Published online before print April 20, 2009, doi: 10.1161/CIRCULATIONAHA.108.807537
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(Circulation. 2009;119:2367-2375.)
© 2009 American Heart Association, Inc.
Vascular Medicine |
Conventional Dendritic Cells at the Crossroads Between Immunity and Cholesterol Homeostasis in Atherosclerosis
From INSERM UMR-S 939, Hôpital de la Pitié (E.L.G., T.H., F.S.-C., B.O., J.P., V.D., M.J.C., P.L.), Paris, France; Université Pierre et Marie Curie, Université Paris 06, UMR-S 939 (E.L.G., T.H., F.S.-C., B.O., J.P., V.D., M.J.C., P.L.), Paris, France; Assistance Publique–Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière, Service d’Endocrinologie-Métabolisme (T.H., M.J.C., P.L.), Paris, France; Department of Medicine, University of California San Diego (E.R.M., J.L.W.), La Jolla, Calif; and Department of Gene and Cell Medicine and Department of Medicine, Mount Sinai School of Medicine (F.G.), New York, NY.
Correspondence to Dr Philippe Lesnik, INSERM U939, Hôpital de la Pitié, 83 Bd de l’hôpital, 75651 Paris 13, France. E-mail philippe.lesnik{at}upmc.fr
Received July 15, 2008; accepted March 6, 2009.
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Abstract |
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Background— Immunoinflammatory mechanisms are implicated in the atherogenic process. The polarization of the immune response and the nature of the immune cells involved, however, are major determinants of the net effect, which may be either proatherogenic or antiatherogenic. Dendritic cells (DCs) are central to the regulation of immunity, the polarization of the immune response, and the induction of tolerance to antigens. The potential role of DCs in atherosclerosis, however, remains to be defined.
Methods and Results— We created a mouse model in which the lifespan and immunogenicity of conventional DCs are enhanced by specific overexpression of the antiapoptotic gene hBcl-2 under the control of the CD11c promoter. When studied in either low-density lipoprotein receptor–deficient or apolipoprotein E–deficient backgrounds, DC-hBcl2 mice exhibited an expanded DC population associated with enhanced T-cell activation, a T-helper 1 and T-helper 17 cytokine expression profile, and elevated production of T-helper 1–driven IgG2c autoantibodies directed against oxidation-specific epitopes. This proatherogenic signature, however, was not associated with acceleration of atherosclerotic plaque progression, because expansion of the DC population was unexpectedly associated with an atheroprotective decrease in plasma cholesterol levels. Conversely, depletion of DCs in hyperlipidemic CD11c–diphtheria toxin receptor/apolipoprotein E–deficient transgenic mice resulted in enhanced cholesterolemia, thereby arguing for a close relationship between the DC population and plasma cholesterol levels.
Conclusions— Considered together, the present data reveal that conventional DCs are central to the atherosclerotic process, because they are directly implicated in both cholesterol homeostasis and the immune response.
Key Words: atherosclerosis • immune system • homeostasis • dendritic cells • lymphocytes
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Introduction |
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Dendritic cells (DCs) are the most potent antigen-presenting cells. Indeed, DCs possess a markedly elevated capacity to stimulate T cells, B cells, and natural killer T cells and to drive T-cell differentiation along both T-helper 1 (Th1) and T-helper 2 (Th2) pathways.1 Moreover, DCs are known to favor tolerance to antigens, possibly via the generation of regulatory T cells.2 As major regulators of immune responses and T-cell polarization, DCs are potentially key players in chronic inflammatory diseases such as atherosclerosis. Indeed, available evidence suggests that immune responses are directly implicated in the pathogenesis of atherosclerosis.3,4 Although the presence of DCs has been reported in atherosclerotic plaques,5–7 no mechanistic insight into the potential central immunoregulatory role of DCs in the immunoinflammatory dimension of atherosclerosis has been provided in atherosclerosis-prone mice. Modulation of the capacity of DCs to induce an immune response may facilitate evaluation of their impact on the pathogenesis of atherosclerosis. Indeed, enhancement of the lifespan of DCs has been reported to increase their immunogenicity in mice.8–11 In this context, it is especially relevant that Bcl-2 has been shown to be a major regulator of DC lifespan and immunogenicity.12 We therefore developed a mouse model in which DC lifespan and immunogenicity are enhanced by overexpression of human Bcl-2 (hBcl-2) under the control of the DC-specific CD11c promoter. This experimental approach allowed us to modulate the half-life and thus the immunogenicity of DCs in vivo with a view to evaluate their impact on the immune response during atherosclerosis.
