PROJECT SUMMARY/ABSTRACT Rheumatic diseases including rheumatoid arthritis, rheumatic heart disease, and systemic lupus erythematosus are associated with increased risk of cardiovascular disease. While inflammation is a critical link between rheumatic diseases and CVD, continued research is needed to determine causal mechanisms. The K/B.g7 model of spontaneous autoantibody-induced arthritis and mitral valve (MV) disease provides a valuable pre- clinical tool to elucidate disease-driving mechanisms related to valvular carditis, which is currently difficult to detect prior to the onset of reduced cardiac function. Prior work from our group demonstrated a role for macrophages in promoting valve disease. Single-cell sequencing data from inflamed MV reported a heterogeneous population of macrophages in this tissue prompting further inquiry into the macrophage subsets and subset-specific roles that drive disease. Preliminary data from our group demonstrated that lymphatic endothelial cells promote early stages of MV disease, but the mechanisms driving the growth of these neo- lymphatic structures remains unclear. We identified the presence of hyaluronan receptor LYVE1+ macrophages near the atrial layer of endothelium and MV LECs in both normal and diseased valves. Gene expression and flow cytometry profiling revealed that these cells are tissue resident macrophages. LYVE1+ macrophages have been identified in other locations in the heart as well as other tissues including the lung, adipose, and peritoneum. In these niches, LYVE1+ macrophages interact with tissue native hyaluronan, regulate extracellular matrix homeostasis, and promotes neovascular growth. Work proposed in this fellowship will test the hypothesis that a) early in disease, LYVE1+ macrophages promote MV disease at least in part by promoting lymphangiogenesis and b) the LYVE1 protein does not simply delineate this population of macrophages but is required for the macrophages to perform these functions. Using RNA sequencing, LYVE1+ and LYVE1- MV macrophage subsets will be compared to one another and across disease time points to identify the gene expression profile unique to LYVE1+ macrophages, which we predict will include lymphangiogenesis- and HA-binding-related transcripts. Beyond characterizing these cells, an inducible model of LYVE1+ macrophage depletion and LYVE1 WT and KO bone marrow chimeras will be used to test the necessity of this cell population and LYVE1 expression on macrophages, respectively, in driving both MV disease and lymphatic growth in diseased valves. Deep-tissue imaging and analysis techniques will be applied to further interrogate whether LYVE1+ macrophages preferentially interact with LECs to promote vessel growth. In all, these studies will elucidate important information about the role of innate immune responses in driving valvular carditis. These data can be used to improve our understanding of the early mechanisms driving human disease and potentially identify...