**2. (Auto-)antigens implicated in atherogenesis and their effects on DCs**

Many (auto-)antigens are involved in atherogenesis, both endogenous and exogenous. Here, we summarize some of the best-studied endogenous self-antigens in relation to DC function.

#### **2.1. Oxidized low density lipoprotein (oxLDL)**

OxLDL is one of the best-studied antigens in atherogenesis. It is considered as a 'neoanti‐ gen', i.e. a self-antigen that has the potential to provoke an auto-immune response upon modification, but that is tolerated by the immune system in its normal (unmodified) form [13]. It has already been shown that oxLDL can induce differentiation of monocytes into phenotypically abnormal cells, when it is added to monocytes during the early stages of dif‐ ferentiation [14]. These cells have functional characteristics of DCs, such as decreased endo‐ cytosis capacity, increased ability to stimulate T cell proliferation and secretion of IL-12, but not IL-10. These findings were consistent with our own study (unpublished data), which showed that monocytes differentiated (at least partly) into DCs, when they were incubated with oxLDL. This was evidenced by a pronounced decrease in the expression of CD14, a typical monocyte/macrophage marker, and increased expression of CD1a, which is mainly expressed on cortical thymocytes and DCs, and CCR-6, a receptor for CCL20 that is ex‐ pressed by resting T cells and DCs (figure 1).

Dendritic Cells in Atherogenesis: From Immune Shapers to Therapeutic Targets http://dx.doi.org/10.5772/52900 57

or primed to become regulatory T cells [8]. In contrast, when infection and tissue damage occur, immature DCs take up antigens in the presence of inflammatory signals, which evokes activation and functional transformation into mature DCs. Meanwhile, they exit the non-lymphoid tissues to migrate via afferent lymph vessels to lymphoid tissues, where they completely mature. Mature DCs present short peptide fragments, which are bound to the surface molecules CD1 or major histocompatibility complex (MHC)-I or MHC-II. Conse‐ quently, they activate (naïve) T and B lymphocytes that recognize the presented antigen [9]. Morphological changes occur as well during the DC life cycle: DC precursors are often small, round-shaped cells that turn into larger cells with an irregular (star-like) shape and cytoplasmic protrusions (dendrites) as the cell matures, while migrating DCs are also called

Following the first observation of DCs in human arteries in 1995 [11], numerous studies sug‐ gest that these cells presumably play a crucial role in directing innate or adaptive immunity against altered self-antigens present in atherosclerosis. Localization of DCs nearby vasa vasorum allows monitoring of the major access pathways to the vessel wall and screening of the tissue environment for the appearance of exogenous and endogenous stressors [12]. Once sufficiently activated, DCs in the arterial wall might present the (modified auto-) anti‐ gens, such as oxidized epitopes on apoptotic cells, oxidized low density lipoproteins

veiled cells, as they possess large cytoplasmic 'veils' rather than dendrites [10].

(oxLDL) or heat shock proteins (Hsp) to T cells and initiate inflammatory responses.

**2.1. Oxidized low density lipoprotein (oxLDL)**

56 Current Trends in Atherogenesis

pressed by resting T cells and DCs (figure 1).

**2. (Auto-)antigens implicated in atherogenesis and their effects on DCs**

Many (auto-)antigens are involved in atherogenesis, both endogenous and exogenous. Here, we summarize some of the best-studied endogenous self-antigens in relation to DC function.

OxLDL is one of the best-studied antigens in atherogenesis. It is considered as a 'neoanti‐ gen', i.e. a self-antigen that has the potential to provoke an auto-immune response upon modification, but that is tolerated by the immune system in its normal (unmodified) form [13]. It has already been shown that oxLDL can induce differentiation of monocytes into phenotypically abnormal cells, when it is added to monocytes during the early stages of dif‐ ferentiation [14]. These cells have functional characteristics of DCs, such as decreased endo‐ cytosis capacity, increased ability to stimulate T cell proliferation and secretion of IL-12, but not IL-10. These findings were consistent with our own study (unpublished data), which showed that monocytes differentiated (at least partly) into DCs, when they were incubated with oxLDL. This was evidenced by a pronounced decrease in the expression of CD14, a typical monocyte/macrophage marker, and increased expression of CD1a, which is mainly expressed on cortical thymocytes and DCs, and CCR-6, a receptor for CCL20 that is ex‐

**Figure 1.** *Effects of oxLDL on monocyte differentiation.* Expression of CD14, CCR-6 and CD1a after 24h incubation of monocytes with 10 µg/mL oxLDL or 50 µg/mL oxLDL points to differentiation to a phenotype with characteristics of DCs (N=3). \*\*\*P<0.001, \*P<0.05 versus control, Repeated Measures ANOVA and Dunnett's post-hoc test.

