**6. Th1/Th2 Cells, Treg Cells, IL-2R, and IL-15**

Charles J. Malemud: Suppression of Pro-Inflammatory Cytokines via Targeting of

**Major Function(s) of STAT-Responsive Genes3** 

Complexes with IL-2Rβ/IL-2Rγ→high affinity IL-2R Activator of JAK3, I-SRE-4/IL-10 gene ↑ Treg Cell Development IL18/IL-18R1

Activator of JAK3

Heterodimer between gp130/LIFR5 forms the OSMR6 ↑Th2 Differentiation

Activator of multiple protein kinases

Activator of JAK2

Activator of JAK3; Activator of p387, JNK8

**Involvement of STAT activator in RA** 

Antiinflammatory Cytokine

↑ T-Cell Growth ↑ T-Cell Development

Inducer of TNF-α, GM-CSF, IFN-γ

Promotes Th17 Cell Production;

↑MMP Synthesis ↑MMP Synthesis

Promotes Th17 Cell Production

Inhibitor of Anti-Inflammatory Cytokine IL-4 and IL-10 Production

↑ Monocyte Trafficking ↑ MMP-2 ↑ VEGF

Activator of NF-κB

**Representative Reference** 

[81]

[73] [207] [127]

[9] [174] [140]

[207]

[140]

[100]

[118]

[86]

[207] [207] [113]

[122] [125]

[200] [86] [186]

**Activated STAT(s)2** 

STAT6

STAT5, STAT5A, STAT5B STAT6

STAT3/STAT1 STAT5 STAT4 STAT3 STAT4

STAT3/STAT4/STAT5A

STAT3/STAT4/STAT5A

STAT1/STAT4/STAT5/STAT6

STAT5 STAT5 STAT4

STAT3

STAT5 STAT3/STAT5 STAT1/STAT3/STAT5

2Activated STAT that becomes a transcription factor for the STAT-responsive cytokine/protein

STAT-Responsive Genes

**STAT Activator1** 

IL-4

IL-2, IL-3

IL-4

IL-6/IL-17/OSM

IL-2

IL-3 IL-2 IL-12

IL-6/IL-19 IL-15 IL-22

**Table 1.** STAT-Responsive Pro-Inflammatory Cytokine Gene Expression

OSM IL-2/IL-3 STAT5

IL-6 IL-6/IL-17 IL-3 IL-12 IL-19 IL-10/IL-13

**STAT Responsive Cytokine/Protein** 

378 Drug Discovery

IL-2Rα

IL-10

IL-18R1

IL-6ST(gp130)4

IL-4

INF-γ IFN-γ

TNF-α IL-3

1Cytokines that activate this STAT protein

5Leukemia Inhibitory Factor Receptor

4IL-6 Signal Transducer

6Oncostatin M Receptor

8C-Jun-N-terminal kinase

7p38 kinase

3Function(s) of STAT-responsive cytokine/protein

Up-regulation of the Th1 and Th17 T-cell subsets and reduced levels of human T-regulatory (Treg) cells are known to occur in autoimmune diseases [16, 68]. In addition, Treg cells are a critical contributor to T-cell development in the thymus as well as being the T-cell subset that regulates the genesis and maintenance of immune tolerance [16].

The IL-2Rα/IL-2Rβ subunits in complex with the common IL-2γ subunit make up the highaffinity IL-2 receptor, whereas homodimeric IL-2Rα results in a low-affinity receptor [69]. The functional significance of blocking the high-affinity IL-2R with the small molecule inhib‐ itor (SMI), SP4206 (Kd ~70nM) in response to IL-2 (Kd~10nM) was that JAK/STAT activation was inhibited [70]. This result could provide the impetus for development of the next gener‐ ation SMI designed to efficiently inhibit the IL-2/IL-2R pathway and this task should be fa‐ cilitated by employing recently developed technologies based on the principles of proteinprotein interactions [71].

