Cystic Fibrosis (CF) is a common and clinically severe genetic disease, caused by mutations in CFTR, a membrane protein that mediates Cl- and fluid secretion in a number of secretory epithelia, including the biliary tree. About 30% of CF patients present biochemical liver abnormalities and about 10% of these develop clinically significant liver disease and hepatobiliary complications characterized by a chronic cholangiopathy that can eventually evolve into sclerosing cholangitis and focal biliary cirrhosis. Cystic Fibrosis liver disease (CFLD) negatively impacts the quality of life and survival of CF patients, and may require liver transplantation, however, the pathogenesis of this condition is not well understood. In Cftr-KO mice, bile flow and biliary secretion are reduced;however the spontaneous development of CFLD is extremely rare, suggesting that genetic and/or acquired factors other than cholestasis are at play. It has been shown that portal endotoxemia, induced by oral administration of dextrans specifically causes biliary damage in Cftr-KO mice, but not in their wild type littermates4. Using this experimental model, we recently showed that biliary damage and inflammation caused by treatment with DSS in Cftr-KO mice were not prevented by restoring biliary secretion with nor-UDCA, and that, exposure of cultured CFTR-defective cholangiocytes to LPS in vitro, significantly increased cytokine secretion and NF-?B activity as compared to WT cells. The increased activation of NF-?B was prevented by inhibition of TLR4. We also found that the activity of Src, a tyrosine kinase involved in cell response to LPS, was upregulated in CF cells and resulted in an increased phosphorylation of TLR4. We propose that Src is the potential molecular link between CFTR and TLRs. In fact, treatment with Src inhibitor PP2 blocked TLR4 phosphorylation and NF-?B activation in response to LPS. We have also found that the expression and distribution of Csk and EBP-50, involved in Src regulation, were altered in Cftr-KO cholangiocytes. In this application, we will further investigate this novel paradigm shifting hypothesis, and in particular we will elucidate 1) the pathogenetic role of different TLRs in CFLD, 2) the role of Src in linking CFTR and TLR- mediated signaling and 3) the therapeutic value of PPAR? agonist as a strategy to inhibit the TLR/NF-?B pathway in CF cholangiocytes. These studies will be performed in Cftr-defective mice and in ferrets, a novel CF model that spontaneously develops CFLD. These studies will change our current understanding of the pathogenesis and treatment of CF-cholangiopathy. Furthermore, better knowledge of the regulation of TLR pathways in the biliary epithelium, will also provide important insights into the pathogenesis of other inflammation-mediated diseases of the epithelium, providing a firm foundation for future studies in the broader field of epithelial immunology.
Cystic Fibrosis liver disease (CFLD) negatively impacts the quality of life and survival of CF patients, and may require liver transplantation, however, the pathogenesis of this condition is not well understood. Our proposal will test the novel hypothesis that CFTR participates to the regulation of epithelial innate immunity and that CFLD results from a dysregulated TLR response of CFTR-defective biliary cells. Using mouse and ferret models, we will also study the therapeutic benefit of nuclear receptor agonists and other treatment strategies targeted on this new interpretation of CF-related liver disease.