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Aims: CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice., Method and Results: Cd40flox/flox and LysM-cre Cd40flox/flox mice on an Apoe-/- background were generated (CD40wt and CD40mac-/-, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced in CD40mac-/- compared to CD40wt mice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of the CD40mac-/- atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b- macrophages in the atherosclerotic aorta of CD40mac-/- compared to CD40wt mice. RNA-sequencing of bone marrow-derived macrophages of CD40mac-/- mice demonstrated upregulation of genes associated with alternatively activated macrophages (including Folr2, Thbs1, Sdc1, and Tns1)., Conclusions: We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis., Competing Interests: Conflicts of interest: C.W., D.A., E.L. and N.G. are supported by the Deutsche Forschungs Gemeinschaft (grants CRC1123, A5, SFB 1123, TRR259, SFB1116). E.L is also supported by the Netherlands CardioVascular Research Initiative, the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development and the Royal Netherlands Academy of Sciences’ for the GENIUS project ‘Generating the best evidence-based pharmaceutical targets for atherosclerosis’ (CVON2011-19), and the ERC Con grant (CD40-INN). E.L. is also the vice chair at the ESC working group on atherosclerosis, serves on the EAS programme committee and the ATVB awards and programme committee and has received payment or honoraria for lectures at Novartis and Novo Nordisk. The remaining authors have nothing to disclose., (© The Author(s) 2022. 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.