As indicated previously, the interaction of IL-2 with the high-affinity IL-2R causes activation of JAK/STAT with STAT5A and STAT5B, the principally activated STAT proteins. However, the eventual change in STAT5-gene responsiveness following IL-2 activation of STAT5 was shown to be dependent on the complexity of the promoter regions of those STAT5-target genes [72]. Interestingly, Tsuji-Takayama et al. [73] showed that IL-2-mediated JAK/STAT activation up-regulated the production of IL-10 by Treg cells. The production of IL-10 arose from the interaction of STAT5 with a STAT5-responsive element within intron 4, designated I-SRE-4 of the IL-10 locus which was considered to be an interspecies conserved enhancer sequence (Table 1). Of note, the clustered CpG regions around I-SRE-4 were under-methy‐ lated in IL-10-producing Treg cells, but not in other T-cell subsets. This result confirmed pre‐ vious results which showed that expression of Foxp3, a member of the forkhead/wingedhelix family of transcription factors and a biomarker for the development and function of Treg cells [47, 74] was also IL-2/STAT5-dependent [75]. Thus, development of Treg cells was regulated by the methylation status of CpG residues because methylation of CpG residues suppressed Foxp3 expression [76].

Chen et al. [77] identified a novel set of IL-4/STAT6-target genes in mice that regulated the proliferation of activated T-cells. In addition, these genes were shown to regulate the pro‐ duction of the Th2 lineage as evinced by the finding that the cells isolated from wild-type mice produced Th2 whereas cells from STAT6-/- mice did not. Later, Lund et al. [78] showed that the IL-4/STAT6 pathway was also critical for the commitment of naïve T-cells to become either the Th1 or Th2 subset. In that regard, the ratio of Th1 to Th2 produced from naïve Tcells was found to be dependent on a set of STAT6-responsive genes which included the transcription factors STATB1, Bcl-6, and TCF7 [78, 79]. Moreover, the IL-4/STAT6-mediated pathway was also shown to be a strong modulator of human Treg cell production from either Th1 or Th17 cells [80].

Wurster et al. [81] were among the first to demonstrate that IL-4-mediated activation of STAT6 could also up-regulate IL-2Rα gene expression (Table 1). Because IL-2 is the major growth-promoting cytokine for T-cells [81], elevated production of IL-2Rα in response to ac‐ tivated STAT6 is considered instrumental in facilitating the proliferation of activated T-cells in cancer as well as in several types of autoimmune diseases. In that regard, the high level of expression of IL-2Rα in tumors correlated with a poor prognosis in cancer patients [82]. Thus, it will be interesting to determine if the same relationship holds true for RA patients as well, including what role IL-2Rα polymorphisms [83, 84] might play in determining the level of the expression of IL-2Rα. For example, IL-15, a pro-inflammatory cytokine which in‐ teracts with two receptor subunits similar to IL-2Rα/β drives the production of the memory CD8+ T-cell phenotype [85]. Experimental therapies focusing on inhibiting the binding of IL-15 to the IL-2Rα/β receptor complex were a decade ago considered to be a potential target for autoimmune diseases [85]. However, since then considerable evidence has accumulated showing a robust relationship between IL-15/IL-2Rα/β-mediated signaling, osteoclastogene‐ sis and boney erosions in RA joints [3]. In addition, González-Alvaro et al. [86] showed that IL-15 stimulated production of TNF-α by monocytes derived from RA patients including, the induction of the CD69 monocyte biomarker, and synthesis of IFN-γ protein by natural killer (NK) cells. Of note, the results of a clinical study showed that IL-15 expression in RA synovial tissue persisted even after TNF-α blockade, the latter treatment resulting in a posi‐ tive clinical response and reduced disease activity [87]. However, treating mononuclear cells *in vitro* with HuMax-IL-15 f (ab')2 neutralized the effect of IL-15 on these cells. Furthermore, treatment with HuMax-IL-15 f(ab')2 caused a significant improvement in RA disease activity as measured by the American College of Rheumatology (ACR) clinical response criteria [88]. This finding may be particularly important for future drug development because the results of a recently completed clinical trial showed that high levels of serum IL-15 in patients with early arthritis predicted a more progressive and severe clinical course which may call for early and aggressive drug therapy [89].