Apart from the induction of monocyte differentiation into DCs, oxLDL can also activate DCs, as demonstrated by several *in vitro* studies. After 24h incubation with high concentra‐ tions of oxLDL (50 µg/mL), expression of activation markers CD40, CD80 and CD83 was sig‐ nificantly upregulated (figure 2), and endocytotic capacity was significantly reduced (figure 3; own unpublished data).

**Figure 2.** *Effects of oxLDL on maturation of monocyte-derived DCs.* Expression of maturation markers CD40, CD80 and CD83 after 24h incubation of immature monocyte-derived DCs with 10 µg/mL or 50 µg/mL oxLDL (N=4). Black bars represent the positive control for DC maturation, monocyte-derived DCs stimulated with lipopolysaccharide (LPS; 0.1 µg/mL). \*\*\*P<0.001, \*\*P<0.01, \*P<0.05 versus control, Repeated Measures ANOVA and Dunnett's post-hoc test.

**Figure 3.** *Effects of oxLDL on endocytotic capacity of monocyte-derived DCs.* Decreased endocytotic capacity of mono‐ cyte-derived DCs 24h after stimulation with oxLDL (10 µg/mL or 50 µg/mL) or the positive control LPS (0.1 µg/mL) provides functional evidence of DC maturation (N=5). \*P<0.05, Repeated Measures ANOVA, Dunnett's post-hoc test.

**Figure 4.** *Effects of oxLDL on morphology of monocyte-derived DCs.* Representative micrographs of immature, mono‐ cyte-derived DC cultures after 24h incubation with medium (A), 0.1 µg/mL lipopolysaccharide (LPS; positive control for DC maturation; B), 10 µg/mL oxLDL (C), or 50 µg/mL oxLDL (D). Phase contrast light microscopy, magnification: 10x

Dendritic Cells in Atherogenesis: From Immune Shapers to Therapeutic Targets

http://dx.doi.org/10.5772/52900

59

Beta2-glycoprotein I (β2-GPI) is a plasma protein involved in the haemostatic system that has been detected in carotid atherosclerotic lesions [18]. A previous study in mice showed that the transfer of lymphocytes obtained from β2-GPI-immunized LDLr-/- mice into synge‐ neic mice resulted in larger fatty streaks within the recipients compared with mice that re‐ ceived lymphocytes from control mice [19]. From that study, it appeared that T cells specific for β2-GPI are able to increase atherosclerosis, suggesting that β2-GPI is a target auto-anti‐

*In vitro* studies have demonstrated that oxidative modification of β2-GPI, either spontane‐ ously or induced by treatment with hydrogen peroxide, rendered the self-antigen able to in‐ duce an autoimmune response. Oxidized β2-GPI caused DC maturation, indicated by increased expression of CD80, CD86, CD83 and HLA-DR [20]. In addition, the interaction between oxidized β2-GPI and DCs led to enhanced secretion of IL-12, IL-1β, IL-6, IL-8, TNFα and IL-10. DCs stimulated with oxidized β2-GPI showed increased allostimulatory ability and induced T-helper (Th)1 polarization [20]. Also, glucose-modified β2-GPI caused phenotypic and functional maturation of iDCs, by activation of the p38 MAPK, ERK and NF-κB pathways. However, DCs stimulated with glucose-modified β2-GPI primed naïve T

(A, D), 20x (B, C).

**2.2. Beta2-Glycoprotein I**

gen in atherosclerosis [19].

cells toward a Th2 polarization [21].

Cell morphology pointed to DC maturation as well: oxLDL-stimulated monocyte-derived DCs became more elongated and were arranged in clusters, when compared to unstimulat‐ ed monocyte-derived DCs. The arrangement in clusters was also more pronounced when cells were stimulated with 50 µg/mL oxLDL as compared to cells stimulated with the lower concentration of oxLDL (10 µg/mL) (figure 4; own unpublished data). Alderman et al. [15] compared the effects of mildly, moderately and highly oxidized LDL and reported a signifi‐ cant upregulation of DC activation markers, including HLA-DR, CD40 and CD86 when cells were incubated with highly oxidized LDL. Furthermore, highly oxidized LDL increased DCinduced T cell proliferation. However, high concentrations of highly oxidized LDL (100 µg/mL) inhibited DC function through increased DC apoptosis [15]. In contrast, another study demonstrated that oxLDL did not trigger maturation of immature DCs [14]. This seems to be a discrepancy, but can easily be explained by a concentration-dependent effect of oxLDL. Perrin-Cocon and colleagues [14] varied the oxLDL concentrations between 2.5-10.0 µg/mL, which could have been insufficient to obtain monocyte-derived DC matura‐ tion. Also Zaguri et al. [16] observed no effect of 10 µg/mL oxLDL on CD86, CD83, and CCR-7 expression on DCs, whereas all those activation markers were upregulated with higher concentrations of oxLDL (50-100 µg/mL). Finally, Nickel et al. [17] reported matura‐ tion and differentiation of DCs by 10 µg/mL, but he investigated other phenotypic out‐ comes, such as the expression of scavenger receptors LOX1 and CD36, the mannose receptor CD205 and the activation of the nuclear factor kappa B (NF-κB) pathway.

