Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice.

Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12-/-Apoe-/-) mice. Compared to F12+/+Apoe-/- controls, atherosclerotic lesion formation was reduced in F12-/-Apoe-/- mice. This was associated with a decrease in serum interleukin (IL)-1ß and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12-/-Apoe-/- mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1ß, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4+ T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.

Deficiency of HIF1α in Antigen-Presenting Cells Aggravates Atherosclerosis and Type 1 T-Helper Cell Responses in Mice.

OBJECTIVE: Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)-mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1α in APCs in atherosclerosis. APPROACH AND RESULTS: We found upregulated HIF1α expression in CD11c(+) APCs within atherosclerotic plaques of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Conditional deletion of Hif1a in CD11c(+) APCs in high-fat diet-fed Ldlr(-/-) mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1α directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1α-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr(-/-) mice. Notably, deletion of Hif1a in LysM(+) bone marrow cells in Ldlr(-/-) mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1α, STAT3, and interleukin-12 protein were found to colocalize with APCs. CONCLUSIONS: Our findings identify HIF1α to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr(-/-) mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.

CD8+ T Cells Regulate Monopoiesis and Circulating Ly6C-high Monocyte Levels in Atherosclerosis in Mice.

RATIONALE: Proinflammatory adaptive immune responses are recognized as major drivers of atherosclerotic lesion formation. Although CD8(+) T cells have recently been proposed as a proatherogenic cell subset, their full scope of actions remains to be elucidated. OBJECTIVE: We here addressed the contribution of CD8(+) T cells to monocyte trafficking in atherosclerosis. METHOD AND RESULTS: We observed that CD8(+) T cells express proinflammatory cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-12) within atherosclerotic lesions and spleens of high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Antibody-mediated CD8(+) T-cell depletion in high-fat diet-fed Ldlr(-/-) mice decreased atherosclerotic plaque formation, associated with decreased macrophage accumulation within lesions. Despite a reduction in vascular chemokine (CC-motif) ligand 2 and chemokine (CXC-motif) ligand 1 expression, CD8(+) T-cell depletion did not directly affect monocyte recruitment to inflamed vessels. However, CD8(+) T-cell depletion decreased chemokine (CC-motif) ligand serum concentrations and circulating Ly6C(high) monocyte counts. We further evidenced that CD8(+) T-cell depletion decreased levels of mature monocytes and myeloid granulocyte-monocyte progenitors in the bone marrow and spleen of hypercholesterolemic mice, effects that were partially reproduced by interferon-γ neutralization, showing a role for interferon-γ. CONCLUSIONS: These data suggest that CD8(+) T cells promote atherosclerosis by controlling monopoiesis and circulating monocyte levels, which ultimately contributes to plaque macrophage burden without affecting direct monocyte recruitment, identifying this cell subset as a critical regulator of proatherogenic innate immune cell responses in atherosclerosis.

Programmed cell death-1 deficiency exacerbates T cell activation and atherogenesis despite expansion of regulatory T cells in atherosclerosis-prone mice.

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3(+) regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4(+) T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr(-/-)Pd1(-/-)) displayed striking increases in systemic CD4(+) and CD8(+) T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3+ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr(-/-)Pd1(-/-) mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr(-/-) mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.

CCR6 selectively promotes monocyte mediated inflammation and atherogenesis in mice.

The chemokine receptor CCR6 is expressed by various cell subsets implicated in atherogenesis, such as monocytes, Th17 and regulatory T cells. In order to further define the role of CCR6 in atherosclerosis, CCR6-deficient (Ccr6-/-) mice were crossed with low-density lipoprotein receptor-deficient (Ldlr-/-) mice to generate atherosclerosis-prone mice deficient in CCR6. Compared to Ldlr-/- controls, atherosclerotic burden in the aortic sinus and aorta were reduced in Ccr6-/-Ldlr-/- mice fed a high fat diet, associated with a profound depression in lesional macrophage accumulation. Local and systemic distributions of T cells, including frequencies of Th1, Th17 and regulatory T cells were unaltered. In contrast, circulating counts of both Gr-1(high) and Gr1(low) monocytes were reduced in Ccr6-/-Ldlr-/- mice. Moreover, CCR6 was revealed to promote monocyte adhesion to inflamed endothelium in vitro and leukocyte adhesion to carotid arteries in vivo. Finally, CCR6 selectively recruited monocytes but not T cells in an acute inflammatory air pouch model. We here show that CCR6 functions on multiple levels and regulates the mobilisation, adhesion and recruitment of monocytes/macrophages to the inflamed vessel, thereby promoting atherosclerosis, but is dispensable for hypercholesterolaemia-associated adaptive immune priming. Targeting CCR6 or its ligand CCL20 may therefore be a promising therapeutic strategy to alleviate atherosclerosis.

Hematopoietic interferon regulatory factor 8-deficiency accelerates atherosclerosis in mice.

OBJECTIVE: Inflammatory leukocyte accumulation drives atherosclerosis. Although monocytes/macrophages and polymorphonuclear neutrophilic leukocytes (PMN) contribute to lesion formation, sequelae of myeloproliferative disease remain to be elucidated. METHODS AND RESULTS: We used mice deficient in interferon regulatory factor 8 (IRF8(-/-)) in hematopoietic cells that develop a chronic myelogenous leukemia-like phenotype. Apolipoprotein E-deficient mice reconstituted with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow displayed an exacerbated atherosclerotic lesion formation compared with controls. The chronic myelogenous leukemia-like phenotype in mice with IRF8(-/-) bone marrow, reflected by an expansion of PMN in the circulation, was associated with an increased lesional accumulation and apoptosis of PMN, and enlarged necrotic cores. IRF8(-/-) compared with IRF8(+/+) PMN displayed unaffected reactive oxygen species formation and discharge of PMN granule components. In contrast, accumulating in equal numbers at sites of inflammation, IRF8(-/-) macrophages were defective in efferocytosis, lipid uptake, and interleukin-10 cytokine production. Importantly, depletion of PMN in low-density lipoprotein receptor or apolipoprotein E-deficient mice with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow abrogated increased lesion formation. CONCLUSIONS: These findings indicate that a chronic myelogenous leukemia-like phenotype contributes to accelerated atherosclerosis in mice. Among proatherosclerotic effects of other cell types, this, in part, is linked to an expansion of functionally intact PMN.