IKKβ and phosphatidylinositol 3-kinase/Akt participate in non-pathogenic gram-negative enteric bacteria-induced RelA phosphorylation and NF-κB activation in both primary and intestinal epithelial cell lines

Pathogenic and enteroinvasive bacteria have been shown to trigger the IκB/NF-κB transcriptional system and proinflammatory gene expression in epithelial cells. In this study, we investigated the molecular mechanism of the commensal Gram-negative Bacteroides vulgatus. induced NF-κB signal transduction in intestinal epithelial cells (IEC). We report that B. vulgatus induced interleukin-1 receptor-associated kinase-1 degradation, IκBα phosphorylation/degradation, RelA and Akt phosphorylation, as well as NF-κB DNA binding and NF-κB transcriptional activity in rat non-transformed IEC-6 cells. B. vulgatus- but not interleukin-1β-mediated NF-κB transcriptional activity was inhibited by dominant negative (dn) toll-like receptor 4. Of importance, B. vulgatus induced IκBα phosphorylation/degradation and IKKα/β and RelA phosphorylation in primary IEC derived from germ-free or mono-associated HLA-B27 transgenic and wild type rats, demonstrating the physiological relevance of non-pathogenic bacterial signaling in IEC. Adenoviral delivery of dn IKKβ or treatment with wortmannin inhibited B. vulgatus-induced endogenous RelA Ser-536 and GST-p65TAD (Ser-529/Ser-536) phosphorylation as well as NF-κB transcriptional activity in IEC-6 cells, suggesting a critical role of IKKβ and phosphatidylinositol 3-kinase/Akt in bacteria-induced RelA phosphorylation and NF-κB activation. Interestingly, B. vulgatus-induced IκBα degradation and NF-κB transcriptional activity in IEC transwell cultures were inhibited in the presence of lymphocytes. We propose that non-pathogenic B. vulgatus activates the NF-κB signaling pathway through both IκB degradation and RelA phosphorylation but that immune cells mediate tolerance of IEC to this commensal bacteria.