title: Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB.
abstract: Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-kappaB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48-96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-kappaB.
authors.affiliation: The Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Xian-Ming
authors.initials: XM
authors.lastname: Chen
authors.forename: Steven P
authors.initials: SP
authors.lastname: O'Hara
authors.forename: Jeremy B
authors.initials: JB
authors.lastname: Nelson
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Splinter
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Small
authors.forename: Pamela S
authors.initials: PS
authors.lastname: Tietz
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Limper
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: LaRusso
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000943:Antigens, Differentiation / Q000276:immunology / Q000378:metabolism; D001652:Bile Ducts / Q000166:cytology / Q000276:immunology*; D015153:Blotting, Western; D002478:Cells, Cultured; D016785:Cryptosporidium parvum / Q000276:immunology*; D004789:Enzyme Activation / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism / Q000382:microbiology*; D006801:Humans; D007150:Immunohistochemistry; D053592:Interleukin-1 Receptor-Associated Kinases; D008856:Microscopy, Fluorescence; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D011971:Receptors, Immunologic / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051193:Toll-Like Receptors / Q000096:biosynthesis / Q000276:immunology*; D014162:Transfection; D023083:beta-Defensins / Q000096:biosynthesis; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; C106641:DEFB4A protein, human; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D011971:Receptors, Immunologic; D051193:Toll-Like Receptors; D023083:beta-Defensins; D011494:Protein Kinases; D053592:Interleukin-1 Receptor-Associated Kinases; D048051:p38 Mitogen-Activated Protein Kinases