**Figure 4.** *Effects of oxLDL on morphology of monocyte-derived DCs.* Representative micrographs of immature, mono‐ cyte-derived DC cultures after 24h incubation with medium (A), 0.1 µg/mL lipopolysaccharide (LPS; positive control for DC maturation; B), 10 µg/mL oxLDL (C), or 50 µg/mL oxLDL (D). Phase contrast light microscopy, magnification: 10x (A, D), 20x (B, C).

#### **2.2. Beta2-Glycoprotein I**

**Figure 3.** *Effects of oxLDL on endocytotic capacity of monocyte-derived DCs.* Decreased endocytotic capacity of mono‐ cyte-derived DCs 24h after stimulation with oxLDL (10 µg/mL or 50 µg/mL) or the positive control LPS (0.1 µg/mL) provides functional evidence of DC maturation (N=5). \*P<0.05, Repeated Measures ANOVA, Dunnett's post-hoc test.

58 Current Trends in Atherogenesis

Cell morphology pointed to DC maturation as well: oxLDL-stimulated monocyte-derived DCs became more elongated and were arranged in clusters, when compared to unstimulat‐ ed monocyte-derived DCs. The arrangement in clusters was also more pronounced when cells were stimulated with 50 µg/mL oxLDL as compared to cells stimulated with the lower concentration of oxLDL (10 µg/mL) (figure 4; own unpublished data). Alderman et al. [15] compared the effects of mildly, moderately and highly oxidized LDL and reported a signifi‐ cant upregulation of DC activation markers, including HLA-DR, CD40 and CD86 when cells were incubated with highly oxidized LDL. Furthermore, highly oxidized LDL increased DCinduced T cell proliferation. However, high concentrations of highly oxidized LDL (100 µg/mL) inhibited DC function through increased DC apoptosis [15]. In contrast, another study demonstrated that oxLDL did not trigger maturation of immature DCs [14]. This seems to be a discrepancy, but can easily be explained by a concentration-dependent effect of oxLDL. Perrin-Cocon and colleagues [14] varied the oxLDL concentrations between 2.5-10.0 µg/mL, which could have been insufficient to obtain monocyte-derived DC matura‐ tion. Also Zaguri et al. [16] observed no effect of 10 µg/mL oxLDL on CD86, CD83, and CCR-7 expression on DCs, whereas all those activation markers were upregulated with higher concentrations of oxLDL (50-100 µg/mL). Finally, Nickel et al. [17] reported matura‐ tion and differentiation of DCs by 10 µg/mL, but he investigated other phenotypic out‐ comes, such as the expression of scavenger receptors LOX1 and CD36, the mannose receptor

CD205 and the activation of the nuclear factor kappa B (NF-κB) pathway.

Beta2-glycoprotein I (β2-GPI) is a plasma protein involved in the haemostatic system that has been detected in carotid atherosclerotic lesions [18]. A previous study in mice showed that the transfer of lymphocytes obtained from β2-GPI-immunized LDLr-/- mice into synge‐ neic mice resulted in larger fatty streaks within the recipients compared with mice that re‐ ceived lymphocytes from control mice [19]. From that study, it appeared that T cells specific for β2-GPI are able to increase atherosclerosis, suggesting that β2-GPI is a target auto-anti‐ gen in atherosclerosis [19].

*In vitro* studies have demonstrated that oxidative modification of β2-GPI, either spontane‐ ously or induced by treatment with hydrogen peroxide, rendered the self-antigen able to in‐ duce an autoimmune response. Oxidized β2-GPI caused DC maturation, indicated by increased expression of CD80, CD86, CD83 and HLA-DR [20]. In addition, the interaction between oxidized β2-GPI and DCs led to enhanced secretion of IL-12, IL-1β, IL-6, IL-8, TNFα and IL-10. DCs stimulated with oxidized β2-GPI showed increased allostimulatory ability and induced T-helper (Th)1 polarization [20]. Also, glucose-modified β2-GPI caused phenotypic and functional maturation of iDCs, by activation of the p38 MAPK, ERK and NF-κB pathways. However, DCs stimulated with glucose-modified β2-GPI primed naïve T cells toward a Th2 polarization [21].

