Identification of a non-canonical chemokine-receptor pathway suppressing regulatory T cells to drive atherosclerosis

CCL17 is produced by conventional dendritic cells, signals through CCR4 on regulatory T (Treg) cells and drives atherosclerosis by suppressing Treg functions through yet undefined mechanisms. Here we show that conventional dendritic cells from CCL17-deficient mice display a pro-tolerogenic phenotype and transcriptome that is not phenocopied in mice lacking its cognate receptor CCR4. In the plasma of CCL17-deficient mice, CCL3 was the only decreased cytokine/chemokine. We found that CCL17 signaled through CCR8 as an alternate high-affinity receptor, which induced CCL3 expression and suppressed Treg functions in the absence of CCR4. Genetic ablation of CCL3 and CCR8 in CD4+ T cells reduced CCL3 secretion, boosted FoxP3+ Treg numbers and limited atherosclerosis. Conversely, CCL3 administration exacerbated atherosclerosis and restrained Treg differentiation. In symptomatic versus asymptomatic human carotid atheroma, CCL3 expression was increased, whereas FoxP3 expression was reduced. Together, we identified a non-canonical chemokine pathway whereby CCL17 interacts with CCR8 to yield a CCL3-dependent suppression of atheroprotective Treg cells. Doring, van der Vorst, Yan, Neideck et al. present a non-canonical chemokine pathway involving CCL17 signaling through CCR8, which induces CCL3 expression independent of CCR4 and suppresses the functions of atheroprotective Treg cells

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity Evidence From Mouse and Human Studies

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2-or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/beta-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=1215) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/\CXCR4, which triggered Akt/WNT/beta-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis

Evaluation of the BDCA2-DTR Transgenic Mouse Model in Chronic and Acute Inflammation

Plasmacytoid dendritic cells (pDCs) are a small subset of dendritic cells and the main producers of type I interferons. Besides their contribution to tolerance, they are known to be involved in autoimmune diseases and have recently been implicated in atherosclerosis. However, their precise involvement, particularly in advanced lesion development, remains elusive. Hence, we investigated the role of pDCs in atherogenesis vs atheroprogression by specifically depleting this cell population using the BDCA2-DTR mouse model bred to Apolipoprotein E (Apoe-/-) deficient mice. Our results revealed that continuous diphtheria toxin-induced pDC depletion in Apoe-/- BDCA2-DTR mice receiving a high-fat diet (HFD) for 4 weeks did not alter lesion size or composition. Instead, these mice displayed increased B cell numbers and altered levels of inflammatory cytokines. Analysis of depletion efficiency showed that complete pDC depletion could only be sustained for one week and reoccurring pDCs sorted after 4 weeks did not express DTR anymore. Consequently, we analyzed lesion development in a model of partial carotid ligation, inducing established lesions after 5 weeks of HFD feeding, and only depleted pDCs during the last week of 5 weeks HFD feeding. Despite short-term, but efficient pDC depletion, we observed no differences in atherosclerotic lesion development, but changes in inflammatory cytokine titers. To assure the functionality of the BDCA2-DTR model in acute settings, we additionally examined the effect of pDC depletion in an indirect acute lung injury (iALI) model. This time, efficient pDC depletion resulted in a significantly reduced macrophage and neutrophil accumulation in the lung 12 hours after LPS challenge, underlining a pro-inflammatory role of pDCs in the innate immune response in iALI. Taken together, the BDCA2-DTR mouse model only allows efficient pDC depletion for one week, which subsequently restricts its usability to more acute but not chronic inflammatory disease model

Resolving Lipid Mediators Maresin 1 and Resolvin D2 Prevent Atheroprogression in Mice

Atheroprogression is a consequence of nonresolved inflammation, and currently a comprehensive overview of the mechanisms preventing resolution is missing. However, in acute inflammation, resolution is known to be orchestrated by a switch from inflammatory to resolving lipid mediators. Therefore, we hypothesized that lesional lipid mediator imbalance favors atheroprogression. To understand the lipid mediator balance during atheroprogression and to establish an interventional strategy based on the delivery of resolving lipid mediators. Aortic lipid mediator profiling of aortas from Apoe(-/-) mice fed a high-fat diet for 4 weeks, 8 weeks, or 4 months revealed an expansion of inflammatory lipid mediators, Leukotriene B4 and Prostaglandin E2, and a concomitant decrease of resolving lipid mediators, Resolvin D2 (RvD2) and Maresin 1 (MaR1), during advanced atherosclerosis. Functionally, aortic Leukotriene B4 and Prostaglandin E2 levels correlated with traits of plaque instability, whereas RvD2 and MaR1 levels correlated with the signs of plaque stability. In a therapeutic context, repetitive RvD2 and MaR1 delivery prevented atheroprogression as characterized by halted expansion of the necrotic core and accumulation of macrophages along with increased fibrous cap thickness and smooth muscle cell numbers. Mechanistically, RvD2 and MaR1 induced a shift in macrophage profile toward a reparative phenotype, which secondarily stimulated collagen synthesis in smooth muscle cells. We present evidence for the imbalance between inflammatory and resolving lipid mediators during atheroprogression. Delivery of RvD2 and MaR1 successfully prevented atheroprogression, suggesting that resolving lipid mediators potentially represent an innovative strategy to resolve arterial inflammatio

The BDCA2-DTR transgene does not influence pDC function in steady state and after 4 weeks HFD and DT injection.

<p>CD45⁺B220⁺440c⁺ pDCs were sorted from bone marrow of <i>Apoe</i>^<i>-/-</i> and <i>Apoe</i>^<i>-/-</i><i>BDCA2-DTR</i> mice (A, B) fed normal chow or (C, D) receiving a HFD and 3 times weekly DT injection for 4 weeks. PDCs were cultured in RPMI1640 +/- CpG (5 μg/ml) for 12 hours at 37°C and (A, C) Ifn-α levels (pg/ml) were analyzed in cell culture supernatants and (B, D) expression of costimulatory molecules CD86 and MHCII on pDCs was measured with FACS. (E) Representative histogramms of intra- and extracellular hDTR (anti-HB-EGF) expression in <i>Apoe</i>^{<i>−/−</i>} (dotted black), <i>Apoe</i>^{<i>−/−</i>}<i>BDCA2-DTR mice</i> (pDC depleted, red) after 4 weeks of DT administration (i.p., 3 times weekly) and <i>Apoe</i>^{<i>−/−</i>}<i>BDCA2-DTR</i> mice (green) without DT treatment. Graphs represent mean±SD; n = 5. Mann-Whitney test. *<i>P</i><0.05</p

Repeated DT administration efficiently depletes pDCs in spleen and bone marrow up to 1 week.

<p>(A) Injection scheme of DT administration. DT was injected once for 24 and 48 hours, 3 times for 1 week and 3 times weekly during 2 and 3 weeks. (B-E) Evaluation of pDC depletion efficiency for different time points: 24 hours, 48 hours, 1 week, 2 weeks and 3 weeks in <i>Apoe</i>^<i>-/-</i> (control) and <i>Apoe</i>^<i>-/-</i><i>BDCA2-DTR</i> mice (pDC depleted) after i.p. DT administration. Representative dot plots of pDC numbers in (B) spleen and (C) bone marrow and quantification of pDCs in (D) spleen and (E) bone marrow are depicted. PDCs are shown as CD45⁺B220⁺mPDCA1⁺440c⁺ cells in % of CD45⁺ leukocytes. Graphs represent mean±SD; n = 3 to n = 5. Mann-Whitney test <i>P</i><0.05 **<i>P</i><0.01 ***<i>P</i><0.001.</p