Clinical Perspective on p 2375
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Methods |
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Transgenic mice expressing hBcl-2 under the murine CD11c promoter were described previously.13 All procedures (bone marrow transplantation, plasma lipid analyses, chimerism, quantification of atherosclerotic plaques, immunohistochemistry, quantification of autoantibodies, analysis of gene expression by quantitative polymerase chain reaction, flow cytometry, cytokine assays, and generation of bone marrow–derived DCs) were performed as described previously13–16 and are detailed in the online-only Data Supplement, along with details about the animal model.
Statistical Analysis
The statistical significance of the differences between groups was evaluated with the unpaired or paired 2-tailed Student t test. P<0.05 was considered significant. Values are expressed as mean±SEM.
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Results |
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Characterization of DC-hBcl2 Mice
Because CD11c is differentially expressed by DC subpopulations, and to gain insight into the specificity of transgene expression, we assessed the expression of hBcl-2 in DC subpopulations and in different types of leukocytes (online-only Data Supplement Figure I). Flow cytometric analysis revealed that splenic conventional DCs (CD11chigh, MHCII+), comprising CD11b+ DCs (CD11chigh, CD11b+) and CD8+ DCs (CD11chigh, CD8+), expressed hBcl-2, whereas plasmacytoid DCs (CD11cint, PDCA-1+) did not. Equally, we demonstrated that splenic B cells, T cells, and macrophages did not express hBcl-2. Another population of leukocytes known to express CD11c, bronchoalveolar macrophages (CD11c+, F4/80+), did not express hBcl-2 in the DC-hBcl-2 mice in the present study (online-only Data Supplement Figure I). This finding may be explained by the nature of the CD11c promoter used, which is a minimal promoter previously described to drive expression only in DCs with high endogenous expression of CD11c.17 In our model, monocytes (CD11b+, F4/80+) did not express hBcl-2, and therefore, monocyte count was similar in control apolipoprotein E–deficient (Apoe–/–) mice and DC-hBcl-2 Apoe–/– mice (online-only Data Supplement Figure II).
hBcl-2 Overexpression in DCs Enhances Their Lifespan and Immunogenicity
DCs generated from bone marrow cells of DC-hBcl-2 mice expressed hBcl-2 protein as expected (online-only Data Supplement Figure IIIA) and displayed enhanced resistance to apoptotic stress (online-only Data Supplement Figure IIIB). Such enhanced survival impacted the relative number of DCs in vivo. Indeed, the DC population was enriched in spleens from DC-hBcl-2 mice (online-only Data Supplement Figure IIIC; P<0.05). Then, because DCs may control lymphocyte homeostasis, we assessed T-cell activation in splenocytes of DC-hBcl-2 and control mice at the basal state. The data revealed that activated T cells, CD3+ and CD4+ cells expressing the activation marker CD69, were significantly increased in DC-hBcl-2 mice compared with controls (online-only Data Supplement Figure IIID; P<0.01 and P<0.001, respectively), whereas expression of CD25 by CD4+ T cells was similar in both groups in the basal state (online-only Data Supplement Figure IIID). These data are consistent with an enhanced immunogenicity of DCs in DC-hBcl-2 mice fed a chow diet. In this regard, it is relevant that on a nonlethal lipopolysaccharide challenge, we reported that DC-hBcl-2 mice equally exhibited significant elevation in the DC population, as well as in activation of T and B cells, compared with their littermate controls.14 Collectively, hBcl-2 overexpression in DCs prolonged their lifespan, led to a significant increase in the DC population, and enhanced T-cell activation in vivo.
Effect of Enhanced DC Lifespan and Immunogenicity on T-Cell Activation in Ldl-r–/– Mice
To evaluate whether DC lifespan and immunogenicity impact both immunity and atherogenesis, irradiated female low-density lipoprotein (LDL) receptor–deficient (Ldl-r–/–) mice were reconstituted with bone marrow cells from DC-hBcl-2 mice or wild-type (WT) littermates. After 4 weeks of recovery, mice were switched to a Western diet for 12 weeks. The efficiency of transplantation was established by the detection of <5% of Ldl-r knockout alleles in bone marrow cells from these mice (online-only Data Supplement Figure IV), thereby indicating a chimerism in the range of 95% to 100%.