than JAK/STAT which resulted in elevated production of IL-1β and TNF-α. Therefore, it has become obvious that suppressing the activity of IL-17 could bring about a reduction of these pro-inflammatory cytokines as well, although this point must be rigorously reexamined in view of the results from Dragon et al. [94] who showed that IL-17A significantly decreased GM-CSF-induced neutrophil/granulocyte apoptosis by suppressing activation of p38 kinase,

Suppression of Pro-Inflammatory Cytokines via Targeting of STAT-Responsive Genes

http://dx.doi.org/10.5772/52506

381

Inhibiting aberrant T-cell survival in RA may ultimately hinge on the development of a ther‐ apeutic strategy directed specifically at STAT3 since STAT3 was shown to inhibit T-cell pro‐ liferation by up-regulating the Class O Forkhead transcriptions factors (Fox) via the binding of STAT3 to FoxO1and FoxO3a promoters [95]. Potentially, STAT3 may also protect T-cells from apoptosis [30, 96] in RA by suppressing IL-2 activity, although the results [95] indicat‐ ed that STAT3 increased T-cell proliferation and their survival through the up-regulation of OX-40 (CD134), a member of the TNFR-superfamily of receptors and bcl-2 and by suppress‐

Perhaps the most intriguing aspect of the clinical studies with tocilizumab performed in RA patients is the extent to which neutralization of the IL-6/IL-6R/gp130 pathway using this drug together with the putative suppression of IL-6 and gp130 gene expression in response to inhibition of the STAT3 activation rebalances the skewed ratio of Th17/Treg in favor of Treg [97, 98], the elevated serum levels of Th17-associated cytokines, IL-17, IL-23, IL-6 and TNF-α, and the depressed level of Treg cells with its associated growth factor, transforming growth factor-β (TGF-β) [99]. What is pertinent to these events are the results of a recent study which showed that treatment of RA patients with tocilizumab in combination with metho‐ trexate resulted in a significant decrease in the percentage of Th17 cells (from 0.9% at base‐ line to 0.45%) and a significant increase in Treg cells (from 3.05% to 3.94%) whilst maintaining

The extent to which gp130 gene expression is altered in response to inhibition of the JAK/STAT pathway activation is also an area of immense importance because deregulat‐ ed over-expression of gp130 in RA patients should not be neutralized by anti-IL-6R ther‐ apy. Importantly, O'Brien and Manolagas [100] showed that IL-6 or oncostatin M (OSM), a member of the IL-6 protein superfamily, stimulated the activity of the gp130 (Table 1) promoter in which the cytokine response element contained a cis-acting motif for activat‐ ed STAT complexes, including activated STAT1 and STAT3 homo- and heterodimers. Furthermore, it can be conjectured that other pro-inflammatory cytokine members of the IL-6 protein superfamily, such as ciliary neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and cardiotropin-1 which use gp130 as their primary signal transducer pro‐ tein [9, 101] may provide an alternate mechanism resulting in constitutive JAK/STAT ac‐ tivation. Under those conditions STAT protein activation may not be inhibited any of the anti-IL-6R agents which retain up-regulated gp130 gene expression. Of note, constitutive activation of STAT proteins is one of the signature events in the development and pro‐ gression of various cancers [102, 103] with a similar phenomenon having been described in RA synovial tissue [15]. Constitutively activated STAT proteins could also be predic‐

extracellular-regulated kinase 1/2 and STAT5B.

ing FasL and Bad expression.

their functional activity [98].

tive of a more aggressive form of RA [96].

