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Unlike conventional dendritic cells (cDC), plasmacytoid DCs (pDC) are poor in antigen presentation and critical for type I interferon response. While proposed to be present in human atherosclerotic lesions, their role in atherosclerosis remains elusive.
To investigate the role of pDC in atherosclerosis.
We show that pDC are scarcely present in human atherosclerotic lesions, and almost absent in mouse plaques. Surprisingly, pDC depletion by 120G8 mAb administration was seen to promote plaque T cell accumulation and exacerbate lesion development and progression in LDLr−/− mice. PDC depletion was accompanied by increased CD4+ T cell proliferation, IFN-γ expression by splenic T cells and plasma IFN-γ levels. Lymphoid tissue pDC from atherosclerotic mice showed increased indoleamine 2,3-dioxygenase (IDO) expression and IDO blockage abrogated the pDC suppressive effect on T cell proliferation.
Our data reveal a protective role for pDC in atherosclerosis, possibly by dampening T cell proliferation and activity in peripheral lymphoid tissue, rendering pDC an interesting target for future therapeutic interventions.
Plasmacytoid dendritic cells (pDC) are a subset of dendritic cells derived from both myeloid and lymphoid precursors in bone marrow, and constitute only 0.1-0.5% of the total leukocyte pool in blood and peripheral lymphoid tissue. As the main type I interferon (IFN) producing cells, pDC have a critical role in detection of and host defense against bacterial and viral infection, but also in sensing RNA/DNA and immune complexes. Upon stimulation, pDC produce large amounts of type I interferons (IFN-α, IFN-β, IFN-ω and IFN-γ) in a toll-like receptor (TLR) 7 and 9 dependent manner, thereby inducing effector T and natural killer (NK) cell activation and linking innate and adaptive immunity.1 PDC differ from conventional dendritic cells (cDC) in that they are poor T cell activators due to low expression of major histocompatibility complex class-II (MHC-II) and co-stimulatory molecules.2,3
The presence of pDC in human atherosclerotic lesions has been documented in 19954 and was subsequently confirmed by C. Weyand and co-workers.5 PDC (CD4+CD45RA+IL-3α(CD123)+ILT3+ILT1−CD11−) were reported to be expressed in the shoulder region of human plaques where they are believed to regulate T cell function, even in the absence of antigen recognition. CpG induced IFN-α release by pDC effected a 10-fold up regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on CD4+ T cell surface, thereby promoting vascular smooth muscle cell (vSMC) and endothelial cell (EC) apoptosis, processes that are generally deemed deleterious for plaque stability. However, these in vitro data leave unaddressed whether pDC are instrumental in plaque destabilization in vivo.
While viral infection associated acute pDC activation results in massive type I IFN release, chronic activation of pDC in the absence of infection was reported to cause severe autoimmune diseases. For instance, in experimental autoimmune encephalomyelitis pDC were shown to exert overt pathogenic activity, mainly by enhancing T helper 17 (Th17) dependent immune responses.6 In patients with systemic lupus erythematosus, pDC activity and IFN-α/β release correlated with disease activity and severity.7,8 Conversely, evidence is culminating that immature and alternatively activated pDC have the capacity to dampen chronic low grade inflammation and autoimmune diseases, including diabetes type I, asthma and transplant rejection, possibly by suppressing CD8+ effector T cell and inducing CD4+CD25+ regulatory T cell (Treg) function by the release of tolerogenic molecules such as indoleamine 2,3-dioxygenase (IDO) or programmed death-ligand 1 (PD-L1).9,10,11,12,13 Altogether, these observations show that pDC are very plastic cells with the capacity to produce high levels of type I IFN and activate the adaptive immune system in infection on one hand and to regulate inflammation by inhibiting effector T cell and inducing regulatory T cell responses on the other hand.
In this study, we addressed the actual role of pDC in atherosclerosis by a loss-of-function approach, providing evidence for an IDO-dependent T cell suppressive activity of pDC in human and mouse atherosclerosis.
An expanded Methods section is available in the Online Supplemental Material.