We first evaluated the impact of enhancement of DC lifespan on the DC population itself and on T-cell activation and Th1 polarization. DCs were enriched in the spleens of DC-hBcl-2
Ldl-r–/– compared with wtLdl-r–/– mice (+56%, P<0.01; Figure 1A). Analysis of splenic T cells revealed an elevation in the proportion of both CD3+ and CD4+ T cells expressing the activation marker CD69 (P<0.0001 for each) and of CD4+ T cells expressing CD25 (+25%, P<0.01) in DC-hBcl-2Ldl-r–/– compared with controls (Figure 1B). Concomitantly, an increment of 20% in the percentage of CD44-expressing CD4+ memory T cells was observed in DC-hBcl-2Ldl-r–/– mice compared with wtLdl-r–/– mice (P<0.0001; Figure 1C). We next quantified the mRNA expression of key mediators of DC function, as well as T-cell responses and polarization in the spleen. Analysis of the expression of genes characteristic of DC function revealed a significant increase in the mRNA of interleukin (IL)-12p40, IL-23p19, and IL-15 in DC-hBcl-2Ldl-r–/– mice, whereas expression of the IL-12p35 and IL-18 genes was unchanged (Figure 1D). This was associated with enhanced expression of interferon (IFN)-
and TIM-3 (T-cell immunoglobulin- and mucin-containing molecule, a transcription factor promoting Th1 development), together with unchanged levels of GATA3 mRNA (a transcription factor promoting Th2 development) in DC-hBcl-2Ldl-r–/– mice (Figure 1E). Expression levels of classic inflammatory genes revealed elevated levels of IL-1β mRNA but similar levels of CD40L and tumor necrosis factor-
mRNAs in the spleens of DC-hBcl-2Ldl-r–/– mice compared with controls (Figure 1E). These findings support the contention that an enhanced DC lifespan leads to elevation in DC immunogenicity and increased T-cell activation with polarization toward a Th1 profile.
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Effect of Enhanced DC Lifespan and Immunogenicity on Regulatory T Cells in Ldl-r–/– Mice
Natural regulatory T cells (Treg) exhibit marked antiatherogenic properties18 as a consequence of their ability to counteract both Th1- and Th2-mediated immune responses. Because DCs might influence the content and function of regulatory T cells, we evaluated whether the Treg population was modified in DC-hBcl-2Ldl-r–/– compared with wtLdl-r–/– mice. As shown in Figure 2A, flow cytometric analysis revealed that spleen CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ T cells were similar in both groups, thereby arguing for the absence of an altered natural Treg population. We next quantified the expression of key markers of Treg cell population and function in the spleens of both groups of mice. Real-time quantitative polymerase chain reaction analysis confirmed the unaltered Foxp3 expression consistent with the absence of elevation in the natural Treg population. In this context, it was relevant that expression levels of transforming growth factor-β and CTLA-4 (cytotoxic T-lymphocyte–associated protein 4) were similar in splenic cells of both DC-hBcl-2Ldl-r–/– and wtLdl-r–/– mice (Figure 2B). By contrast, a marked 7-fold increment in IL-10 mRNA expression was observed in DC-hBcl-2Ldl-r–/– mice (Figure 2B; P=0.01). Overall, these data indicate that Treg cells are not markedly altered in DC-hBcl-2Ldl-r–/– compared with wtLdl-r–/– mice.
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Effect of Enhanced DC Lifespan and Immunogenicity on B-Cell Activation and Circulating Levels of Autoantibodies Against Oxidation-Specific Epitopes in Ldl-r–/– Mice
Several recent studies have emphasized the protective role of B lymphocytes in atherosclerosis.15,19,20 These findings led us to question whether the increment in the DC population in our mouse model might affect levels of antibodies directed against oxidation-specific epitopes, the titer of the atheroprotective EO6 antibody idiotype, and the polarization of the humoral response (Th2-driven IgG1 versus Th1-driven IgG2c/IgG3 isotype production). We first evaluated B-cell activation by measuring the proportion of B cells bearing the activation marker CD86. A minor increment in B-cell activation was observed in DC-hBcl-2Ldl-r–/– compared with wtLdl-r–/– mice (Figure 3A; P<0.05). Quantification of anti-malondialdehyde-LDL and anti-oxidized LDL IgG1, IgG2c, IgG3, and IgM antibody production revealed significant elevation in the IgG2c fraction of both anti-MDA-LDL and anti-oxidized LDL antibodies (2-fold; Figure 3B and 3C; P<0.0005 for each) in DC-hBcl-2Ldl-r–/– compared with control mice, whereas levels of IgG1, IgG3, and IgM fractions were comparable between groups (Figure 3B and 3C). Moreover, titers of the EO6 antibody were markedly elevated in DC-hBcl-2Ldl-r–/– compared with wtLdl-r–/– mice (Figure 3D; P<0.0005). In conclusion, the increment in IgG2c titer in DC-hBcl-2Ldl-r–/– mice, which is characteristic of a Th1-driven immune response, is consistent with the cytokine expression profile observed in the spleens of these animals and indicates that expansion of the DC population favors Th1 polarization in an atherogenic environment.
