Project Summary Alcohol associated health problems are a major medical burden in industrialized countries. Patients with alcohol-associated liver disease show intestinal bacterial dysbiosis and increased intestinal permeability. Although there is considerable progress in understanding the interaction between the host and intestinal bacteria, the role of the intestinal fungal microbiome (also called mycobiome) in alcohol-associated liver disease is not very well understood. Results from our laboratories indicate a proportional increase of Candida albicans (C. albicans) and Malassezia restricta (M. restricta) in patients with alcohol use disorder. Results from chronic ethanol administration in mice or chronic alcohol abuse in patients show that C. albicans-specific T cell responses occur in the intestine. CD4+ T cells re-circulate to the liver, where they re-activate by translocated C. albicans antigens, produce interleukin 17 (IL17) and contribute to progression of ethanol-induced steatohepatitis. In addition, products from M. restricta translocate from the gut lumen to the systemic circulation and liver. M. restricta induces liver inflammation via ligation with the Dectin-2 (Clec4n) receptor on Kupffer cells and augments ethanol-induced liver disease in mice. The testable central hypothesis of this proposed collaborative and multidisciplinary research application implicates disturbances in the gut fungal mycobiota as an important etiological factor in the modulation of adaptive and innate immunity in the liver. Through the proposed study, we will characterize the host gut mycobiome and immune response in a human cohort. We will mechanistically test our hypothesis in a mouse model of ethanol-induced liver disease. Towards this goal, we will use pharmacological interventions, supplementation of fungi and genetically modified mice. We predict that two pathogenic factors contribute to dysfunction of the gut-liver axis in alcohol-associated liver disease: C. albicans overgrowth drives Th17 cell expansion contributing to liver inflammation and damage (Aim 1). Binding of M. restricta to Dectin-2 induces hepatic inflammation and exacerbates alcohol-associated liver disease (Aim 2). We believe these studies will provide important insights into alcohol-mediated changes of the intestinal mycobiome that result in an immune response contributing to alcohol-associated liver disease. Eventually this approach might lead to new therapeutic targets for patients with alcohol-associated liver disease.