#### **6.1. IL-6/gp130/IL-17**

The IL-6/IL-6R/gp130 pathway is one of the strongest inducers of STAT3 activation [9] (Ta‐ ble 1) so much so that many studies have been devoted to the activation of the JAK/STAT pathway by IL-6 because IL-6 is critical to the progression of joint damage in RA [16]. In fact, the development of the anti-IL-6R monoclonal antibody, tocilizumab, appears to have been predicated on this emerging evidence such that this drug is now considered useful in the armamentarium of drug therapies for RA [90, 91]. Most compelling was recent evidence that tocilizumab in conjunction with methotrexate retarded the progression of joint damage in RA patients [92], an effect of this drug regimen that was apparently independent of the ca‐ pacity of tocilizumab to modify several clinical biomarkers of inflammation and concomi‐ tant RA disease activity.

Recent results have also emerged which have focused attention on the extent to which other pro-inflammatory cytokines, such as IL-17, activate JAK/STAT and the mechanism by which IL-17 modifies the production of IL-6 and other pro-inflammatory cytokines [9]. In that re‐ gard, the results of a study by Jovanovic et al. [93] was extremely informative because it pro‐ vided evidence that IL-17 was capable of activating additional signaling pathways other than JAK/STAT which resulted in elevated production of IL-1β and TNF-α. Therefore, it has become obvious that suppressing the activity of IL-17 could bring about a reduction of these pro-inflammatory cytokines as well, although this point must be rigorously reexamined in view of the results from Dragon et al. [94] who showed that IL-17A significantly decreased GM-CSF-induced neutrophil/granulocyte apoptosis by suppressing activation of p38 kinase, extracellular-regulated kinase 1/2 and STAT5B.

growth-promoting cytokine for T-cells [81], elevated production of IL-2Rα in response to ac‐ tivated STAT6 is considered instrumental in facilitating the proliferation of activated T-cells in cancer as well as in several types of autoimmune diseases. In that regard, the high level of expression of IL-2Rα in tumors correlated with a poor prognosis in cancer patients [82]. Thus, it will be interesting to determine if the same relationship holds true for RA patients as well, including what role IL-2Rα polymorphisms [83, 84] might play in determining the level of the expression of IL-2Rα. For example, IL-15, a pro-inflammatory cytokine which in‐ teracts with two receptor subunits similar to IL-2Rα/β drives the production of the memory

 T-cell phenotype [85]. Experimental therapies focusing on inhibiting the binding of IL-15 to the IL-2Rα/β receptor complex were a decade ago considered to be a potential target for autoimmune diseases [85]. However, since then considerable evidence has accumulated showing a robust relationship between IL-15/IL-2Rα/β-mediated signaling, osteoclastogene‐ sis and boney erosions in RA joints [3]. In addition, González-Alvaro et al. [86] showed that IL-15 stimulated production of TNF-α by monocytes derived from RA patients including, the induction of the CD69 monocyte biomarker, and synthesis of IFN-γ protein by natural killer (NK) cells. Of note, the results of a clinical study showed that IL-15 expression in RA synovial tissue persisted even after TNF-α blockade, the latter treatment resulting in a posi‐ tive clinical response and reduced disease activity [87]. However, treating mononuclear cells *in vitro* with HuMax-IL-15 f (ab')2 neutralized the effect of IL-15 on these cells. Furthermore, treatment with HuMax-IL-15 f(ab')2 caused a significant improvement in RA disease activity as measured by the American College of Rheumatology (ACR) clinical response criteria [88]. This finding may be particularly important for future drug development because the results of a recently completed clinical trial showed that high levels of serum IL-15 in patients with early arthritis predicted a more progressive and severe clinical course which may call for