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Effect of DC Lifespan on Atherosclerotic Lesion Progression, Plasma Lipids, and Lipoprotein Profile in Ldl-r–/– Mice
The impact of enhanced DC lifespan and immunogenicity on the progression of atherosclerosis was evaluated in DC-hBcl-2 transgenic and control mice on an Ldl-r–deficient background. As shown in Figure 4A, the lesion area in the aortic root of Ldl-r–/– recipients reconstituted with DC-hBcl-2 marrow cells was unchanged compared with their wild-type reconstituted controls after 12 weeks of Western diet (247.0±17.1x103 µm2 versus 294.9±26.0x103 µm2, respectively; P=0.2). Furthermore, macrophage areas were comparable in lesions of DC-hBcl-2Ldl-r–/– and wtLdl-r–/– mice (Figure 4B; P=0.5). Because lesion formation and progression are predominantly dependent on plasma cholesterol levels and its distribution among the different lipoprotein subclasses, we then assessed whether elevated numbers of DCs might alter cholesterol homeostasis. Compared with controls, Ldl-r–/– mice transplanted with DC-hBcl-2 bone marrow cells displayed a significant reduction in plasma total cholesterol (547±61 versus 426±64 mg/dL, respectively; P<0.005) and in free cholesterol levels (198±45 versus 149±33 mg/dL, respectively; P<0.05); by contrast, triglyceride levels were similar in both groups of mice (192±48 versus 170±42 mg/dL, respectively; Table). Analysis of cholesterol distribution among plasma lipoprotein subclasses revealed that the lower total cholesterol level in DC-hBcl-2Ldl-r–/– compared with wtLdl-r–/– mice was due to a reduction in the abundance of VLDL and LDL subclasses (–59% and –57%, respectively), whereas the HDL fraction was decreased to a lesser degree (–33%; Figure 5A). Because a larger DC population in DC-Bcl-2 animals was associated with lower circulating cholesterol levels, thereby revealing that the role of DC is important in the setting of hypercholesterolemia, we sought to determine whether the opposite mechanism (ie, acute depletion of DCs) was associated with an enhanced degree of cholesterolemia. We took advantage of the DT receptor (DTR)/diphtheria toxin (DT) system, which allows depletion of DCs after DT injection in transgenic mice expressing DTR under the CD11c promoter.21 First, Ldl-r–/– mice were irradiated, transplanted with CD11c-DTR bone marrow cells, and submitted to a 4-week recovery period. These mice were fed a Western diet for 2 weeks, and then half of the mice were injected with DT whereas the other half were treated with vehicle. Plasma total cholesterol level was measured 24 hours after treatment (Figure 5B) and revealed an increment of 34% in CD11c-DTRLdl-r–/– mice treated with DT compared with vehicle-treated animals (333±36 versus 446±32 mg/dL, respectively; P<0.05).