#### **2.3. Heat shock proteins**

Another category of auto-antigens that have been implicated in atherosclerosis are the stress-induced heat shock proteins (HSPs) [22]. HSPs are responsible for the repair or degra‐ dation of denatured proteins and, by maintaining protein conformation, they enhance the cell's ability to survive under conditions of metabolic or oxidative stress [23]. The mRNA ex‐ pression level of several HSPs, including HSP40 and HSP70, has been shown to be signifi‐ cantly increased in carotid endarterectomy specimens as compared to healthy arteries [24]. HSP70 seems to be homogenously distributed throughout the intima and media in healthy aortas, and a strong increase in its immunostaining intensity is observed in aortic athero‐ sclerotic plaques [25]. They appear to stimulate an immune response leading to the develop‐ ment and progression of atherosclerosis [26]. A number of studies indicate that HSPs are associated with DC function and might trigger DC activation and maturation. DCs seem to overexpress HSP70 in atherosclerotic plaques and the latter protein is presumably an impor‐ tant trigger for DC activation [27]. Gp96 (of the HSP90 family) and HSP70 have indeed been shown to stimulate bone marrow-derived DCs *in vitro* to secrete cytokines [28] and to ex‐ press antigen-presenting (MHC II) and costimulatory molecules (B7.2) [29]. However, To‐ dryk and colleagues [30] reported that HSP70 targets immature DCs to make them significantly more able to capture antigens. The presence of HSP70 inhibited DC maturation induced by tumour cell lysates from parental B16 cells and maintained the DC precursor population in a more poorly differentiated phenotype. Thus, there is still controversy on whether HSPs activate DCs or keep them in an immature state, and data are lacking to ro‐ bustly support a conclusion.

related pathways revealed an upregulation of several important antioxidant genes during differentiation of monocytes into DCs, including catalase, peroxiredoxin 2 (PRDX2) and glu‐ tathione peroxidase 3 (GPX3). Catalase encodes the enzyme that catalyses the decomposi‐ tion of hydrogen peroxide to water and oxygen. GPX3 and PRDX2 are genes encoding enzymes that can detoxify hydrogen peroxide and lipid hydroperoxides [34,35]. However, PRDX2 is more efficient in neutralizing hydrogen peroxide than catalase or GPXs [36,37]. Immunoblotting or immunohistochemistry showed that the upregulated transcription of PRDX2 and GPX3 was translated in a significant increase at the protein level. Especially PRDX2 appears to be an important factor in the neutralization of ROS induced by *tert*-BHP [31]. Previously, and in accordance with our recent findings, two studies that used different detection methods reported high expression of antioxidant enzymes in monocyte-derived DCs. A functional study indicated indirectly that monocyte-derived DCs might show en‐ hanced activity of catalase [38]. A proteomic analysis showed higher expression of superox‐ ide dismutase (SOD)2, PRDX1 and PRDX2 in monocyte-derived DCs when compared to precursor monocytes [39]. The latter study also stated that DCs were more resistant than monocytes to apoptosis induced by high amounts of oxLDL [39]. It is conceivable that the good survival skills of monocyte-derived DCs in oxidative stress environments are crucial in atherosclerotic plaques, enabling these professional antigen-presenting cells to exert their

Dendritic Cells in Atherogenesis: From Immune Shapers to Therapeutic Targets

http://dx.doi.org/10.5772/52900

61

As discussed above, DCs process and present self and foreign antigens to T cells and are therefore important inducers of adaptive immune responses. However, 'the' DC does not exist, as DCs comprise a network of subsets that are phenotypically, functionally, and de‐ velopmentally distinct [40,41]. It is essential to understand the diversity in DC subtypes to target DCs for immunomodulating therapies. Most studies on DC subsets have been per‐ formed in mice, because lymphoid tissue is easier to obtain from mice than from humans. Mature mouse DCs are identified based on their expression of the integrin alpha X chain CD11c, the costimulatory molecules CD40, CD80 and CD86, and high surface levels of the antigen-presenting molecule MHC II [42,43,40]. The T cell markers CD4 and CD8 (in the form of a αα-homodimer) are also expressed on mouse DCs, and can be used to distin‐ guish different subtypes [44]. In general, three DC subsets can be characterized in mouse

DCs; 2) CD8α-

gans, whereas the CD8α- CD4+ DCs are found in the marginal zones. Yet, upon stimulation by microbial products, such as lipopolysaccharide, the latter can also migrate to the T cell zones [45,46]. Other markers that can be used to further subdivide mouse DC subsets include the integrin alpha M chain CD11b and the endocytosis receptor CD205

DCs are also CD205+ CD11b-

the thymus, and at moderate levels in lymph nodes [40]. Lymph nodes further contain, in

DCs are mainly localized in the T cell areas of lymphoid or‐

CD11b+ CD205mid DCs which are considered as the mature

CD4+ DCs; and 3) CD8α-

and they are mainly present in

CD4-

function(s).

