Toll-like receptors in atherosclerosis: a 'Pandora's box' of advances and controversies

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Immune cell census in murine atherosclerosis: cytometry by time of flight illuminates vascular myeloid cell diversity

Aims: Atherosclerosis is characterised by the abundant infiltration of myeloid cells starting at early stages of disease. Myeloid cells are key players in vascular immunity during atherogenesis. However, the subsets of vascular myeloid cells have eluded resolution due to shared marker expression and atypical heterogeneity in vascular tissues. We applied the high-dimensionality of mass cytometry to the study of myeloid cell subsets in atherosclerosis. Methods and Results: Apolipoprotein E-deficient (ApoE-/-) mice were fed a chow or a high fat (western) diet for 12 weeks. Single cell aortic preparations were probed with a panel of 35 metal-conjugated antibodies using Cytometry by time of flight (CyTOF). Clustering of marker expression on live CD45+ cells from the aortas of ApoE-/- mice identified 13 broad populations of leucocytes. Monocyte, macrophage, type 1 and type 2 conventional dendritic cell (cDC1 and cDC2), plasmacytoid dendritic cell (pDC), neutrophil, eosinophil, B cell, CD4+ and CD8+ T cell, γδ T cell, natural killer (NK) cell and innate lymphoid (ILC) cell populations accounted for approximately 95% of the live CD45+ aortic cells. Automated clustering algorithms applied to the Lin-CD11blo-hi cells revealed 20 clusters of myeloid cells. Comparison between chow and high fat fed animals revealed increases in monocytes (both Ly6C+ and Ly6C-), pDC and a CD11c+ macrophage subset with high fat feeding. Concomitantly, the proportions of CD206+ CD169+ subsets of macrophages were significantly reduced as were cDC2. Conclusions: A CyTOF-based comprehensive mapping of the immune cell subsets within atherosclerotic aortas from ApoE-/- mice offers tools for myeloid cell discrimination within the vascular compartment and it reveals that high fat feeding skews the myeloid cell repertoire towards inflammatory monocyte-macrophage populations rather than resident macrophage phenotypes and cDC2 during atherogenesis

Myeloid cell regulation by Cd200 signalling in atherosclerosis

Atherosclerosis, the major risk factor for cardiovascular disease and the leading cause of death worldwide, is a multifactorial chronic inflammatory disease. Deletion of CD200 in vivo increases myeloid cell numbers and activation resulting in enhanced susceptibility to autoimmune diseases and infection. However, the importance of CD200 in atherosclerosis development is still unknown. To understand the role of CD200 signalling, both the effect of CD200 deletion and provision were assessed in a murine model of atherosclerosis. Firstly, the role of CD200R ligation was examined in a murine model of carotid injury. Apolipoprotein E deficient (ApoE -/-) mice underwent surgery for placement of a perivascular collar and were treated with 10mg/kg of a CD200-Fc fusion protein. Three weeks post injury, carotid arteries were removed and neointima formation was assessed. CD200-Fc fusion protein treatment attenuated neointima development and increased M2 anti-inflammatory macrophage accumulation. Secondly, CD200-deficient (CD200-/-) mice were crossed with ApoE-/- mice. CD200 deficiency accelerated advanced atherosclerotic lesion formation in the aortic roots, as shown by the morphometric measurement of aortic root atherosclerotic lesion development. Moreover, CD200 regulated hypercholesterolemia-induced monocytosis, as its deletion increased the numbers of CCR2-expressing Ly6Chi monocytes in the blood and in the plaques. To further understand the mechanism of CD200-mediated monocytosis in the plaques, the role of the haematopoietic system was assessed by flow cytometry and in vitro culture systems. CD200 deficiency was shown to regulate GM-CSF-mediated myelopoiesis. CD200 does not only affect myeloid cells in the blood, but also in the spleen and in the aorta. In the plaques, CD200 deficiency skewed the phenotype of macrophages towards iNOS+CD86+MHCII+ inflammatory macrophages and enhanced CD4+ T cell activation. Therefore, CD200 regulates macrophage phenotypes and possibly their antigen presenting abilities at advanced stages of atherosclerosis. Finally, a highly multiparametric technique (CyTOF) enabled the detection of CD200 predominantly in stromal cells in the plaques suggesting a key role for stromal-immune cell interactions in CD200-mediated athero-protection. Collectively, these findings identify a new role for CD200 expression in modulating myeloid cell function and phenotype in advanced stages of atherosclerosis.</p