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Effect of DC Lifespan and Immunogenicity on Immune Response in Apoe–/– Mice
We next evaluated whether the impact of DC lifespan and immunogenicity on immunity that we documented in Ldl-r–/– mice, and more especially that which involved T-cell activation and B-cell responses, was equally manifest in an alternative atherosclerotic model (ie, Apoe–/– mice). We first showed that the DC population was significantly expanded in chow-fed 20-week-old DC-hBcl-2 Apoe–/– mice compared with Apoe–/– controls (Figure 6A; P<0.05). Flow cytometric analysis revealed that both the CD3+- and CD4+-activated T-cell populations were larger in DC-hBcl-2 Apoe–/– mice than in controls (Figure 6B; P<0.001 each), whereas the CD25-expressing CD4+ T-cell population was unaltered (Figure 6B). With regard to the B-cell compartment, no change was observed in CD86 expression by B cells in DC-hBcl-2 Apoe–/– mice compared with controls (Figure 6C). The main alteration in the antibody response observed on the Ldl-r–/– background (ie, elevation in circulating levels of both anti-malondialdehyde-LDL and anti-oxidized LDL IgG2c antibodies in DC-hBcl-2 mice) was equally observed on an Apoe–/– background (Figure 6D and 6E; P<0.05), whereas no statistically significant differences were observed for the other isotypes. Finally, levels of the E06 antibody were not affected in DC-hBcl-2 Apoe–/– mice compared with controls. To further evaluate whether changes in the polarization of the immune response observed in DC-hBcl-2Ldl-r–/– were equally present in the Apoe–/– background, additional experiments were conducted in DC-hBcl-2 Apoe–/– and Apoe–/– mice fed a Western diet. We thus confirmed activation of key mediators of DC function and T-cell responses in the spleen of DC-hBcl-2 in this background (ie, IL-12p40 and IL-23p19 mRNAs; online-only Data Supplement Figure VA). In addition, we characterized the regulatory response in the Apoe–/– background and confirmed the absence of an effect on natural Treg (nTreg) as shown by the absence of major changes in the expression of key genes involved in nTreg function and development (CD25, GITR, ICOS, neuropilin-1, and Drosha; online-only Data Supplement Figure VB). Although a major increase in IL-10 mRNA levels was observed in spleens of DC-hBcl-2Ldl-r–/– mice (Figure 2B), a trend for higher IL-10 mRNA levels in Western diet–fed-DC-hBcl-2 Apoe–/– mice was detectable (online-only Data Supplement Figure VA). However, in assays of restimulated splenocytes, CD4+ T cells from DC-hBcl-2 mice produced significantly more IL-10 than control CD4+ T cells, whereas DCs from DC-hBcl-2 mice produced less IL-10 than controls (Figure 7A and 7B). Such data suggest that changes in DC function and population are associated with increased production of IL-10 by CD4+ T cells, most likely T regulatory type-1 (Tr-1) T cells. Finally, we observed a 4.2-fold increase in the percentage of CD4+IL-17+ cells compared with control Apoe–/– mice in restimulated splenocytes (Figure 7C). Moreover, intracellular staining for IL-12 and IFN- in CD4+ T cells (Figure 7D and 7E) confirmed the Th1 signature, which indicates that both Th1 and Th17 phenotypes were upregulated in our model.
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In conclusion, the main changes in immune system activation observed on the Ldl-r–/– background (ie, enhanced Th1 activation along with Th1-driven IgG2c anti-oxidized LDL and anti-malondialdehyde-LDL production) were equally present on an Apoe–/– background. Additionally, we observed an increase in the activity of the Th17 pathway and a potential increase in Tr-1 regulatory T cells.
Effect of DC Lifespan and Immunogenicity on Atherosclerotic Lesion Progression, Plasma Lipids, and Lipoprotein Profile in Apoe–/– Mice
We analyzed lesion area in chow-fed 20-week-old DC-hBcl-2 Apoe–/– and Apoe–/– mice and reported the absence of a significant difference between the 2 groups of animals (Figure 8A; 149.5±20.9 versus 181.4±17.5x103 µm2, respectively; P=0.3). Moreover, comparison of plaque burden in DC-hBcl-2 Apoe–/– and Apoe–/– mice fed a Western diet for 8 weeks revealed no difference in lipid deposition between groups (Figure 8B; 184.1±21.7 versus 247.4±35.3x103 µm2, respectively; P=0.13). Taken together, and despite marked elevation in T-cell activation and a Th1-polarized immune response in DC-hBcl-2 mice compared with controls, no significant difference in lesion areas was found between groups. On the contrary, we observed a consistent trend toward attenuated lesion progression in DC-hBcl-2 mice compared with controls in all mouse models studied. We next compared plasma lipid levels in Apoe–/– and DC-hBcl-2 Apoe–/– fed either a chow or a Western diet and observed that plasma total and free cholesterol levels were significantly decreased in DC-hBcl-2 Apoe–/– mice compared with controls in both conditions, whereas triglyceride levels were unchanged (Table). Analysis of cholesterol distribution among plasma lipoprotein classes in DC-hBcl-2 Apoe–/– animals fed the Western diet revealed a reduction in the cholesterol content of particles in the size range of both VLDL and LDL (–37% and –22%, respectively; Figure 8C). It is interesting to note that we did not observe such changes in plasma lipid levels in DC-Bcl-2 mice on a wild-type background maintained on a chow diet (online-only Data Supplement Figure VI).
