title: Loss of CFTR affects biliary epithelium innate immunity and causes TLR4-NF-κB-mediated inflammatory response in mice.
abstract: OBJECTIVE
  Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins.
  
  
  METHODS
  Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftrtm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor κB (NF-κB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs.
  
  
  RESULTS
  DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF-κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-κB.
  
  
  CONCLUSIONS
  CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-κB. These findings might be used to develop therapies for CF-associated cholangiopathy.
authors.affiliation: Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Romina
authors.initials: R
authors.lastname: Fiorotto
authors.forename: Roberto
authors.initials: R
authors.lastname: Scirpo
authors.forename: Michael
authors.initials: M
authors.lastname: Trauner
authors.forename: Luca
authors.initials: L
authors.lastname: Fabris
authors.forename: Rafaz
authors.initials: R
authors.lastname: Hoque
authors.forename: Carlo
authors.initials: C
authors.lastname: Spirli
authors.forename: Mario
authors.initials: M
authors.lastname: Strazzabosco
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001652:Bile Ducts / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D002756:Cholagogues and Choleretics / Q000494:pharmacology; D002761:Cholangitis / Q000139:chemically induced / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology / Q000517:prevention & control; D003092:Colitis / Q000139:chemically induced / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D016207:Cytokines / Q000378:metabolism; D016264:Dextran Sulfate; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D057809:HEK293 Cells; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D018836:Inflammation Mediators / Q000378:metabolism*; D053539:Keratin-19 / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018181:Mice, Inbred CFTR; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D010766:Phosphorylation; D011112:Polymyxin B / Q000494:pharmacology; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014580:Ursodeoxycholic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D019061:src-Family Kinases
chemical_list: D000900:Anti-Bacterial Agents; D002756:Cholagogues and Choleretics; D016207:Cytokines; D018836:Inflammation Mediators; D053539:Keratin-19; D008070:Lipopolysaccharides; D016328:NF-kappa B; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014580:Ursodeoxycholic Acid; D016264:Dextran Sulfate; C081331:24-norursodeoxycholic acid; D019061:src-Family Kinases; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse; D009355:Neomycin; D011112:Polymyxin B