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 lkappaB/NF-kappaB 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-kappaB signal transduction in intestinal epithelial cells (IEC). We report that B. vulgatus induced interleukin-1 receptor-associated kinase-1 degradation, IkappaBalpha phosphorylation/degradation, RelA and Akt phosphorylation, as well as NF-kappaB DNA binding and NF-kappaB transcriptional activity in rat non-transformed IEC-6 cells. B. vulgatus- but not interleukin-1beta-mediated NF-kappaB transcriptional activity was inhibited by dominant negative (dn) toll-like receptor 4. Of importance, B. vulgatus induced IkappaBalpha phosphorylation/degradation and IKKalpha/beta 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 IKKbeta or treatment with wortmannin inhibited B. vulgatus-induced endogenous RelA Ser-536 and GST-p65TAD (Ser-529/Ser-536) phosphorylation as well as NF-kappaB transcriptional activity in IEC-6 cells, suggesting a critical role of IKKbeta and phosphatidylinositol 3-kinase/Akt in bacteria-induced RelA phosphorylation and NF-kappaB activation. Interestingly, B. vulgatus-induced IkappaBalpha degradation and NF-kappaB transcriptional activity in IEC transwell cultures were inhibited in the presence of lymphocytes. We propose that non-pathogenic B. vulgatus activates the NF-kappaB signaling pathway through both IkappaB degradation and RelA phosphorylation but that immune cells mediate tolerance of IEC to this commensal bacteria.