ABSTRACT Alcohol-associated hepatitis (AH) is the most severe manifestation of alcohol-associated liver disease that is characterized by hepatocyte dysfunction, systemic inflammation and poor clinical outcomes. We recently reported heterogeneity in neutrophils in AH with over-activated, high-density (HDN), and functionally exhausted, low density (LDN) neutrophil populations that contribute to liver damage. Here we hypothesize that alcohol-induced extracellular traps (NETs) play a role in neutrophil heterogeneity and also contribute to hepatocyte damage, inflammation and fibrosis in AH. Our preliminary results suggest that alcohol-induced neutrophil activation and NET release are associated with increased activation and phosphorylation of the Bruton tyrosine kinase (BTK), a non-receptor tyrosine kinase. We postulate that BTK is a master regulator in AH by promoting both inflammation and hepatocyte dysfunction. Given that BTK can interact with the NLRP3 inflammasome as well as the STING/IRF3 pathways that play key roles in alcohol-induced inflammation, hepatocyte damage and steatosis, we propose to explore the role of these interactions in AH. Finally, we propose that therapeutic inhibition of NETs, BTK, or both will ameliorate neutrophil mediated inflammation and liver damage. The aims of this proposal are #1: To characterize neutrophil phenotypes, populations and the role alcohol-induced NETs in AH by: a) Characterizing neutrophil phenotypes and populations from AH patients using single cell RNAseq; b) delineating mechanisms by which alcohol-induced NETs activate monocytes/macrophages, hepatic stellate cells and hepatocytes; c) Defining the role of BTK activation in alcohol-induced neutrophil NET induction and LDN generation. Aim 2 will evaluate the role of cell-specific BTK phosphorylation in AH by assessing: a) Mechanisms of BTK activation by alcohol and defining the impact of acute and chronic alcohol on BTK activation in the liver; b) A BTK phosphorylation on the phenotypes and NLRP3 inflammasome activation in circulating monocytes of patients with AH; c) The effect of myeloid- and neutrophil-specific deletion of BTK in a mouse model of AH. Aim 3 will evaluate inhibition of NETs or BTK as potential therapeutic interventions in preclinical models of AH in mice. Results from these experiments will unravel unique heterogeneity of neutrophils in AH that will lead to better understanding of the controversial role of neutrophils in AH. Our results will characterize the functional impact of NETs on inflammation and fibrosis in alcohol-induced liver damage. The proposed studies will provide novel insights into the role of BTK at the molecular and cell-specific level and define its interactions with other key signal transduction pathways and explore BTK and/or NETs as new therapeutic targets for amelioration of AH.