Search

Abstract Background Atherosclerosis, the main underlying pathology of cardiovascular disease, is a chronic inflammatory disease characterized by lipid accumulation and immune cell responses in the vascular wall, resulting in plaque formation. It is well-known that atherosclerosis prevalence and manifestation vary by sex. However, sexual dimorphism in the immune landscape of atherosclerotic plaques has up to date not been studied at high-resolution. In this study, we investigated sex-specific differences in atherosclerosis development and the immunological landscape of aortas at single-cell level in aged Ldlr −/− mice. Methods We compared plaque morphology between aged male and female chow diet-fed Ldlr −/− mice (22 months old) with histological analysis. Using single-cell RNA-sequencing and flow cytometry on CD45+ immune cells from aortas of aged Ldlr −/− mice, we explored the immune landscape in the atherosclerotic environment in males and females. Results We show that plaque volume is comparable in aged male and female mice, and that plaques in aged female mice contain more collagen and cholesterol crystals, but less necrotic core and macrophage content compared to males. We reveal increased immune cell infiltration in female aortas and found that expression of pro-atherogenic markers and inflammatory signaling pathways was enriched in plaque immune cells of female mice. Particularly, female aortas show enhanced activation of B cells (Egr1, Cd83, Cd180), including age-associated B cells, in addition to an increased M1/M2 macrophage ratio, where Il1b + M1-like macrophages display a more pro-inflammatory phenotype (Nlrp3, Cxcl2, Mmp9) compared to males. In contrast, increased numbers of age-associated Gzmk+CD8+ T cells, dendritic cells, and Trem2 + macrophages were observed in male aortas. Conclusions Altogether, our findings highlight that sex is a variable that contributes to immunological differences in the atherosclerotic plaque environment in mice and provide valuable insights for further preclinical studies into the impact of sex on the pathophysiology of atherosclerosis.

Abstract Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4 (LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4 biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr−/− mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+ myeloid cells was observed, including Ly6Clo and Ly6Chi monocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression.

BackgroundInterleukin‐23 (IL‐23) has been implicated in inflammatory and autoimmune diseases by skewing CD4+ T helper cells towards a pathogenic Th17 phenotype. In this study we investigated the presence of IL‐23 receptor (IL‐23R)‐expressing cells in the atherosclerotic aorta and evaluated the effect of IL‐23R deficiency on atherosclerosis development in mice. Methods and ResultsWe used heterozygous Ldlr−/−Il23reGFP/WT knock‐in mice to identify IL‐23R‐expressing cells by flow cytometry and homozygous Ldlr−/−Il23reGFP/eGFP (Ldlr−/− Il23r−/−) mice to investigate the effect of lack of IL‐23R in atherosclerosis. We demonstrate the presence of relatively rare IL‐23R‐expressing cells in lymphoid tissue and aorta (≈0.1–1% IL23R+ cells of all CD45+ leukocytes). After 10 weeks on a high‐fat diet, production of IL‐17, but not interferon‐γ, by CD4+ T cells and other lymphocytes was reduced in Ldlr−/−Il23r−/− compared with Ldlr−/−controls. However, Ldlr−/− and Ldlr−/−Il23r−/− mice had equivalent amounts of aortic sinus and descending aorta lesions. Adoptive transfer of IL‐23R‐deficient CD4+ T cells to lymphopenic Ldlr−/−Rag1−/− resulted in dramatically reduced IL‐17‐producing T cells but did not reduce atherosclerosis, compared with transfer of IL‐23R‐sufficient CD4+ T cells. ConclusionsThese data demonstrate that loss of IL‐23R does not affect development of experimental atherosclerosis in LDLr‐deficient mice, despite a role for IL‐23 in differentiation of IL‐17‐producing T cells.