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Mechanisms of enteroaggregative escherichia coli flagellin-induced IL-8 secretion from epithelial cells Khan, Mohammed Aatif Shah


Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea in a number of clinical settings. EAEC diarrhea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. The molecular pathogenesis of EAEC diarrhea is not understood completely. Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, has been shown to induce IL-8 release from several epithelial cell lines. The aim of this study is to elucidate the mechanisms of IL-8 secretion by FliC-EAEC in epithelial cells lines. Toll-like receptors (TLRs) have been described as important component of the innate immune response in humans. It is known that host response to Gram-negative flagellin is mediated by TLR5, which signals through NF-KB to induce transcription of pro-inflammatory cytokines. Our results show that FliC-EAEC activates Akt (PKB) and 1-KB degradation (downstream targets of PI-3K) as observed in Western blots. Experiments with pharmacological inhibitors and dominant negative PI-3K indicated that PI-3K/Akt signaling is not necessary for FliC-EAEC induced IL-8 secretion. p38 MAP kinase is one of a family of stress-related kinases that influence a diverse range of cellular functions including inflammatory responses to microbial products. To determine whether this enzyme mediates IL-8 release, a pharmacological inhibitor of p38 MAP kinase, SB 203580, was used to inhibit FliC-EAEC induced IL-8 release. We found that IL-8 secretion is regulated by p38 MAP kinase, which undergoes activation in Caco-2 and THP-1 cells, as determined by Western analysis. Exposure of HEp-2 cells transiently expressing TLR5 resulted in p38 MAP kinase dependent IL-8 secretion. Activation of IRAK was also observed in TLR5-transfected HEp-2 cells after treatment with FliC-EAEC. These results suggest that FliC-EAECinduced IL-8 secretion is regulated by p38 MAP kinase and involves TLR5 in epithelial cells.

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