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To confirm whether depletion of DCs in the Apoe–/– background would also result in an increase in cholesterol levels as seen in the LDL-r–deficient background (Figure 5B), cholesterol-fed CD11c-DTR Apoe–/– mice and Apoe–/– controls were injected with DT. An increment of 63% in plasma cholesterol concentration was observed in DT-treated CD11c-DTR Apoe–/– mice compared with DT-treated Apoe–/– controls after 24 hours (1102±110 versus 676±64 mg/dL, respectively, P=0.01; Figure 8D). Next, we analyzed the time course of changes in plasma cholesterol levels in another set of CD11c-DTR Apoe–/– mice fed a normal chow and observed a transient increase (Figure 8E) that was statistically significant 24 and 48 hours after DT injection. This time course is consistent with published data on the duration of CD11c-positive cell depletion in DT treated-CD11c-DTR mice.21 Such depletion was specific to conventional DCs and did not affect plasmacytoid DCs (online-only Data Supplement Figure VII). Considered together, these results reveal that modulation of conventional DC number impacts plasma cholesterol levels under conditions of hypercholesterolemia.
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Discussion |
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A central question in the pathogenesis of atherosclerosis concerns the potential impact of DCs. Although DCs are present in human and murine atherosclerotic lesions,5–7 a paucity of experimental data exists on the role of DCs in atherosclerotic plaque progression. Therefore, we designed genetically modified mouse models to address this question. For this purpose, we examined the relationship between DC lifespan and progression of atherosclerosis and made several novel findings: (1) The extended lifespan of DCs impacts on T-cell activation status and polarization of the immune response toward the Th1 pathway; (2) DCs markedly alter the degree of cholesterolemia in mice; and (3) the hypocholesterolemic action of DCs compensates for the proatherogenic Th1 response, thereby resulting in the absence of modification in atherosclerotic lesion size in DC-hBcl-2 mice compared with controls.
DCs undergo accelerated clearance from lymphoid organs after interaction with antigen-specific T cells,22 which indirectly indicates that their lifespan may influence the duration of their ability to stimulate lymphocytes. Indeed, in several mouse models in which apoptosis of DCs was inhibited (including overexpression of p35 or deletion of Bim), enhanced DC lifespan was associated with expansion of the DC population and enhanced immunogenicity, as revealed by a major impact on T-cell activation.9,10 This is consistent with the increase in CD25+, CD69+, and CD44+ CD4 T cells we observed in DC-hBcl-2 mice. Moreover, in our model, we observed that DCs profoundly modulated both IL-12p40 and thereby IFN- expression. Such activated T cells and Th1 cytokines are proatherogenic, because they promote both lesion progression and plaque destabilization.4,23 Moreover, we showed that IL-12p35 expression was not altered in DC-hBcl-2 in either the Ldl-r–/– or the Apoe–/– background, whereas IL-23p19 mRNA levels were markedly elevated. Because the IL-12p40 subunit is common to both IL-12 (which is formed of the p40 and p35 subunits) and IL-23 (formed of the p40 and p19 subunits), such an expression profile is consistent with a marked increase in activity of the IL-23 pathway and suggests that the effect of DCs on immune system activation may also involve the IL-23 axis. This recently discovered pathway has been shown to drive the differentiation of Th17 cells, which are known to be triggers of autoimmune-driven inflammation.24 Interestingly, we report activation of the Th17 pathway in our model. Nevertheless, to date, this pathway has not been implicated in plaque progression but might represent another proatherogenic arm of the immune system.
