Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells.

Enteropathogenic Escherichia coli (EPEC) virulence requires a type III secretion system (TTSS) to deliver effector molecules in host cells. Although the TTSS is crucial to EPEC pathogenesis, its function in EPEC-induced inflammation is not known. The aim of this study was to investigate the role of the TTSS in EPEC-induced inflammation. HT-29 intestinal epithelial cells were infected with wild-type (WT) EPEC or select mutant strains or exposed to corresponding filter-sterilized supernatants (SN), and interleukin-8 (IL-8) secretion was determined by ELISA. EPEC SN stimulated significantly greater IL-8 production than EPEC organisms. Flagellin, as well as a TTSS-independent >50-kDa nonflagellin protein, was found to significantly contribute to this response. Dose-response studies showed that increasing concentrations of WT SN proportionally increased IL-8, whereas increasing multiplicity of infection of EPEC inversely correlated with IL-8 secretion, suggesting that EPEC dampens this host response. Infection with DeltaescN (nonfunctional TTSS) markedly increased IL-8 compared with WT, indicating that a functional TTSS is required for this anti-inflammatory property; complementation of escN restored the attenuated response. Mutation of espB also enhanced the IL-8 response, and complementation returned IL-8 to near WT levels, suggesting involvement of this effector. The anti-inflammatory effect extends to both bacterial and host-derived proinflammatory stimuli, since prior infection with EPEC suppressed the IL-8 response to tumor necrosis factor-alpha, IL-1beta, and enterohemorrhagic E. coli flagellin. These findings indicate that EPEC-induced inflammation is a balance between pro- and anti-inflammatory proteins; extracellular factors, including flagellin and an unidentified TTSS-independent, >50-kDa protein, trigger inflammation while intracellular TTSS-dependent factors, including EspB, attenuate this response.