**4. DC subtypes in mice and men**

lymphoid tissue (table 1): 1) CD8α+ CD4-

CD4-

DCs [44]. The CD8α+ CD4-

(DEC205). The CD8α+ CD4-

contrast to spleen, CD8α-

#### **3. Survival of DCs in oxidative stress environments**

Atherosclerosis is a disease that is associated with strong oxidative stress, and the creation of neo-epitopes is one of the consequences of this situation. As mentioned in section 2, the presence of reactive oxygen species (ROS) in atherosclerotic plaques may lead to the forma‐ tion of oxLDL and oxidized β2-GPI, which might affect DC phenotype and function. Indeed, oxidative stress has been shown to alter the capacity of antigen-presenting cells to process antigens and to initiate a primary T-cell response. In this respect, it is interesting to unravel whether DCs show phenotypic adaptations in order to function under oxidative stress situa‐ tions. In a recent study, we demonstrated that DCs appear to be resistant to the detrimental effects of oxidative stress. We showed by confocal live cell imaging that monocyte-derived DCs, which were generated as described earlier [31], were better capable of neutralizing ROS induced by tertiary-butylhydroperoxide (*tert*-BHP) in comparison to their precursor monocytes [31]. *Tert*-BHP was selected to induce ROS because it acutely evokes oxidative stress, resulting in cell toxicity [32]. Decomposition of *tert*-BHP to alkoxyl or peroxyl radicals accelerates lipid peroxidation chain reactions [33]. By means of a neutral red viability assay, we observed that *tert*-BHP induced significant and rapid cell death in both monocytes and DCs. Yet, monocyte-derived DCs were more resistant to *tert*-BHP-induced cell death than their precursor cells [31]. A PCR profiler array specific for oxidative stress and antioxidantrelated pathways revealed an upregulation of several important antioxidant genes during differentiation of monocytes into DCs, including catalase, peroxiredoxin 2 (PRDX2) and glu‐ tathione peroxidase 3 (GPX3). Catalase encodes the enzyme that catalyses the decomposi‐ tion of hydrogen peroxide to water and oxygen. GPX3 and PRDX2 are genes encoding enzymes that can detoxify hydrogen peroxide and lipid hydroperoxides [34,35]. However, PRDX2 is more efficient in neutralizing hydrogen peroxide than catalase or GPXs [36,37]. Immunoblotting or immunohistochemistry showed that the upregulated transcription of PRDX2 and GPX3 was translated in a significant increase at the protein level. Especially PRDX2 appears to be an important factor in the neutralization of ROS induced by *tert*-BHP [31]. Previously, and in accordance with our recent findings, two studies that used different detection methods reported high expression of antioxidant enzymes in monocyte-derived DCs. A functional study indicated indirectly that monocyte-derived DCs might show en‐ hanced activity of catalase [38]. A proteomic analysis showed higher expression of superox‐ ide dismutase (SOD)2, PRDX1 and PRDX2 in monocyte-derived DCs when compared to precursor monocytes [39]. The latter study also stated that DCs were more resistant than monocytes to apoptosis induced by high amounts of oxLDL [39]. It is conceivable that the good survival skills of monocyte-derived DCs in oxidative stress environments are crucial in atherosclerotic plaques, enabling these professional antigen-presenting cells to exert their function(s).

## **4. DC subtypes in mice and men**

**2.3. Heat shock proteins**

60 Current Trends in Atherogenesis

bustly support a conclusion.

**3. Survival of DCs in oxidative stress environments**

Atherosclerosis is a disease that is associated with strong oxidative stress, and the creation of neo-epitopes is one of the consequences of this situation. As mentioned in section 2, the presence of reactive oxygen species (ROS) in atherosclerotic plaques may lead to the forma‐ tion of oxLDL and oxidized β2-GPI, which might affect DC phenotype and function. Indeed, oxidative stress has been shown to alter the capacity of antigen-presenting cells to process antigens and to initiate a primary T-cell response. In this respect, it is interesting to unravel whether DCs show phenotypic adaptations in order to function under oxidative stress situa‐ tions. In a recent study, we demonstrated that DCs appear to be resistant to the detrimental effects of oxidative stress. We showed by confocal live cell imaging that monocyte-derived DCs, which were generated as described earlier [31], were better capable of neutralizing ROS induced by tertiary-butylhydroperoxide (*tert*-BHP) in comparison to their precursor monocytes [31]. *Tert*-BHP was selected to induce ROS because it acutely evokes oxidative stress, resulting in cell toxicity [32]. Decomposition of *tert*-BHP to alkoxyl or peroxyl radicals accelerates lipid peroxidation chain reactions [33]. By means of a neutral red viability assay, we observed that *tert*-BHP induced significant and rapid cell death in both monocytes and DCs. Yet, monocyte-derived DCs were more resistant to *tert*-BHP-induced cell death than their precursor cells [31]. A PCR profiler array specific for oxidative stress and antioxidant-

