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+
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.CREx
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+
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−/−
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.