Female LDLr−/− mice, obtained from The Jackson Laboratoy and backcrossed at least 11 times to C57Bl/6, were placed on high fat diet containing 0.25% cholesterol (Special Diets Services, Witham, Essex, UK). Two weeks later, atherosclerotic lesions were induced in the carotid artery by bilateral placement of semi-constrictive collars.1 To study effects of pDC depletion on atherosclerosis development, a pDC depleting antibody, 120G8 (250μg/mouse/injection, Bioceros, Utrecht, The Netherlands), was administered 4 times per week i.p. for 3,5 weeks starting at the time of collar placement (n=19) after which mice were sacrificed. From these mice, also the aortic root was analyzed to study effects of pDC depletion on natural atherosclerosis development. To study effects of pDC depletion on progression of atherosclerosis, 120G8 (250μg/mouse/injection, Bioceros, Utrecht, The Netherlands) was administered 4 times per week i.p for 3 weeks starting at 4 weeks after collar placement, once initial lesions had formed (n=17). As control antibody, an isotype control (GL113) was used. All animal work was approved by the regulatory authority of Maastricht University and performed in compliance with the Dutch government guidelines.
LDLr−/− mice on high fat diet treated either with 120G8 or GL113 received BrdU i.p. injections (0.8mg/day) for 5 consecutive days. BrdU incorporation in CD4+ and CD8+ T cells was assessed using a FITC BrdU flow kit (BD) according to manufacturer’s instructions. Cells were analyzed using FACS CANTO II.
LDLr−/− recipient mice on high fat diet received CFSE-labeled purified OVA-specific OT-1/CD45.1 or OT-2/CD90.1 cells together with non-transgenic purified CD8+/CD90.1 or CD4+/CD45.1 T cells that served as an internal control. The next day, mice received i.v. irradiated (1500 rad) Kb−/−-actmOVA or C3H-actmOVA cells. Three days later, OT-1/CD90.1 and OT-2/CD45.1 proliferation and expansion were determined based on CFSE dilution and the ratio of OT-1/CD45.1 to CD90.1 control CD8+ T cells and OT-2/CD90.1 to CD45.1 control CD4+ T cells. In parallel, splenocytes were restimulated with OVA 257-264 or OVA 323-339 peptide (A&A labs, San Diego, CA) in the presence of Brefeldin A for 5 hours. Surface staining for CD8/CD4 (BD) and CD45.1/CD90.1 (eBioscience) was performed.
PDC were isolated from spleens of Bl6 FLt3L treated high fat diet fed LDLr−/− mice using PDCA-1 microbeads (Miltenyi). Splenic CD3+ T cells from pDC depleted LDLr−/− mice on high fat diet were isolated after staining with biotinylated CD4 (Biolegend) and CD8 (Biolegend) antibodies using a biotin isolation kit (Miltenyi). PDC and CD3+ T cells were co-cultured for 3 days at a 1:6 ratio in the presence or absence of 1-MT (Sigma Aldrich, 100μM) or anti-PD-L1 (20μg/ml, Bioceros). CD3+ T cell proliferation was assessed by 3[H]-thymidine incorporation and plotted as relative proliferation index, defined as the ratio of T cell proliferation in the presence and absence of pDC. In parallel cultures, purified CD4+ T cells were CFSE labeled prior to stimulation and proliferation was determined by flow cytometry.
Human atherosclerotic lesions were isolated from carotid endarterectomy patients and graded for progression stage according to Virmani et al..14 All human work was approved by the Ethical Committee of the University Hospital Maastricht. Signed informed consent for participation in the study was obtained from all individuals.
Data are expressed as mean ± SEM and are considered statistically significant when P<0.05.