Another category of auto-antigens that have been implicated in atherosclerosis are the stress-induced heat shock proteins (HSPs) [22]. HSPs are responsible for the repair or degra‐ dation of denatured proteins and, by maintaining protein conformation, they enhance the cell's ability to survive under conditions of metabolic or oxidative stress [23]. The mRNA ex‐ pression level of several HSPs, including HSP40 and HSP70, has been shown to be signifi‐ cantly increased in carotid endarterectomy specimens as compared to healthy arteries [24]. HSP70 seems to be homogenously distributed throughout the intima and media in healthy aortas, and a strong increase in its immunostaining intensity is observed in aortic athero‐ sclerotic plaques [25]. They appear to stimulate an immune response leading to the develop‐ ment and progression of atherosclerosis [26]. A number of studies indicate that HSPs are associated with DC function and might trigger DC activation and maturation. DCs seem to overexpress HSP70 in atherosclerotic plaques and the latter protein is presumably an impor‐ tant trigger for DC activation [27]. Gp96 (of the HSP90 family) and HSP70 have indeed been shown to stimulate bone marrow-derived DCs *in vitro* to secrete cytokines [28] and to ex‐ press antigen-presenting (MHC II) and costimulatory molecules (B7.2) [29]. However, To‐ dryk and colleagues [30] reported that HSP70 targets immature DCs to make them significantly more able to capture antigens. The presence of HSP70 inhibited DC maturation induced by tumour cell lysates from parental B16 cells and maintained the DC precursor population in a more poorly differentiated phenotype. Thus, there is still controversy on whether HSPs activate DCs or keep them in an immature state, and data are lacking to ro‐

> As discussed above, DCs process and present self and foreign antigens to T cells and are therefore important inducers of adaptive immune responses. However, 'the' DC does not exist, as DCs comprise a network of subsets that are phenotypically, functionally, and de‐ velopmentally distinct [40,41]. It is essential to understand the diversity in DC subtypes to target DCs for immunomodulating therapies. Most studies on DC subsets have been per‐ formed in mice, because lymphoid tissue is easier to obtain from mice than from humans. Mature mouse DCs are identified based on their expression of the integrin alpha X chain CD11c, the costimulatory molecules CD40, CD80 and CD86, and high surface levels of the antigen-presenting molecule MHC II [42,43,40]. The T cell markers CD4 and CD8 (in the form of a αα-homodimer) are also expressed on mouse DCs, and can be used to distin‐ guish different subtypes [44]. In general, three DC subsets can be characterized in mouse lymphoid tissue (table 1): 1) CD8α+ CD4- DCs; 2) CD8α- CD4+ DCs; and 3) CD8α- CD4- DCs [44]. The CD8α+ CD4- DCs are mainly localized in the T cell areas of lymphoid or‐ gans, whereas the CD8α- CD4+ DCs are found in the marginal zones. Yet, upon stimulation by microbial products, such as lipopolysaccharide, the latter can also migrate to the T cell zones [45,46]. Other markers that can be used to further subdivide mouse DC subsets include the integrin alpha M chain CD11b and the endocytosis receptor CD205 (DEC205). The CD8α+ CD4- DCs are also CD205+ CD11b and they are mainly present in the thymus, and at moderate levels in lymph nodes [40]. Lymph nodes further contain, in contrast to spleen, CD8α- CD4- CD11b+ CD205mid DCs which are considered as the mature

form of tissue interstitial DCs [40,42,43] (table 1). Another DC subtype, which is langer‐ inhigh CD11b+ CD8αlow CD205high, is only found in skin-draining lymph nodes and consid‐ ered as the mature form of epidermal Langerhans cells. These cells are also positive for MHC II and CD40, CD80 and CD86, suggesting that they are fully activated [42].

[53]. As a result, it remains unclear which subtype represents the human equivalent of ma‐

formed on blood, due to the limited availability of human spleen tissue. Moreover, human blood DCs are mainly immature and heterogeneous in their expression of a range of mark‐ ers. It might be that part of the heterogeneity reflects differences in the maturation or activa‐ tion state of DCs, rather than that they all represent separate sub-lineages. Yet, one subtype that is similar to its mouse counterpart is the human Langerhans cell, which expresses CD1a

In human blood, the first made classification is often the distinction between plasmacytoid (p)DCs, and myeloid or conventional DCs (cDCs) (table 1). Freshly isolated pDCs resemble plasma cells and have a morphology typical of that of large, round cells with a diffuse nucleus and few dendrites. These type I IFN-producing cells (IPCs) are specialized in innate antiviral immune responses by producing copious amounts of type I interferons. pDCs express CD303 (blood dendritic cell antigen (BDCA) 2), CD304 (BDCA 4) and CD123 (IL 3Rα), whereas cDCs are characterized by their expression of CD1c (BDCA 1) and CD11c [54] (table 1). In addition, pDCs and cDCs also express different sets of Toll-like receptors (TLRs). In brief, pDCs express mainly TLR7 and TLR9, whereas cDCs exhibit strong expression of TLR1, TLR2, TLR3, TLR4, and TLR8. Accordingly, pDCs mainly recognize viral components with subsequent produc‐ tion of a large amount of IFN-α. In contrast cDCs recognize bacterial components and produce

