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American Liver Foundation Liver Scholar Award
Yale University, New Haven, Connecticut
“Dysregulation of TLR4-dependent epithelial innate immunity in cystic fibrosis cholangiopathy”
Cystic Fibrosis (CF) is an autosomic recessive systemic disorder that affects secretory epithelia, including the bile duct epithelium of the liver. Defective function of CFTR reduces bile flow and results in ductal cholestasis, and eventually in a sclerosing cholangiopathy that may progress to focal biliary cirrhosis. The pathogenesis of the disease is believed to be a consequence of ductal cholestasis; however, while the biliary secretory defect is present in every CF patients, only 30% of them develop clinical significant liver disease. Cftr-KO mice do not usually develop liver disease, unless exposed to dextran sodium sulfate colitis. In this condition, homozygous, but not WT or heterozygous mice show biliary damage and inflammation. Interesting, the liver damage/inflammation is not attenuated by restoration of biliary secretion with norUDCA, but is improved by intestinal decontamination with antibiotics. Dr. Fiorotto also found that when exposed to LPS cultured CFTR-defective cholangiocytes secrete significantly higher amount of NF-κB-dependent cytokines as, compared to WT cells. Since LPS response is specifically mediated by TLR4 she hypothesized that lack of CFTR alters TLR4-mediated innate immunity of the biliary epithelium. This project will study the mechanistic relationship between CFTR and TLR4 signaling focusing on three specific aims: 1) She will demonstrate that CFTR modulates the TLR4-mediated response of the biliary epithelium and in particular that CFTR expression is necessary for a controlled TLR4 signaling; 2) She will investigate the role of Src kinase, as a molecular link between CFTR deficiency and TLR4 stimulation; 3) based on preliminary data showing differential expression of miRNAs between WT and CFTR-defective cells, she will study if certain miRNAs, and in particular the let-7a miRNA, alters the post-trascriptional regulation of TLR4in CFTR-defective cells. These studies will be conducted using well established mouse cholangiocyte primary cell lines (both WT and Cftr-KO) and SEAP Reporter 293 cells (HEK-BlueTM –hTLR4 cells) expressing the human TLR4 gene. The role of CFTR in regulating TLR4 will be demonstrated using different CFTR constructs (WT and different mutations). Confocal microscopy and co-immunoprecipitation will be used to show the spatial and physical interaction of CFTR-Src-TLR4. Also, functional studies using luciferase reporter assay, transfection of miRNA precursors or miRNA antisense will be used to validate the role of let-7a in the regulation of TLR4 signaling. Dr. Fiorotto expects to clarify important mechanisms regulating the innate immune responses in cholangiocytes: the importance of CFTR channel as regulator of TLR4 singnaling, the molecular interaction of Src with CFTR and TLR4 and the role of let-7a as a post-transcriptional regulator of TLR4. These will potentially transform our understanding of the pathogenesis of CF liver disease and directly impact the management of liver disease in CF patients.