Concordant with previous observations,5 CD123+ pDC were seen to be present in human atherosclerotic lesions and their presence increases with plaque progression (P<0.05) (Figure 1A). Of note, CD123 stained cells were found to show considerable co-localization with macrophages (Figure 1B) and vSMC (Figure 1C), supporting earlier findings from van Vré et al. showing that CD123 is not a specific marker for human pDC.15 Next, we considered BDCA-4 as a more selective human pDC marker16. Flow cytometry on human whole blood samples confirmed the high specificity of this marker for human pDC, as it was virtually absent on circulating monocyte, B cell, T cell and granulocyte subsets (Online Figure IA). Surprisingly, BDCA-4+ staining revealed the scanty presence of pDC in human plaques; moreover BDCA-4+ cell expression did not differ between stable and unstable advanced atherosclerotic lesions (Figure 1D). In agreement, micro-array (Figure 1E and Online Figure IB) and real-time PCR analysis (Online Figure IC) failed to demonstrate differential expression of established pDC markers during plaque progression. Likewise, pDC were almost absent in mouse carotid and aortic artery lesions of LDLr−/− and ApoE−/− mice as well (data not shown), although we did observe few scattered pDCs in the adventitia (Figure 1F), which is in line with our human data (Online Figure ID).
Next, we addressed the specificity of pDC depletion by 120G8 mAb, which recognizes PDCA-1 (also referred to as bone marrow stromal cell antigen 2 (BST2)), a marker specifically expressed on mouse pDC. 120G8 significantly depleted (>90%) pDC numbers in blood and spleen (P<0.05) (Figure 2A). PDC repopulation started already 24 hours after a single 120G8 administration and full recovery was obtained after 72 hours (Online Figure IIA), necessitating a every two daily 120G8 dose regimen for effective and persistent pDC depletion. It has been reported that in vitro PDCA-1 expression is upregulated at mRNA level in other cell types in response to viral infection or exposure to inflammatory stimuli, which theoretically could thwart the depletion specificity.17 In our study, 120G8 treatment did neither affect cDC (Figure 2B), nor B cell numbers (Figure 2C). Also monocyte and granulocyte levels were unchanged (Online Figure IIB and C). Moreover, assessment of PDCA-1 expression by flow cytometry showed that in high fat diet fed LDLr−/− mice PDCA-1 expression is completely restricted to pDC (Figure 2D). These data demonstrate that in our mouse model of atherosclerosis pDC depletion by 120G8 mAb was effective and specific. To investigate whether prolonged antibody administration by itself could modulate immune responses, we compared the T cell activation status between GL113 and PBS treated mice and did not find a difference in the number of CD44high T cells (Online Figure IID). At a functional level 120G8 treatment almost abrogated CpG induced pDC activation in vivo, as judged by the 6-fold attenuated induction in plasma IFN-α release upon CpG injection in 120G8 treated versus control mice (P<0.05; Figure 2E). While pDC activity and TLR 7/9 function appear to be intact, under conditions of hyperlipidemia, baseline plasma IFN-α levels remained unchanged after pDC depletion, both in the plaque initiation and progression study. This suggests that an atherogenic stimulus per se does not increase IFN-α release by peripheral pDCs and/or that pDC are under these conditions not the major source of circulating IFN-α.
To address the role of pDC in atherosclerosis, we examined lesion development and progression in LDLr−/− mice fed a high fat diet. For plaque initiation, 120G8 treatment was started at time of collar placement, whereas for plaque progression, it was started at week 4 after collar placement, once initial lesions had formed. 120G8 treatment did not affect body weight, nor did it lead to overt pathogenic responses. 120G8 treatment tended to decrease plasma cholesterol levels initially (1384 ± 78.53 vs 1126 ± 46.28 pg/ml in control and 120G8 treated mice, respectively), but this effect was blunted at later stages of plaque development (1097 ± 44.83 vs 1231 ± 72.31 pg/ml in control and 120G8 treated mice, respectively) (Table 1). To our surprise, atherosclerosis considerably deteriorated after pDC depletion. Plaque volume was 2-fold increased in the plaque initiation study (1.4×107 ± 2.6×106 vs 2.7×107 ± 4.7×106 μm3 in control and 120G8 treated mice, respectively) (P<0.05) (Figure 3A), while we observed a 3-fold increase in plaque progression compared to plaques at baseline (1.4×107 ± 2.5×106 vs 5.4×107 ± 7.2×106 μm3 in control baseline and 120G8 treated mice, respectively) (P<0.0005) versus only 2-fold for the GL113 Ab treated mice (1.4×107 ± 2.5×106 vs 3.3×107 ± 7.5×106 μm3 in control baseline and GL113 treated mice, respectively) (P<0.05) (Figure 3B). Likewise, pDC depletion induced a more unstable plaque phenotype in the progression study, characterized by necrotic core expansion (Figure 3C) and diminished cap vSMC content (Figure 3D). There was no difference in plaque collagen content (data not shown). In addition to the carotid artery, we also examined atherosclerosis development in the aortic root, showing an essentially similar aggravation of lesion formation after pDC depletion (P<0.05; Figure 3E).