Furthermore, cDCs and pDCs also differ in migration behaviour. Generally it is assumed that myeloid (m)DCs are the conventional DCs that infiltrate peripheral tissues, while pDCs migrate directly from the blood into lymphoid organs [54]. Finally, a small third population of blood DCs expressing CD11c and BDCA-3 (CD141) but not BDCA-1, CD123 or BDCA-2 can be distinguished (table 1). Of particular importance is their superior antigen cross-pre‐ sentation capacity and expression of the XC chemokine receptor 1 (XCR1), suggesting that

myeloid DC subset that is characterized by high expression of TLR3, production of IL-12 and IFN-β, and a superior capacity to induce T helper-1 cell responses, when compared to

Only in a few recent studies, human DCs have been isolated from lymphoid tissues, which allow direct comparison with mouse DC subtypes. Mittag and colleagues [41] identified four DC subsets in human spleen that resemble DCs found in human blood. These include three cDC subtypes and one pDC subtype (table 1). The cDCs are all negative for lineage markers and positive for HLA-DR and CD11c, and they differ in their expression of CD1b/c (= BDCA-1), CD141 (= BDCA-3) and CD16. The pDCs express high levels of CD304 (=

sets, which include IL-12p70 secretion and cross-presentation, appeared to be not restricted

cDCs as thought earlier, but shared by CD1b/c+

BDCA-4), but not CD11c [41]. Moreover, the hallmark functions of mouse CD8α<sup>+</sup>

DCs. They emerge as a distinctive

DC sub‐

and CD16<sup>+</sup>

and langerin and is characterized by the presence of Birbeck granules [40].

pro-inflammatory cytokines such as IL-12p70, TNF-α, and IL-6 [54,7].

they represent the human counterpart of mouse CD8α<sup>+</sup>

DCs. Another important barrier is that most human studies are per‐

Dendritic Cells in Atherogenesis: From Immune Shapers to Therapeutic Targets

http://dx.doi.org/10.5772/52900

63

ture mouse CD8α<sup>+</sup>

BDCA-1+

mDCs [54,7].

to the equivalent human CD141+

DC subsets [41].

The numerous DC subtypes in mouse lymphoid organs are all able to present antigens to T cells, however, they differ in other aspects of DC-T cell communication [40]. CD8α<sup>+</sup> DCs mainly induce Th1/Th17-polarizing cytokine responses in CD4+ effector T cells, whereas CD8α- DCs are able to induce Th2-biased cytokine responses [47,48,49,50]. CD8α<sup>+</sup> DCs also seem to be specialized for the uptake and cross-presentation of exogenous antigens on MHC I and consequently stimulate CD8+ cytotoxic T cells, whereas CD8α- DCs mainly stimulate CD4+ T helper cells [51,52].


cDC = conventional dendritic cell, pDC = plasmacytoid dendritic cell

lineage = cocktail of CD3, CD14, CD16, CD19, CD20, CD56; CD1c = BDCA-1; CD303 = BDCA-2; CD141 = BDCA-3; CD304 = BDCA-4

BDCA = blood dendritic cell antigen

**Table 1.** Markers used for characterization of DC subtypes in mice and men

It has to be noticed that the association between mouse and human DC subsets remains elu‐ sive, making translation of the above-mentioned findings difficult. One of the major barriers in comparing mouse and human DC subsets is the lack of CD8α expression on human DCs [53]. As a result, it remains unclear which subtype represents the human equivalent of ma‐ ture mouse CD8α<sup>+</sup> DCs. Another important barrier is that most human studies are per‐ formed on blood, due to the limited availability of human spleen tissue. Moreover, human blood DCs are mainly immature and heterogeneous in their expression of a range of mark‐ ers. It might be that part of the heterogeneity reflects differences in the maturation or activa‐ tion state of DCs, rather than that they all represent separate sub-lineages. Yet, one subtype that is similar to its mouse counterpart is the human Langerhans cell, which expresses CD1a and langerin and is characterized by the presence of Birbeck granules [40].

form of tissue interstitial DCs [40,42,43] (table 1). Another DC subtype, which is langer‐ inhigh CD11b+ CD8αlow CD205high, is only found in skin-draining lymph nodes and consid‐ ered as the mature form of epidermal Langerhans cells. These cells are also positive for

The numerous DC subtypes in mouse lymphoid organs are all able to present antigens to T cells, however, they differ in other aspects of DC-T cell communication [40]. CD8α<sup>+</sup> DCs mainly induce Th1/Th17-polarizing cytokine responses in CD4+ effector T cells, whereas

DCs are able to induce Th2-biased cytokine responses [47,48,49,50]. CD8α<sup>+</sup>

