Severe alcoholic liver disease (ALD) has a high morbidity and mortality. Recent studies demonstrated that probiotics reversed alcohol-induced hepatic steatosis and inflammation, and improved liver enzymes in animal models and in patients. Our laboratory showed that administration of a probiotic strain, Lactobacillus rhamnosus Gorbach-Goldin (LGG), significantly improved liver enzymes and histology in alcohol treated mice. However, the beneficial mechanisms of action of LGG, either in viable bacteria form or in culture supernatant form, are not clear. Our overall hypothesis is that LGG increases intestinal cathelicidin-related antimicrobial peptide (CRAMP), intestinal trefoil factor (ITF) expression and intestinal short chain fatty acids (SCFAs), leading to improved gut microbiota homeostasis, intestinal barrier integrity and hepatic fat metabolism, and these are likely the mechanisms by which LGG supplementation attenuates alcohol induced liver injury. We evaluate mechanisms of LGG action with 4 specific aims: Aim 1 will determine the role of CRAMP in the alcohol-mediated changes in gut microbiota and the role of CRAMP in the beneficial effects of LGG in experimental ALD. We will use CRAMP knockout mouse model of ALD to determine the role of CRAMP and LGG in alcohol-induced changes in gut microbiota using a metagenomic approach. Aim 2 will determine the role of LGG in promoting ITF expression and evaluate whether ITF positively modulates intestinal tight junctions as a mechanism for improved intestinal barrier integrity in experimental ALD. We will use an ITF-secreting goblet cell line to evaluate the role of LGG in the production of ITF and the role of ITF in the modulation of intestinal tight junctions in an epithelial cell line. We will also examine th strategy that oral administration of recombinant human ITF attenuates alcohol-induced intestinal barrier and liver injury. Aim 3 will determine the role and mechanisms of butyric acid in the beneficial effects of LGG on intestinal tight junctions in experimental ALD. A metabolomics approach will be used to analyze the changes in SCFAs induced by alcohol and LGG intervention in the intestinal lumen. We will explore the potential mechanisms underlying the role of butyric acid in the regulation of occludin expression involving intestinal histone deacetylases (HDACs), miR122a, and HIF-?-mediated signaling in vitro and in vivo. Aim 4 will evaluate and compare the effectiveness of LGG supernatant (LGGs) with viable LGG (vLGG) and heat-inactivated LGG (hiLGG) in the prevention/therapy of ALD in mouse models. These three LGG preparations will be tested for the effectiveness in the prevention/therapy of ALD in three experimental models of ALD of steatosis, steatohepatitis and fibrosis. This study will have a major impact on the development of probiotic-based new therapeutic strategy for the prevention and treatment of ALD.