RESEARCH PROJECT 1 - ABSTRACT Alcoholic hepatitis (AH) is a devastating spectrum of alcoholic liver disease (ALD) which manifests itself as acute on chronic liver failure with high short-term mortality and infection as a common complication. Although liver transplantation is successfully performed for AH patients who do not respond to prednisolone treatment, there is an urgent need to identify drivers which cause a drastic shift to AH with intense PMN infiltration. Our unbiased global search for AH drivers in a mouse model and a cross-analysis with AH patient data, have identified CASP4 in man and Casp11 in mouse as one of the differentially upregulated genes in AH. CASP4/11 is a non-canonical inflammasome caspase which is activated by intracellular pathogens or LPS. Active CASP4/11 in turn cleaves pro-Gasdermin-D (GSDMD) to release a 30kD active N-terminal fragment which interacts with plasma membrane to form pores and induce lytic cell death called pyroptosis. Indeed, CASP11 and GSDMD are activated in mouse AH but not in the precursor stage of chronic ASH. Casp11 null mice are protected from GSDMD activation, liver bacterial load, and severe AH. Conversely, the deficiency of IL-18, the key anti-microbial cytokine, aggravates CASP11 and GSDMD activation, liver bacterial load, and AH. Ectopic expression of the active GSDMD in hepatocytes induces extensive hepatocellular death and worsens AH in mice. Importantly, both CASP4 and GSDMD are robustly and consistently activated in AH patient livers but not in healthy livers. Fecal bacterial 16S rRNA sequencing identifies E Coli as one of the most prominently increased bacteria in AH patients compared to alcoholics without liver disease or healthy subjects. Based on these findings, we hypothesize that pyropotosis of hepatocytes and hepatic macrophages caused by translocating gut bacteria via the CASP4/11-GSDMD pathway, underlies intense neutrophilic inflammation and systemic bacterial infection and inflammation in AH. We also propose pyroptosis of intestinal epithelium determines the host's susceptibility for bacterial translocation and liver cell pyroptosis. To test these hypotheses, we will pursue the following two main specific aims: Aim 1. To determine the effects of conditional knockout of Gsdmd in intestinal epithelium, hepatocytes, or macrophages on key phenotypic parameters of AH, bacterial translocation and dissemination, intestinal and systemic inflammation. Aim 2. To test the effects of colonization of AH patient fecal materials (Aim 2-1) or their most dominant Gram-negative bacterium (Escherichia coli) (Aim 2-2) on the CASP11/4-GSDMD pyroptotic pathway in the intestine and liver of the mouse AH model.