The IL-6/IL-6R/gp130 pathway is one of the strongest inducers of STAT3 activation [9] (Ta‐ ble 1) so much so that many studies have been devoted to the activation of the JAK/STAT pathway by IL-6 because IL-6 is critical to the progression of joint damage in RA [16]. In fact, the development of the anti-IL-6R monoclonal antibody, tocilizumab, appears to have been predicated on this emerging evidence such that this drug is now considered useful in the armamentarium of drug therapies for RA [90, 91]. Most compelling was recent evidence that tocilizumab in conjunction with methotrexate retarded the progression of joint damage in RA patients [92], an effect of this drug regimen that was apparently independent of the ca‐ pacity of tocilizumab to modify several clinical biomarkers of inflammation and concomi‐

Recent results have also emerged which have focused attention on the extent to which other pro-inflammatory cytokines, such as IL-17, activate JAK/STAT and the mechanism by which IL-17 modifies the production of IL-6 and other pro-inflammatory cytokines [9]. In that re‐ gard, the results of a study by Jovanovic et al. [93] was extremely informative because it pro‐ vided evidence that IL-17 was capable of activating additional signaling pathways other

CD8+

380 Drug Discovery

early and aggressive drug therapy [89].

**6.1. IL-6/gp130/IL-17**

tant RA disease activity.

Inhibiting aberrant T-cell survival in RA may ultimately hinge on the development of a ther‐ apeutic strategy directed specifically at STAT3 since STAT3 was shown to inhibit T-cell pro‐ liferation by up-regulating the Class O Forkhead transcriptions factors (Fox) via the binding of STAT3 to FoxO1and FoxO3a promoters [95]. Potentially, STAT3 may also protect T-cells from apoptosis [30, 96] in RA by suppressing IL-2 activity, although the results [95] indicat‐ ed that STAT3 increased T-cell proliferation and their survival through the up-regulation of OX-40 (CD134), a member of the TNFR-superfamily of receptors and bcl-2 and by suppress‐ ing FasL and Bad expression.

Perhaps the most intriguing aspect of the clinical studies with tocilizumab performed in RA patients is the extent to which neutralization of the IL-6/IL-6R/gp130 pathway using this drug together with the putative suppression of IL-6 and gp130 gene expression in response to inhibition of the STAT3 activation rebalances the skewed ratio of Th17/Treg in favor of Treg [97, 98], the elevated serum levels of Th17-associated cytokines, IL-17, IL-23, IL-6 and TNF-α, and the depressed level of Treg cells with its associated growth factor, transforming growth factor-β (TGF-β) [99]. What is pertinent to these events are the results of a recent study which showed that treatment of RA patients with tocilizumab in combination with metho‐ trexate resulted in a significant decrease in the percentage of Th17 cells (from 0.9% at base‐ line to 0.45%) and a significant increase in Treg cells (from 3.05% to 3.94%) whilst maintaining their functional activity [98].

The extent to which gp130 gene expression is altered in response to inhibition of the JAK/STAT pathway activation is also an area of immense importance because deregulat‐ ed over-expression of gp130 in RA patients should not be neutralized by anti-IL-6R ther‐ apy. Importantly, O'Brien and Manolagas [100] showed that IL-6 or oncostatin M (OSM), a member of the IL-6 protein superfamily, stimulated the activity of the gp130 (Table 1) promoter in which the cytokine response element contained a cis-acting motif for activat‐ ed STAT complexes, including activated STAT1 and STAT3 homo- and heterodimers. Furthermore, it can be conjectured that other pro-inflammatory cytokine members of the IL-6 protein superfamily, such as ciliary neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and cardiotropin-1 which use gp130 as their primary signal transducer pro‐ tein [9, 101] may provide an alternate mechanism resulting in constitutive JAK/STAT ac‐ tivation. Under those conditions STAT protein activation may not be inhibited any of the anti-IL-6R agents which retain up-regulated gp130 gene expression. Of note, constitutive activation of STAT proteins is one of the signature events in the development and pro‐ gression of various cancers [102, 103] with a similar phenomenon having been described in RA synovial tissue [15]. Constitutively activated STAT proteins could also be predic‐ tive of a more aggressive form of RA [96].
