Project 1: Regulation of CD9+ Monocyte & Macrophage Immune Functions in Atherosclerosis

Project 1 Summary In the last cycle of this PPG, we identified eight subsets of human monocytes in healthy individuals using high dimensional mass cytometry. These subsets have now been confirmed in other studies. We discovered that some monocyte subsets expressed the tetraspanin CD9, and that these CD9+ monocyte subsets positively correlated with cardiovascular disease (CAD) severity in humans. The tetraspanin CD9 facilitates the organization and clustering of receptors within membranes. CD9 regulates MHCII trafficking in myeloid cells, which is critical for T cell activation, and aids in macrophage uptake of oxLDL by organizing plasma membrane clustering of the scavenger receptor CD36. Thus, CD9 location in membranes allows for the modulation of immune receptor activity. Functional pathway analysis of CD9+ monocytes revealed induction of genes involved in inflammation, leukocyte migration, and complement activation pathways. Although such characteristics would be important for the homeostatic functions of monocytes in clearing viruses, tumors, and damaged host cells, we surmise that these functions could instead become deleterious in the vessel wall, and promote atherosclerosis. We also have recent evidence to suggest that CD9+ monocytes give rise to CD9+ macrophages in the aortic wall. Here, we hypothesize that immunomodulatory CD9+ monocytes and CD9+ macrophages promote atherosclerosis progression by driving a pro-inflammatory cytokine program, regulating plaque macrophage lipid content, and interacting with T cells and B cells to drive plaque progression. We will test this hypothesis in two specific aims, where we study both humans with CAD and mouse models of atherosclerosis. Aim 1 will functionally study CD9+ monocyte subsets in human subjects from well-characterized clinical cohorts, who have clinically-documented low and high CAD, and in subjects who are participating in a longitudinal study of plaque progression. Aim 2 will mechanistically study the functions and metabolism of CD9+ macrophage subsets, and will directly test the role of monocyte and macrophage CD9 expression in regulating atherosclerosis progression. We will utilize unique monocyte- specific mouse models (mice lacking nonclassical monocytes (E2)) and mice lacking classical monocytes (Ccr2-/-) to functionally examine how CD9 in either classical or nonclassical monocytes modulates atherosclerosis development. Together, these completed aims in this renewal could lead to new biomarkers and therapies for immunomodulation of monocytes in atherosclerosis.