1 PROJECT SUMMARY 2 Cardiovascular disease (CVD) remains the leading cause of death in the United States and worldwide even with 3 advances in the understanding of risk factors for CVD, the development of prevention interventions to reduce 4 the risks, and the improvements in therapy for patients with CVD. There has been an increasing attention in the 5 contribution of the gut microbiota to age-associated CVD, as both human and mouse studies associate changes 6 in the gut microbiome with disease progression. Aging is the dominant risk factor for atherosclerotic CVD, and 7 the process of aging such as inflammation and cellular senescence can be influenced by the gut microbiome. 8 Atherosclerosis is a chronic, low grade inflammatory response that attracts cells of the innate and adaptive 9 immune systems into the atherosclerotic plaque. Both aging and atherosclerosis have been linked with 10 alterations in mucosal-associated invariant T (MAIT) cells ー a subset of innate like T cells that localize 11 preferentially to the gastrointestinal tract and recognize microbial-derived vitamin B metabolite antigens. Germ- 12 free (GF) mice are deficient in MAIT cells, and colonization of feces from conventionally-raised mice into GF 13 mice restores the MAIT frequencies in tissues, suggesting that gut microbes are necessary for the complete 14 development of MAIT cells. Our collaborator Dr. Clement has recently found that elderly subjects and patients 15 with metabolic syndrome exhibit a decreased frequency and functional defects of MAIT and that this 16 phenomenon is more pronounced in patients with CVD. Recent developments of tools and rodent models have 17 provided insight into the mode of action and characterization of MAIT cells in diseases, but their roles in aging 18 and atherosclerosis remains to be elucidated. Moreover, the contribution of human gut microbiota to the induction 19 of MAIT cells are still unknown. Using a gnotobiotic mouse model with human fecal samples, our preliminary 20 data showed that mice colonized with feces from a high MAIT subject had significantly higher abundance of 21 MAIT cells compared to mice with feces from a low MAIT subject. We now propose to extend this study to 22 characterize the frequency and function of MAIT cells during atherosclerosis development (Aim 1a); to elucidate 23 the role of MAIT cells on atherosclerosis using MR1 deficient mice (Aim 1b); and to test the contribution of distinct 24 human gut microbiota to MAIT cell induction (Aim 2a) and atherosclerosis (Aim 2b). Combining novel 25 experimental tools in studying MAIT cell, such as antigen-loaded MR1 tetramers, a magnetic bead-based 26 enrichment method of murine MAIT cells, and MAIT cell-deficient MR1-/- mice, with innovative experimental 27 approaches to investigate the role of the gut microbiota on atherosclerosis (i.e., gnotobiotic atherosclerosis- 28 prone mice colonized with human gut microbiota), we anticipate to discover novel mechanisms...