DCs had similarly been known to favor tolerance to antigens, and several studies suggest that this process may involve the generation of regulatory T cells.2,25 In this context, we examined whether in atherosclerotic-prone mice, DCs may be critical for maintaining immune tolerance through their impact on the regulatory T-cell population. Treg cells are of particular importance in atherosclerosis, because recent evidence suggests that they are associated with protection against atherogenesis.18,26 In an atherosclerotic context, we found no significant changes in the percentage of the splenic natural Treg population (CD4+ Foxp3+ CD25+) in DC-hBcl-2 mice, consistent with previous studies showing that DC lifespan and immunogenicity did not alter the natural Treg compartment.9,10 Nevertheless, expression of IL-10 mRNA was upregulated in the spleen of DC-hBcl-2Ldl-r–/– compared with control mice, and IL-10–producing CD4+ T cells were increased in the spleen of DC-hBcl-2 Apoe–/– mice. Such enhancement would predict protection against lesion development, as suggested by studies in which the IL-10 axis was modulated.23 Moreover, expansion of CD4+ IL-10+ T cells, also termed Tr-1 cells or adaptive Tregs, could exert an antiatherogenic effect in our model. Indeed, this specific T-cell compartment has been described as a potent antiatherogenic population that may help to combat Th1 proatherogenic bias.27
Th2 or Th1 responses are associated with an immunoglobulin class switching to IgG1 or IgG2c, respectively.28,29 We therefore quantified titers of serum antibodies directed against oxidation-specific epitopes. Statistically significant increases occurred in titers of anti-malondialdehyde-LDL IgG2c and anti-oxidized LDL IgG2c in DC-hBcl-2 mice on Ldl-r- or Apoe-deficient backgrounds, which corroborates the development of a Th1 bias immune response in these mice observed at the level of cytokine expression (IFN-, IL-12, IL-15, and TIM-3).
As a major result of the present study, we unexpectedly observed that elevation in the DC population led to markedly decreased plasma cholesterol levels in both the Ldl-r–/– and Apoe–/– backgrounds. Using a mouse model that allowed a reverse approach (ie, specific depletion of DCs), we observed that conventional DC elimination induced elevation in plasma cholesterol levels, thereby arguing that conventional DCs may contribute to correction of hyperlipidemia and that such cells may be implicated in cholesterol homeostasis. Interestingly, the impact of expansion of the DC population on plasma cholesterol levels is consistent with other observations in both mice and humans supporting a role for mononuclear phagocytes (macrophages, DCs, and Kupffer cells) in cholesterol homeostasis. For example, granulocyte-macrophage colony-stimulating factor, a key factor for DC growth and differentiation,30 has been reported to exhibit a cholesterol-lowering effect in patients with aplastic anemia,31 a finding later confirmed in rabbits32 and in patients with coronary artery disease.33 Similarly, the hematopoietic growth factor macrophage colony-stimulating factor was also reported to lower cholesterol levels in rabbits and nonhuman primate models.34,35 In mice, the opposite effect was observed in the op/op strain mutated for macrophage colony-stimulating factor, in which monocytes and tissue macrophage populations such as Kupffer cells are reduced.36 Indeed, when bred on an Apoe–/– background, the op/op Apoe–/– mice present a 3-fold increase in cholesterol levels.37 Considered together, these data indicate a strong relationship between the mononuclear phagocyte system and the potential control of cholesterol homeostasis. In the present study, we report for the first time the implication of conventional DCs as a cell type able to favor cholesterol lowering in a hyperlipidemic environment. The precise mechanisms that underlie the decrement in plasma cholesterol levels in our mouse model were not explored in the framework of the present study; however, because they are present in many tissues (spleen, liver, gut, and intestine), DCs might favor lipoprotein uptake and clearance from the circulation. Of note, Stoneman et al38 reported no significant change in cholesterol levels in CD11b-DTR Apoe–/– mice treated with DT. In this latter model, DT-induced CD11b+ cell depletion was restricted to monocyte/macrophages, neutrophils, and CD11b+ conventional DCs, thereby indicating that depletion of these myeloid cells does not reproduce the effect observed on cholesterol levels in the CD11c-DTR Apoe–/– mice in the present study.
In addition to the role of DCs in facilitating a Th1-polarized immune response, the prevailing paradigm that underlies the proatherogenic effects of T cells, the present study revealed that the size of the conventional DC population was closely associated with regulation of cholesterol homeostasis. Overall, these antagonistic responses balanced each other out, with a null effect on atherosclerotic plaque progression. Clearly then, our findings identify the DC as a key player in atherosclerosis through its impact on both immune response regulation and cholesterol homeostasis.
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Acknowledgments |
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Sources of Funding
This work was funded by INSERM, Fondation de France, Leducq Foundation, and National Institutes of Health HL086559 (Dr Witztum). Dr Gautier was supported by a Fellowship from the Fondation pour la Recherche Médicale. Drs Lesnik, Huby, and Chapman are recipients of "Contrat d’Interface" from Assistance Publique–Hôpitaux de Paris/INSERM.
Disclosures
None.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.807537/DC1.
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