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Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-α, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-α, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-κB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) activate antimicrobial gene expression in response to detection of specific bacterial products. Relatively little is known about TLR5, the only TLR thought to be preferentially expressed by epithelial cells, beyond that it confers activation of the transcription factor NF-κB in a MyD-88 dependent manner in response to fiagellin. Because TLRs, in general, are also thought to signal through members of the MAPK family, we examined fiagellin-induced MAPK activation (via examining its phosphorylation status) and its subsequent role in expression of the chemokine IL-8 in polarized intestinal epithelia. Flagellin, like other proinflammatory stimuli (TNF-α, Salmonella typhimurium), activated p38 MAPK in a TLR5-dependent manner, whereas aflagellate bacteria or EGF did not activate this kinase. Although ERK1 and -2 were also observed to be activated in response to flagellin, their activation was not restricted to proinfiammatory stimuli because they were also potently activated by aflagellate bacteria (S. typhimurium or Escherichia coli) and EGF (neither of which activate NF-κB in these cells). Pharmacological inhibition of p38 MAPK (by SB-203580) potently (IC[sub 50] = 10 nM) reduced expression of IL-8 protein (maximal inhibition, 75%) but had no effect on NF-κB activation, only slightly attenuated upregulation of IL-8 mRNA levels in response to flagellin, and did not effect IL-8 mRNA stability. Together, these results indicate that epithelial TLR5 mediates p38 activation and subsequently regulates flagellin-induced IL-8 expression independently of NF-κB, probably by influencing IL-8 mRNA translation. [ABSTRACT FROM AUTHOR]