Thus our data point to an unexpected protective role of pDC in atherosclerosis, which is in contrast to the prevailing notion that pDC might promote atherosclerosis by activating T cells in a type-I IFN dependent manner.5,18 Moreover, given the fact that atherosclerotic lesions are virtually devoid of pDC, they most likely exert their atheroprotective effect by modulating immune responses in the periphery and/or adventitia.
As a next step, we examined effects of pDC depletion on plaque composition to address the potential mechanisms responsible for the protective effects of pDC in atherosclerosis. We found that pDC depletion led to an increase in lesional T cell accumulation (P<0.05; Figure 4A and Online Figure IIIA). The scarce presence of pDC in mouse atherosclerotic lesions suggests that the increased T cell infiltration into the lesions most likely reflects peripheral modulation of T cell function. Indeed, blood and spleen T cell content was increased in pDC depleted mice (P<0.05; Figure 4B), suggesting that pDC interfere with T cell homeostasis. As pDC have been reported to induce regulatory T cell expansion,11,19 we investigated whether the increase in plaque CD3+ T cell content is owing to decreased Treg numbers or function and an associated failure to control T cell responses. However, we did not observe a difference in blood and spleen Treg numbers between 120G8 and GL113 treated LDLr−/− mice (Online Figure IIIB). In keeping, IL-10 plasma levels did not differ as well (Online Figure IIIC). To address whether the increase in T cells resulted from increased proliferation or survival, we examined T cell proliferation in vivo. Spleens of pDC depleted mice were enriched in BrdU+ CD4+ (P<0.05; Figure 4C) but not CD8+ T cells (Online Figure IIID). Moreover, T cell proliferation appeared to be antigen-dependent in that ovalbumin (OVA) challenge led to augmented proliferation of OT-2 CD4+ T cells (P<0.05; Figure 4D) but not OT-1 CD8+ T cells (Online Figure IIIE) in pDC depleted versus non-depleted mice. In addition, these findings also show that the increased proliferation is not due to intrinsic T cell effects, as both innate and administered T cell proliferation was increased, but to changes in the environment in pDC depleted mice. In parallel to increased T cell proliferation, IFN-γ (P<0.0005; Figure 4E) as well as IL-6 and MCP-1 plasma levels (Online Figure IIIF) were seen to be increased after pDC depletion. FACS sorted CD3+ T cells (purity 95%) from spleens of pDC depleted LDLr−/− mice tended to have increased IFN-γ expression (P=0.07; Figure 4F). In addition, CD3+ T cells isolated from atherosclerotic (high fat diet fed) LDLr−/− mice expressed higher levels of GATA-3 (Th2 marker) as well as of t-bet (Th1 marker) compared to T cells isolated from non-atherosclerotic mice (chow diet fed) mice (Online Figure IIIG). PDC depletion abrogated this effect for GATA-3 but not t-bet, suggesting that pDC might help to dampen the high fat diet induced Th1 shift in LDLr−/− mice. Overall, these data further substantiate a tolerogenic activity of pDC under atherogenic conditions, probably by suppressing T cell proliferation and function.