**according Phenotype DC subsets**

CD8α+ CD4-

CD8α-

CD8α- CD4-

**Markers**

CD83

**Table 1.** Markers used for characterization of DC subtypes in mice and men

cDC = conventional dendritic cell, pDC = plasmacytoid dendritic cell

CD40 CD80 CD86

**to localization MOUSE HUMAN**

circulating pDC PDCA-1+ CD11c+ CD11b- CD303+ CD304+ CD123+

lineage = cocktail of CD3, CD14, CD16, CD19, CD20, CD56; CD1c = BDCA-1; CD303 = BDCA-2; CD141 = BDCA-3; CD304

It has to be noticed that the association between mouse and human DC subsets remains elu‐ sive, making translation of the above-mentioned findings difficult. One of the major barriers in comparing mouse and human DC subsets is the lack of CD8α expression on human DCs

CD205+ CD11b- lineage-

CD205mid CD11b+ lineage-

 CD11b+ CD11chigh CD1c+ CD11c+ CD8α+ CD205+ CD11c+ CD141+ CD11c+ XCR1+

CD11c-

CD304+

CD4+ lineage-

seem to be specialized for the uptake and cross-presentation of exogenous antigens on MHC

cytotoxic T cells, whereas CD8α-

DCs also

DCs mainly stimulate

HLA-DR+ CD11c+ CD1b/c+

HLA-DR+ CD11c+ CD141+

HLA-DR+ CD11c+ CD16+

MHC II and CD40, CD80 and CD86, suggesting that they are fully activated [42].

CD8α-

CD4+

**DC subtype**

Activated (mature) DCs

= BDCA-4

I and consequently stimulate CD8+

**Subdivision**

circulating cDC CD8α-

T helper cells [51,52].

62 Current Trends in Atherogenesis

cDCs lymphoid organ-

pDCs lymphoid organ-

resident cDC

resident pDC

Costimulatory molecules

Activation molecules

BDCA = blood dendritic cell antigen

**DC activation status**

In human blood, the first made classification is often the distinction between plasmacytoid (p)DCs, and myeloid or conventional DCs (cDCs) (table 1). Freshly isolated pDCs resemble plasma cells and have a morphology typical of that of large, round cells with a diffuse nucleus and few dendrites. These type I IFN-producing cells (IPCs) are specialized in innate antiviral immune responses by producing copious amounts of type I interferons. pDCs express CD303 (blood dendritic cell antigen (BDCA) 2), CD304 (BDCA 4) and CD123 (IL 3Rα), whereas cDCs are characterized by their expression of CD1c (BDCA 1) and CD11c [54] (table 1). In addition, pDCs and cDCs also express different sets of Toll-like receptors (TLRs). In brief, pDCs express mainly TLR7 and TLR9, whereas cDCs exhibit strong expression of TLR1, TLR2, TLR3, TLR4, and TLR8. Accordingly, pDCs mainly recognize viral components with subsequent produc‐ tion of a large amount of IFN-α. In contrast cDCs recognize bacterial components and produce pro-inflammatory cytokines such as IL-12p70, TNF-α, and IL-6 [54,7].

Furthermore, cDCs and pDCs also differ in migration behaviour. Generally it is assumed that myeloid (m)DCs are the conventional DCs that infiltrate peripheral tissues, while pDCs migrate directly from the blood into lymphoid organs [54]. Finally, a small third population of blood DCs expressing CD11c and BDCA-3 (CD141) but not BDCA-1, CD123 or BDCA-2 can be distinguished (table 1). Of particular importance is their superior antigen cross-pre‐ sentation capacity and expression of the XC chemokine receptor 1 (XCR1), suggesting that they represent the human counterpart of mouse CD8α<sup>+</sup> DCs. They emerge as a distinctive myeloid DC subset that is characterized by high expression of TLR3, production of IL-12 and IFN-β, and a superior capacity to induce T helper-1 cell responses, when compared to BDCA-1+ mDCs [54,7].

Only in a few recent studies, human DCs have been isolated from lymphoid tissues, which allow direct comparison with mouse DC subtypes. Mittag and colleagues [41] identified four DC subsets in human spleen that resemble DCs found in human blood. These include three cDC subtypes and one pDC subtype (table 1). The cDCs are all negative for lineage markers and positive for HLA-DR and CD11c, and they differ in their expression of CD1b/c (= BDCA-1), CD141 (= BDCA-3) and CD16. The pDCs express high levels of CD304 (= BDCA-4), but not CD11c [41]. Moreover, the hallmark functions of mouse CD8α<sup>+</sup> DC sub‐ sets, which include IL-12p70 secretion and cross-presentation, appeared to be not restricted to the equivalent human CD141+ cDCs as thought earlier, but shared by CD1b/c+ and CD16<sup>+</sup> DC subsets [41].
