SUMMARY Cystic Fibrosis (CF) is a common and clinically severe genetic disease, caused by mutations in CFTR, a membrane protein that mediates chloride and fluid secretion in a number of secretory epithelia, including the biliary tree. Cystic Fibrosis liver disease (CFLD) is a chronic cholangiopathy that can eventually evolve into sclerosing cholangitis and focal biliary cirrhosis. The pathogenesis of this condition is not well understood and treatment is limited to the administration of choleretic bile acids, or in selected cases, liver transplantation. CFLD has been classically considered a consequence of the impaired bile secretion caused by the defective CFTR channel function. However, while biliary secretion is universally reduced in CF, the spontaneous development of CFLD is less frequent, suggesting that genetic and/or acquired factors are at play. Our previous studies suggested that reduced tolerance of the biliary innate immune system to endotoxins plays a major pathogenetic role in CFLD. We showed that cholangiocytes isolated from Cftr-KO mice have higher NF-κB activity and secrete a larger amount of inflammatory cytokines, when exposed to the TLR4-ligand LPS. We have also demonstrated that in CF-defective cholangiocytes, TLR4 is activated by the unrestrained function of Src family kinases (SFK), a consequence of defective CFTR. This mechanism is present also in cholangiocytes derived from human iPSC homozygous for the ΔF508 mutation. In addition, novel exciting preliminary data show that the gut microbiota in CFTR-KO mice is already different from WT littermates at birth and it is skewed towards the prevalence of a more pro-inflammatory flora. In this application we will test the hypothesis that CFLD may result from the combination of a genetic mutation affecting biliary epithelial innate immunity along with changes in microbiota composition and increased intestinal permeability. In particular, (1) we will use iPSC technology to dissect the impact of functionally different CFTR mutations on the mechanisms leading to a pro-inflammatory phenotype in human cholangiocytes and (2) we will study whether changes in the gut microbiota play a causal role in the development of liver disease in mouse models of CF. Our study will discover novel aspects of secretory epithelia physiology and innate immunity and clarity if changes in the gut microbiota play a possible causal role in CFLD. These studies represent a paradigm-shift in the understanding of the pathogenesis of CFLD and imply that treatment for CFLD should also control inflammation and the impact of the intestinal microbiota. The outcome of this project will lay the foundation for novel intervention strategies that will have an impact on the management of CFLD and other cholangiopathies. !