Responsiveness Of Intestinal Epithelial Cell Lines To Lipopolysaccharide Correlates With Toll-Like Receptor (Tlr)-4, But Not Tlr-2 Or Cd14 Expression.

Background: We have previously reported that some avirulent Salmonella strains have the ability to attenuate inflammatory responses in colonized epithelial cells by inhibiting the IKBubiquitination step of the NF-KB pathway (Neish, AS et al.Science.289:1560).The eukaryotic or prokaryotic determinants that mediate this phenomenon are unknown.Objective: To investigate the mechanism by which anti-inflammatory Salmonella strains inhibit NF-KB activation.Methods: Model polarized intestinal epithelia were prepared by culturing T-84 cells on permeable supports.ELISA was used to measure IL-8 secretion from colonized monolayers.Activation of NF-KB was assessed by electrophoretic mobility shift assay (EMSA) and immunofluorescent microscopy.Results: Colonization of model epithelia with anti-inflammatory strains of Salmonella (PhoF ~ and S. pu//orum) attenuated IL-8 secretion in response to TNF-e, while virulent strains (wild type) or avirulent strains with structural defects in the type lit secretion apparatus (invA-, invG-) did not reduce TNF induced IL-8 secretion.In the presence of prokaryotic protein synthesis inhibitors (gentamicin or chloramphenicol), the anti-inflammatory effects on IL-8 secretion were not affected.When apically applied cultures of antHnflammatory strains were physically separated from the T-84 monolayer by a .22#m filter that allowed passage of putative soluble bacterial factors, the observed anti-inflammatory effects on epithelial IL-8 secretion were abolished, suggesting that intimate contact is required.By EMSA, antiinflammatory strains prevented TNF-induced NF-KB activation.Immunofluorescent microscopy revealed that the p65 subunit of NF-KB formed aggregates in cells colonized by the antiinflammatory Salmonella strains but not in cells colonized by wild-type or type III secretiondeficient strains.Conclusions: Anti-inflammatory effects of nonpathogenic Salmonella require bacterial-epithelial contact, are protein synthesis-independent, and likely involve type litmediated secretion.Furthermore, anti-inflammatory bacteria may block NF-KB translocation by inducing formation of inactive NF-KB aggregates.