PROJECT SUMMARY/ABSTRACT Systemic sclerosis (SSc) is a prototypic fibrotic illness affecting virtually every organ. Genetic and environmental factors both contribute to disease. In addition to fibrosis, vascular injury and gut dysbiosis are prominent; however, how these distinct processes are governed by gene-environment interactions, and how they are linked together in pathogenesis is largely unknown, precluding development of disease-modifying therapy. Based on remarkable recent data from our lab and others, we now propose a novel paradigm for the elusive gene- environment interaction in SSc that ties gut microbial metabolism to vascular injury and fibrosis and opens the door for innovative therapy: 1) gut microbiota exposed to a Western diet generate trimethylamine (TMA), which is converted in the host to trimethylamine N-oxide (TMAO) by the enzyme flavin-containing monooxygenase (FMO3). Elevated TMAO is associated with endothelial cell injury, promotion of fibrotic cellular phenotypes, and tissue fibrosis; 2) genetic variants of FMO3 show highly significant association with SSc; and 3) expression of FMO3 is significantly upregulated in SSc skin fibroblasts. Our hypothesis is that choline-rich diets via a metaorganismal axis generate elevated TMAO, which promotes vascular injury and organ fibrosis via endothelial-mesenchymal transition (endoMT) and other pathways implicated in SSc pathogenesis. We propose that the fibrotic propensity can be mitigated by selectively inhibiting gut TMA lyase, the microbial enzyme exclusively responsible for TMA generation. This represents a distinct and transformative treatment paradigm. During the first two years (R61 phase), we will determine if and how diet-dependent chronic TMAO elevation impacts fibrosis in distinct in vivo disease models and explanted cells. We will then evaluate if a translationally- relevant novel compound that selectively inhibits TMA lyase in the gut modifies these responses. We will determine whether endoMT represents a key mechanism linking diet-associated TMAO elevation and vascular injury and fibrosis. In Year 3 (R33 phase), undertaken upon achieving our predefined milestones, we will define the role of FMO3 in diet-induced fibrosis propensity, and determine if circulating TMAO is a potential diagnostic and prognostic biomarker of SSc and its endotypes in both cross-sectional and longitudinal studies. This project seeks to validate an entirely novel SSc paradigm that links the environment/diet and genetic risk (FMO3 variants) in a metaorganismal pathway that underlies SSc pathogenesis and can be selectively targeted for therapy.