To address the underlying mechanism for the T cell suppressive capacity of pDC in atherosclerosis, we compared the expression of known key regulators of pDC tolerogenicity, such as indoleamine 2,3-dioxygenase (IDO), IL-10, programmed death ligand-1 (PD-L1) and inducible costimulator-ligand (ICOS-L), by pDC isolated from chow (non-atherosclerotic mice) versus high fat diet (atherosclerotic mice) fed LDLr−/− mice (purity 98.3%). Expression of PD-L1 and IDO (P<0.05; Figure 5A), but not IL-10 and ICOS-L (data not shown), was significantly elevated in pDC from high fat diet versus chow fed mice. Importantly, CD3+ T cells isolated from spleen of pDC depleted atherosclerotic mice, co-cultured with pDC in the presence of 1-methyl-trypthophan (1-MT), an IDO blocker, but not anti-PD-L1, displayed markedly induced T cell proliferation (P<0.05; Figure 5B), suggesting that pDC suppress T cell proliferation in an IDO dependent manner. The 1-MT induced CD4+ T cell mitogenic response was confirmed by flow cytometry (Figure 5C), showing a similar increment in CD4+ T cell proliferation after co-culture with pDC in the presence of 1-MT. Baseline levels of IFN-α in plasma of high fat diet versus chow fed LDLr−/− mice were unaltered (Figure 5D), as well as IFN-α expression by pDC (Figure 5E), firmly establishing that in mice pro-atherogenic conditions per se do not promote IFN-α release and immunogenic activity. Moreover, we extend these findings to the human context, as like in LDLr−/− mice, IFN-α plasma levels were also seen to be unchanged (Figure 5F) and IFN-α expression by pDC even significantly lowered in atherosclerotic patients versus healthy controls (P<0.05; Figure 5G). Altogether, our data indicate that both in mice and man, conditions of chronic atherosclerosis do not trigger pDC immunogenic activity, IFN-α release and ensuing T cell activation and proliferation. Rather, this milieu may even consolidate pDC’s innate tolerogenic capacity to suppress T cell proliferation.20
In this study, we are the first to demonstrate a contributory role for pDC in atherosclerosis. Despite the scarce presence of pDC in mouse atherosclerotic lesions, depletion of pDC in LDLr−/− mice by 120G8mAb aggravated atherosclerosis development and progression. Lesions of pDC depleted mice were characterized by increased T cell accumulation and a more unstable plaque phenotype, which, as we show, is likely linked to a deficiency in pDC associated epitope specific dampening of T cell response.
We demonstrate selective and almost complete pDC depletion by the use of 120G8 mAb in LDLr−/− mice. PDCA-1 expression was exclusively restricted to the pDC population and no other leukocyte subsets other than pDC were depleted. These findings confirm previous reports which highlight the specificity of the 120G8 mAb, all showing selective depletion of pDC in blood, bone marrow, LN, thymus and non-lymphoid organs of C57Bl6 mice but not of CD4/CD8 T cells, DX5+ NK en CD19+ B cells.21,22,23,24 Alternative pDC ablation or depletion models currently available such as IKAROS and IRF8 (mutant) all suffer from major effects on non-pDC subsets, while the CD11c.CRExE22f/- and the BDCA-2.hDTR mice are interesting new models for future ablation studies.
Our data point to an unexpected atheroprotective activity of pDC, which is in contrast to previous findings pointing towards a pro-atherogenic function.5 This notion was largely based on guilt by association, in that 1) plaques were seen to express CD123+ and IFN-α+ cells, in particular when progressed to an unstable phenotype, 2) CpG induced pDC activation in vitro led to type I IFN release, and 3) type I IFN were recently reported to contribute to atherosclerosis in ApoE−/− and LDLr−/− mice by stimulating macrophage recruitment.18 The data presented in this study justify a minor adjustment of this assumption. First, unlike BDCA-4, CD123 staining may not be entirely reflective of the plaque’s pDC content as macrophages and vSMCs appear to express this marker as well and as CD123+ cells often lack characteristic plasmacytoid morphology. This observation concurs with recent findings by van Vré et al., showing that CD123 is not a specific pDC marker staining also for endothelial cells in human atherosclerotic lesions.15 As a result, the actual plaque pDC content may not only be lower than originally envisioned, but also does not markedly increase with progression of disease. This also implies that pDC effects may be precipitated primarily in the periphery rather than within the plaque itself. Second, we show here that pDC are not the prime source of plasma IFN-α at baseline, and that IFN-α release by pDC into the circulation is boosted by CpG treatment, however not by atherogenic conditions. Apparently, atherogenic stimuli per se do not induce pDC activation. Moreover, in atherosclerotic mice, circulating IFN-α originates from other cell types than pDC but may be derived from macrophages. Third, we failed to demonstrate progressively increased expression of IFN-α (by micro-array or real-time PCR analysis) by circulating pDC from atherosclerotic mice and by human pDC from patients with stable versus unstable disease and by unstable versus stable endarterectomy lesions, confirming that in chronic inflammatory processes such as atherosclerosis TLR7/9 activation of pDC is not very prominent. Collectively, our data indicate that pDC exert their atheroprotective effect primarily by modulating extravascular immune responses.