Myeloid cell regulation by Cd200 signalling in atherosclerosis

Atherosclerosis, the major risk factor for cardiovascular disease and the leading cause of death worldwide, is a multifactorial chronic inflammatory disease. Deletion of CD200 in vivo increases myeloid cell numbers and activation resulting in enhanced susceptibility to autoimmune diseases and infection. However, the importance of CD200 in atherosclerosis development is still unknown. To understand the role of CD200 signalling, both the effect of CD200 deletion and provision were assessed in a murine model of atherosclerosis. Firstly, the role of CD200R ligation was examined in a murine model of carotid injury. Apolipoprotein E deficient (ApoE -/-) mice underwent surgery for placement of a perivascular collar and were treated with 10mg/kg of a CD200-Fc fusion protein. Three weeks post injury, carotid arteries were removed and neointima formation was assessed. CD200-Fc fusion protein treatment attenuated neointima development and increased M2 anti-inflammatory macrophage accumulation. Secondly, CD200-deficient (CD200-/-) mice were crossed with ApoE-/- mice. CD200 deficiency accelerated advanced atherosclerotic lesion formation in the aortic roots, as shown by the morphometric measurement of aortic root atherosclerotic lesion development. Moreover, CD200 regulated hypercholesterolemia-induced monocytosis, as its deletion increased the numbers of CCR2-expressing Ly6Chi monocytes in the blood and in the plaques. To further understand the mechanism of CD200-mediated monocytosis in the plaques, the role of the haematopoietic system was assessed by flow cytometry and in vitro culture systems. CD200 deficiency was shown to regulate GM-CSF-mediated myelopoiesis. CD200 does not only affect myeloid cells in the blood, but also in the spleen and in the aorta. In the plaques, CD200 deficiency skewed the phenotype of macrophages towards iNOS+CD86+MHCII+ inflammatory macrophages and enhanced CD4+ T cell activation. Therefore, CD200 regulates macrophage phenotypes and possibly their antigen presenting abilities at advanced stages of atherosclerosis. Finally, a highly multiparametric technique (CyTOF) enabled the detection of CD200 predominantly in stromal cells in the plaques suggesting a key role for stromal-immune cell interactions in CD200-mediated athero-protection. Collectively, these findings identify a new role for CD200 expression in modulating myeloid cell function and phenotype in advanced stages of atherosclerosis.</p

Toll-Like Receptors in Atherosclerosis

Atherosclerosis, the leading cause of cardiovascular disease (CVD), is driven by inflammation. Increasing evidence suggests that toll-like receptors (TLRs) are key orchestrators of the atherosclerotic disease process. Interestingly, a distinct picture is being revealed for individual receptors in atherosclerosis. TLRs exhibit a complex nature enabling the detection of multiple motifs named danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Activation of these receptors triggers an intracellular signalling cascade mediated through MyD88 or TRIF, leading to the production of pro- and anti-inflammatory cytokines. In this review we explore key novel findings pertaining to TLR signalling in atherosclerosis, including recently described endosomal TLRs and future directions in TLR research

Interferon regulatory factor-5-dependent CD11c+ macrophages contribute to the formation of rupture-prone atherosclerotic plaques

AIMS: Inflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability.METHODS AND RESULTS: Bulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE-/- mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE-/-Irf5-/- mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts.CONCLUSION: Using complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture.KEY QUESTION: The transcription factor interferon regulatory factor-5 (IRF5) is a master regulator of macrophage activation that has been shown to have a role in murine atherogenesis. Its role in human atherosclerosis and its complications is unknown.KEY FINDING: Interferon regulatory factor-5 is linked to plaque vulnerability and symptoms in human carotid endarterectomies. In a murine model of inducible carotid artery plaque rupture, IRF5 drives plaque rupture. Interferon regulatory factor-5 modulates macrophage phenotype and it colocalises with CD11c+ macrophages at the plaque shoulder.TAKE-HOME MESSAGE: We demonstrate a mechanistic link between the IRF5, plaque macrophages, and plaque vulnerability to rupture. Interferon regulatory factor-5 is a potential candidate therapeutic target in human atherosclerosis