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

Jennifer E Cole,Claudia Monaco,Christina Kassiteridi,Pasquale Maffia,David J Ahern,Michael E Goddard,Inhye Park,Dina Danso Abeam,Patricia Green

doi:10.1093/cvr/cvy109

Abstract

AimsAtherosclerosis 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 resultsApolipoprotein 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.ConclusionsA 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.

Highlights

Inflammation underlies the pathogenesis of atherosclerosis, the most common cause of cardiovascular disease (CVD)[1]
In addition to a small population of plasmacytoid dendritic cells (SiglecHþB220þ), two subsets of conventional dendritic cells were found. cDC1 cells were CD11blowCD11cþMHCIIhiCD172a-CD103þ whereas cDC2 cells (CD11bþCD11cþMHCIIhiCD172aþ) expressed CD172a and higher levels of CD11b
We showed that when ApoE-/- mice are fed a high fat diet, cDC1 remain unchanged whereas cDC2 arterial content is decreased and plasmacytoid dendritic cells (pDC) are significantly increased

Summary

Introduction

Inflammation underlies the pathogenesis of atherosclerosis, the most common cause of cardiovascular disease (CVD)[1]. Mapping immune cell subsets and their activation in CVD will allow discrimination between pathogenic and reparative subtypes, aiding selective diagnostic and therapeutic approaches and uncoupling of homeostatic and tissue damaging cell function. Development of atherosclerosis is defined by the presence of mononuclear phagocytes in the subendothelial space. Monocytes in the circulation adhere to the activated endothelium of arteries and migrate into the subendothelial space where they differentiate into macrophages under the effects of growth factors. Macrophages, play critical roles in the progression and regression of atherosclerosis including lipid uptake, antigen presentation, dead cell removal and cytokine production and atherosclerotic mice lacking macrophages are protected from disease development[4,5,6]. Tissue resident macrophage populations are present in the vessel wall[7]

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