Myeloid cell-specific Irf5 deficiency stabilizes atherosclerotic plaques in Apoe mice

Abstract

Objective Interferon regulatory factor (IRF) 5 is a transcription factor known for promoting M1 type macrophage polarization in vitro . Given the central role of inflammatory macrophages in promoting atherosclerotic plaque progression, we hypothesize that myeloid cell-specific deletion of IRF5 is protective against atherosclerosis. Methods Female Apoe –/– Lysm Cre/+ Irf5 fl/fl and Apoe −/− Irf5 fl/fl mice were fed a high-cholesterol diet for three months. Atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependent bone marrow-derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Mixed bone marrow chimeras were generated to determine intrinsic IRF5-dependent effects on macrophage accumulation in atherosclerotic plaques. Results Myeloid cell-specific Irf5 deficiency blunted LPS/IFNγ-induced inflammatory gene expression in vitro and in the atherosclerotic aorta in vivo . While atherosclerotic lesion size was not reduced in myeloid cell-specific Irf5 -deficient Apoe –/– mice, plaque composition was favorably altered, resembling a stable plaque phenotype with reduced macrophage and lipid contents, reduced inflammatory gene expression and increased collagen deposition alongside elevated Mertk and Tgfβ expression. Irf5- deficient macrophages, when directly competing with wild type macrophages in the same mouse, were less prone to accumulate in atherosclerotic lesion, independent of monocyte recruitment. Irf5 -deficient monocytes, when exposed to oxidized low density lipoprotein, were less likely to differentiate into macrophage foam cells, and Irf5 -deficient macrophages proliferated less in the plaque. Conclusion Our study provides genetic evidence that selectively altering macrophage polarization induces a stable plaque phenotype in mice.

Therapeutic strategies targeting inflammation and immunity in atherosclerosis: how to proceed?

“…In mouse models of CVD, global or myeloid-specific IRF5 deficiency reduced atherosclerosis and improved plaque stability 204 , 205 , and IRF5 inhibition with nanoparticles decreased myocardial infarct size 206 . The transcription factor IRF5 induces a pro-inflammatory phenotype in mouse and human macrophages 207 .…”

Macrophages in Atheromatous Plaque Developmental Stages

“…The traditional view was Ly6C high monocytes differentiate into macrophages with M1 type features during the progression of atherosclerosis, releasing pro-inflammatory cytokines (TNF-α, IL-12, IL-6) and reactive oxygen species, while differentiation into alternatively activated M2 type macrophages facilitates plaque regression ( 74 ). Recent single cell analyses of atheromatous plaque immune cells using mass cytometry and RNA sequencing refuted the dichotomous M1/M2 classification and instead identified macrophage subsets with mixed phenotypes specialized in inflammation, lipid handling, and homeostasis ( 26 , 28 , 30 ). For example, in mice the expression of transcription factor Interferon regulatory factor 5 (IRF 5), mediating classical M1 type macrophage polarization in vitro , is not restricted to the inflammatory macrophage subpopulation in the atherosclerotic aorta ( 30 ).…”

“…Recent single cell analyses of atheromatous plaque immune cells using mass cytometry and RNA sequencing refuted the dichotomous M1/M2 classification and instead identified macrophage subsets with mixed phenotypes specialized in inflammation, lipid handling, and homeostasis ( 26 , 28 , 30 ). For example, in mice the expression of transcription factor Interferon regulatory factor 5 (IRF 5), mediating classical M1 type macrophage polarization in vitro , is not restricted to the inflammatory macrophage subpopulation in the atherosclerotic aorta ( 30 ). Loss of Irf5 in myeloid cells limits lipid and macrophage accumulation in the plaque, decreases IL-12 and increases TGFβ, MerTK, and CD206 expression, promoting a stable plaque phenotype ( 30 , 75 , 76 ).…”

Platelet Bone Morphogenetic Protein-4 Mediates Vascular Inflammation and Neointima Formation after Arterial Injury

“…The cells were washed in PBS, Fc-Receptors were blocked with an antiCD16/CD32 antibody, and the cells were incubated with the indicated antibodies (all from eBioscience, Thermo Fisher Scientific, Waltham, MA, USA, 1:200) before quantification on a flow cytometer (BD FACS Canto II, BD, San Diego, CA, USA). The gating strategy for the monocyte and neutrophil granulocyte populations were based on previous protocol by Leipner et al [14]. Specifically, the monocyte population was identified as CD45 + CD11b + CD115 + Ly6C high/low and the platelet-monocyte complex (PMC) as the CD41 + subgroup [15][16][17].…”

“…Specifically, the monocyte population was identified as CD45 + CD11b + CD115 + Ly6C high/low and the platelet-monocyte complex (PMC) as the CD41 + subgroup [15][16][17]. The neutrophil granulocyte population was characterized as CD45 + CD11b + CD115 -Ly6C intermediate based on previous protocol by Leipner et al [14] and the platelet-neutrophil complex (PNC) as the CD41 + subgroup [15][16][17]. For the measurement of levels of P-selectin and active αIIbß3, resting or thrombin activated platelets were incubated with a FITC-labelled anti-P-selectin antibody (BD Bioscience, San Diego, CA, USA) or Oregon Green 488-labelled fibrinogen (Thermo-Fisher Scientific, Karlsruhe, Germany) for 30 min at room temperature.…”