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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., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology.)

Macrophages are integral to the pathogenesis of atherosclerosis, but the contribution of distinct macrophage subsets to disease remains poorly defined. Using single cell technologies and conditional ablation via a LysM Cre+ Clec4a2 flox/DTR mouse strain, we demonstrate that the expression of the C-type lectin receptor CLEC4A2 is a distinguishing feature of vascular resident macrophages endowed with athero-protective properties. Through genetic deletion and competitive bone marrow chimera experiments, we identify CLEC4A2 as an intrinsic regulator of macrophage tissue adaptation by promoting a bias in monocyte-to-macrophage in situ differentiation towards colony stimulating factor 1 (CSF1) in vascular health and disease. During atherogenesis, CLEC4A2 deficiency results in loss of resident vascular macrophages and their homeostatic properties causing dysfunctional cholesterol metabolism and enhanced toll-like receptor triggering, exacerbating disease. Our study demonstrates that CLEC4A2 licenses monocytes to join the vascular resident macrophage pool, and that CLEC4A2-mediated macrophage homeostasis is critical to combat cardiovascular disease., (© 2022. The Author(s).)

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Aims: Aortic adaptive immunity plays a role in atherosclerosis; however, the precise mechanisms leading to T-cell activation in the arterial wall remain poorly understood., Methods and Results: Here, we have identified naïve T cells in the aorta of wild-type and T-cell receptor transgenic mice and we demonstrate that naïve T cells can be primed directly in the vessel wall with both kinetics and frequency of T-cell activation found to be similar to splenic and lymphoid T cells. Aortic homing of naïve T cells is regulated at least in part by the P-selectin glycosylated ligand-1 receptor. In experimental atherosclerosis the aorta supports CD4+ T-cell activation selectively driving Th1 polarization. By contrast, secondary lymphoid organs display Treg expansion., Conclusion: Our results demonstrate that the aorta can support T-cell priming and that naïve T cells traffic between the circulation and vessel wall. These data underpin the paradigm that local priming of T cells specific for plaque antigens contributes to atherosclerosis progression., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Aims: Atherosclerosis is characterized 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 cell (ILC) 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 toward inflammatory monocyte-macrophage populations rather than resident macrophage phenotypes and cDC2 during atherogenesis.

Background: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions., Methods: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE -/- ) mice and ApoE -/- mice with a genetic deletion of IRF5 (ApoE -/- Irf5 -/- ) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation., Results: Both lesion and necrotic core size were significantly reduced in ApoE -/- Irf5 -/- mice compared with IRF5-competent ApoE -/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c + macrophages was evident in ApoE -/- Irf5 -/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c + macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c - macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-β3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ., Conclusions: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c + macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis., (© 2017 The Authors.)

Atherosclerosis is the major cause of cardiovascular disease (CVD), the leading cause of death worldwide. Despite much focus on lipid abnormalities in atherosclerosis, it is clear that the immune system also has important pro- and antiatherogenic functions. The enzyme indoleamine-2,3-dioxygenase (IDO) catalyses degradation of the essential amino acid tryptophan into immunomodulatory metabolites. How IDO deficiency affects immune responses during atherogenesis is unknown and we explored potential mechanisms in models of murine and human atherosclerosis. IDO deficiency in hypercholesterolemic ApoE(-/-) mice caused a significant increase in lesion size and surrogate markers of plaque vulnerability. No significant changes in cholesterol levels were observed but decreases in IL-10 production were found in the peripheral blood, spleen and lymph node B cells of IDO-deficient compared with IDO-competent ApoE(-/-) mice. 3,4,-Dimethoxycinnamoyl anthranilic acid (3,4-DAA), an orally active synthetic derivative of the tryptophan metabolite anthranilic acid, but not l-kynurenine, enhanced production of IL-10 in cultured splenic B cells. Finally, 3,4-DAA treatment reduced lesion formation and inflammation after collar-induced arterial injury in ApoE(-/-) mice, and reduced cytokine and chemokine production in ex vivo human atheroma cell cultures. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation. We show that the IDO pathway induces production of IL-10 in B cells in vivo and in vitro, suggesting that IDO may induce immunoregulatory functions of B cells in atherosclerosis. The favorable effects of anthranilic acid derivatives in atherosclerosis indicate a novel approach toward therapy of CVD.

Seminal research over the past 20 years has revealed atherosclerosis to be a chronic inflammatory process that shares features with traditional inflammatory diseases including rheumatoid arthritis. More recently, emphasis has been placed on the role of innate immunity in the development and progression of atherosclerosis. In particular, pattern recognition receptors, including Toll-like receptors (TLRs), have been the focus of much attention as modulators of atherogenesis. This review provides an update on the developments in this area of research in the past 2 years, with a specific focus on the current controversies and how these may affect the design of therapeutics. Specifically, we will address the recent evidence that TLRs elicit both protective and detrimental effects in atherosclerosis and the emerging observation that the outcome of TLR signaling is dependent on the agonist and responding cell type., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Atherosclerosis is an inflammatory disease with a strong involvement of innate immunity. Toll-like receptors (TLRs) are the best-characterized pattern recognition receptors of the innate immune system. Almost all cell types in lesions, inflammatory leukocytes and resident vascular cells alike express TLRs. TLRs are able to sense modified lipids, enhance foam cell formation, induce leukocyte recruitment, and increase cytokine and matrix metalloproteinase production within atherosclerotic lesions. As such, TLRs represent an important link between atheroma and inflammation, making them attractive targets for the treatment of cardiovascular disease. Novel TLR-specific biologics are being developed and tested in other inflammatory diseases. This article will describe the exciting potential of TLRs as therapeutic targets for the treatment of atherosclerosis and will also highlight the potential challenges in the clinical application of TLR-based therapeutics in cardiovascular disease.

Inflammation drives atherosclerosis. Both immune and resident vascular cell types are involved in the development of atherosclerotic lesions. The phenotype and function of these cells are key in determining the development of lesions. Toll-like receptors are the most characterised innate immune receptors and are responsible for the recognition of exogenous conserved motifs on pathogens, and, potentially, some endogenous molecules. Both endogenous and exogenous TLR agonists may be present in atherosclerotic plaques. Engagement of toll-like receptors on immune and resident vascular cells can affect atherogenesis as signalling downstream of these receptors can elicit proinflammatory cytokine release, lipid uptake, and foam cell formation and activate cells of the adaptive immune system. In this paper, we will describe the expression of TLRs on immune and resident vascular cells, highlight the TLR ligands that may act through TLRs on these cells, and discuss the consequences of TLR activation in atherosclerosis.

Macrophages, a significant component of atherosclerotic plaques vulnerable to acute complications, can be pro-inflammatory (designated M1), regulatory (M2), lipid- (Mox) or Heme-induced (Mhem). We showed previously that low (LSS) and oscillatory (OSS) shear stress cause thin-cap fibroatheroma and stable smooth muscle cell-rich plaque formation respectively in ApoE-knockout (ApoE −/− ) mice. Here we investigated whether different shear stress conditions relate to specific changes in macrophage polarization and plaque morphology by applying a shear stress-altering cast to the carotid arteries of high fat-fed ApoE −/− mice. The M1 markers iNOS and IRF5 were highly expressed in macrophage-rich areas of LSS lesions compared to OSS lesions 6 weeks after cast placement, while the M2 marker Arginase-1, and Mox/Mhem markers HO-1 and CD163 were elevated in OSS lesions. Our data indicates shear stress could be an important determinant of macrophage polarization in atherosclerosis, with low shear promoting M1 programming. [ABSTRACT FROM AUTHOR]