bacte-

potentially effective anticancer agents. Clin Cancer Res. 2001;7(4):759–60.

trum of diseases not related to cancer. Mol Med. 2011;17(5–6):333–52.

4833–42.

218 Lupus

3):737–49.

Trends Genet. 2003;19(5):286–93.

Microbes. 2016;7(2):181–94.

rium. Sci Rep. 2016;6:27572.

comes of age. Immunol Rev. 2012;249(1):5–13.

microbiota. Pharmacol Res. 2013;69(1):52–60.

inflammatory bowel disease. Gut. 2016–312135.

lators. Oncogene. 2007;26(37):5450–67.

to mice and men. Nat Rev Mol Cell Biol. 2008;9(3):206–18.


[172] Regna NL, Chafin CB, Hammond SE, Puthiyaveetil AG, Caudell DL, Reilly CM. Class I and II histone deacetylase inhibition by ITF2357 reduces SLE pathogenesis in vivo. Clin

[173] Reilly CM, Mishra N, Miller JM, Joshi D, Ruiz P, Richon VM, et al. Modulation of renal disease in MRL/lpr mice by suberoylanilide hydroxamic acid. J Immunol.

[174] Reilly CM, Thomas M, Gogal R, Jr., Olgun S, Santo A, Sodhi R, et al. The histone deacetylase inhibitor trichostatin A upregulates regulatory T cells and modulates autoimmu-

[175] Cantley MD, Haynes DR. Epigenetic regulation of inflammation: progressing from broad acting histone deacetylase (HDAC) inhibitors to targeting specific HDACs.

[176] Garcia BA, Busby SA, Shabanowitz J, Hunt DF, Mishra N. Resetting the epigenetic histone code in the MRL-lpr/lpr mouse model of lupus by histone deacetylase inhibition.

[177] Lu ZP, Ju ZL, Shi GY, Zhang JW, Sun J. Histone deacetylase inhibitor Trichostatin A reduces anti-DNA autoantibody production and represses IgH gene transcription.

[178] Balasubramanian S, Verner E, Buggy JJ. Isoform-specific histone deacetylase inhibitors:

[179] Witt O, Lindemann R. HDAC inhibitors: magic bullets, dirty drugs or just another tar-

[180] Ma J, Yu J, Tao X, Cai L, Wang J, Zheng SG. The imbalance between regulatory and IL-17-secreting CD4+ T cells in lupus patients. Clin Rheumatol. 2010;29(11):1251–8. [181] Schmidt A, Elias S, Joshi RN, Tegner J. In vitro differentiation of human CD4+FOXP3+ induced regulatory T cells (iTregs) from naive CD4+ T cells using a TGF-beta-containing

[182] Tao R, de Zoeten EF, Ozkaynak E, Chen C, Wang L, Porrett PM, et al. Deacetylase inhibition promotes the generation and function of regulatory T cells. Nat Med.

[183] Liu Y, Upadhyaya B, Fardin-Kia AR, Juenemann RM, Dey M. Dietary resistant starch type 4-derived butyrate attenuates nuclear factor-kappa-B1 through modulation of his-

tone H3 trimethylation at lysine 27. Food Funct. 2016;7(9):3772–81.

nity in NZB/W F1 mice. J Autoimmun. 2008;31(2):123–30.

Inflammopharmacology. 2013;21(4):301–7.

Biochem Biophys Res Commun. 2005;330(1):204–9.

the next step? Cancer Lett. 2009;280(2):211–21.

geted therapy. Cancer Lett. 2009;280(2):123–4.

J Proteome Res. 2005;4(6):2032–42.

protocol. J Vis Exp. 2016;(118).

2007;13(11):1299–307.

Immunol. 2014;151(1):29–42.

2004;173(6):4171–8.

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## *Edited by Wahid Ali Khan*

Lupus is a new book that will serve as perfect update to lupus for all those who are studying/working at different levels starting from graduate students up to senior investigators. This book provides a comprehensive guide to the latest issues and new developments in the field of lupus. The contents are based on different authors extensive research experience in lupus whether for basic or clinical specialties. Divided into four sections, each one covers the basic concept that will be equally valuable both for the expert and for those who are beginners in this field.

Lupus

Lupus

*Edited by Wahid Ali Khan*

Photo by photographer / iStock