Project Summary Alcohol-associated hepatitis (AH) is a severe, potentially life-threatening form of alcohol-associated liver disease (ALD) with limited therapeutic options. Cholestasis and biliary dysfunction are common in AH patients and can worsen AH prognosis. The central hypothesis is that alcohol consumption reshapes the hepatic microenvironment and predisposes biliary epithelial cells (BECs) to additional injury, causing cholestatic liver injury and contributing to the progression of ALD. The overall objectives for this K01 proposal are to leverage a novel mouse model presenting alcohol-associated cholestasis and hepatitis (AlChoHep) to 1) determine the mechanism by which alcohol consumption predisposes livers to cholestatic liver injury, and 2) elucidate the role of cholestasis in the development of AH. The overall objectives of this application will be attained by pursuing two specific aims: (1) determine the mechanism of cholestasis in ALD and (2) elucidate the role of cholestasis in AH. Under the first aim, the working hypothesis will be determined by fecal microbiome transplant, bile acid depletion, liver organoid cultures, RNA sequencing, and ATAC-seq. Alteration of gut-modified bile acids and gut microbiome will be analyzed. The role of HMGB1 will be studied in liver-specific Hmgb1-knockout mice. The working hypothesis of the second aim will be tested by scFFPE-seq, spatial transcriptomics, metabolomics, leukotriene pathway inhibition, and liver organoid cultures. Upon successful completion of the proposed research, the expectation is to have defined the roles of the gut-liver axis and hepatocellular HMGB1 in the development of alcohol-associated cholestasis (Aim 1), and to have elucidated the impact of cholestasis on the liver microenvironment and how this contributes to the pathogenesis of AH (Aim 2). This K01 application will promote the development of the applicant into a multidisciplinarily trained independent principal investigator. The applicant has assembled a Mentoring Committee composed of outstanding scientists, including three mentors (Drs. Xiao-Ming Yin, Wenke Feng, and Xiaojiang Xu) and two key collaborators (Drs. Jay Kolls and Suthat Liangpunsakul), who are renowned hepatologists or well-recognized scientists in the field of ALD, gut-liver axis, inflammation, and bioinformatics, with an outstanding record of training junior scientists to be independent and successful in academia. They will direct the applicant’s career development and provide full support to the implementation of the proposed experiments. Upon completion of the studies, the applicant will receive additional training from the Mentoring Committee to gain expertise in several areas, including gut-liver axis, the pathophysiology of AH, liver inflammation, and bioinformatics. The successful completion of the proposed project will yield significant data for a follow-on R01 level application.