title: Potentiation of flagellin responses in gut epithelial cells by interferon-gamma is associated with STAT-independent regulation of MyD88 expression.
abstract: Flagellin acting via TLR5 (Toll-like receptor 5) is a key regulator of the host response to the gut microbial flora in both health and disease. The present study has investigated regulation of flagellin-TLR5 signalling in human colonocytes (HT29-19A) by IFNgamma (interferon-gamma), a cytokine released early in the inflammatory process which has multiple effects on gut epithelial function that may facilitate abnormal responses to enteric bacteria. Flagellin induced a dose-dependent secretion of chemokines CXCL8 and CCL2 in the human colonocyte line, HT29-19A. Exposure to IFNgamma did not induce chemokine secretion, but markedly potentiated responses to flagellin, increasing CXL8 gene expression and protein secretion by approx. 4-fold. Potentiation by IFNgamma was independent of changes in TLR5 and was associated with a rapid, sustained increase in expression of the downstream adaptor molecule MyD88 (myeloid differentiation factor 88). Knockdown of MyD88 expression using siRNA (small interfering RNA) abolished flagellin-dependent CXCL8 secretion and the potentiating effect of IFNgamma. Exposure of non-transformed mouse and human colonocytes to IFNgamma also increased MyD88 expression. STAT (signal transducer and activator of transcription) 1 knockdown and use of the broad-spectrum JAK (Janus kinase)-STAT inhibitor AG490 had no effect on IFNgamma-mediated up-regulation of MyD88. The findings of the present study suggest that IFNgamma sensitizes colonic epithelial cells to bacterial flagellin via a largely STAT-independent up-regulation of MyD88 expression leading to increased secretion of immunomodulatory factors. These results indicate that epithelial responses to flagellin are potentiated by IFNgamma, most likely mediated by increased MyD88 expression. The present study adds to our understanding of the spectrum of effects of this cytokine on gut epithelium that may contribute to bacterial-driven inflammation in the gut.
authors.affiliation: Salford Royal NHS Foundation Trust, UK.
authors.forename: Ciara
authors.initials: C
authors.lastname: Bannon
authors.forename: Pam J
authors.initials: PJ
authors.lastname: Davies
authors.forename: Andrew
authors.initials: A
authors.lastname: Collett
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Warhurst
mesh_terms: D000818:Animals; D018932:Chemokine CCL2 / Q000378:metabolism; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D005408:Flagellin / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D016209:Interleukin-8 / Q000378:metabolism; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D008297:Male; D051379:Mice; D053594:Myeloid Differentiation Factor 88 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D050791:STAT Transcription Factors / Q000502:physiology*; D051198:Toll-Like Receptor 5 / Q000235:genetics / Q000378:metabolism
chemical_list: D018932:Chemokine CCL2; D016209:Interleukin-8; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D034741:RNA, Small Interfering; D050791:STAT Transcription Factors; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D005408:Flagellin; D007371:Interferon-gamma