Our studies also provide a plausible mechanism by which pDC suppress CD4+ T cell proliferation under conditions of atherosclerosis. PDC isolated from spleens from atherosclerotic mice had a 2-fold increase in expression of tolerogenic molecules IDO and PD-L1 compared to pDC isolated from non-atherosclerotic mice. IDO is an intracellular tryptophan catabolizing enzyme which has been attributed suppressive activity on cDCs and stimulatory activity on Tregs.25 PD-L1 is an inhibitory co-stimulatory molecule which interacts with programmed death-1 (PD-1) on CD8+ T cells to suppress their viability and activity.26
Moreover, co-culture of pDC with T cells in the presence of 1-MT, an IDO blocker, but not anti-PD-L1, showed increased T cell proliferation, suggesting that pDC suppress T cell proliferation in an IDO dependent manner. These observations correspond with previous reports in which pDC were shown to induce tolerance in other low grade chronic inflammatory and autoimmune diseases.9,10,25,26,27 The tolerogenic function of pDC was seen to depend on cytokine/ligand activation. For instance, B7-1 (CD80) engagement by Cytotoxic T-lymphocyte Antigen-4 (CTLA-4Ig), that of CD200R1 by CD200Ig and B7-1/B7-2 (CD80/CD86) by CD28Ig all have been shown to be able to induce the release of IDO by pDC, leading to the suppression of T cells.28 It remains to be established which activation pathway is involved in atherosclerosis. Thus, in analogy, during atherosclerosis pDC not only maintain their immature tolerogenic state, but even invigorate their inborn dampening activity so that they can control T cell activity. If the same also holds for brief episodes of fulminant plaque inflammation (acute myocardial infarction), remains to be established.
In conclusion, this manuscript is the first to unveil a protective role for pDC in an established mouse model of atherosclerosis, throughout disease progression. Given the virtual absence of pDC in the plaque itself, pDC most likely exert their activity extravascularly, by dampening T cell proliferation and function in an IDO dependent manner. While these findings identify pDC as an interesting new target for therapeutic intervention studies, they warrant further study to elucidate the actual pathways underlying the augmented tolerogenic activity of pDC under conditions of atherosclerosis.
What is known?
PDCs differ from classical conventional dendritic cells (cDCs) in that they are poor T cell activators and are critical for type I interferon responses. Although pDCs are present in human atherosclerotic lesions, their role in atherosclerosis has not been determined. Unexpectedly, we show thatpDCs are scarcely present in both human and mouse atherosclerotic lesions. However, pDC depletion by administration of 120G8 increase atherosclerosis development and progression in LDLr−/− mice. We found that pDC reductions of atherosclerosis we asscoaited with suppression of CD4+ T cell proliferation and activity via the release of tolerogenic molecule indoleamine 2,3-dioxygenase (IDO). This study is the first to demonstrate a protective role for pDCs in atherosclerosis. These findings identify pDCs as an interesting new target for therapeutic intervention studies in atherosclerosis.
We thank Dr. W. Buurman from Hycult Biotechnology for supplying us the 440c antibody, and Vigdis Bjerkeli and Cassandra Hennies for technical assistance.
Sources of funding
Supported by the Dutch Heart Foundation (NHS2005B294 to Dr. E. Biessen) and National Institute of Health/National Cancer Institute (Grant CA138617 to Dr. E. M. Janssen).
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