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Enterotoxin produced by enterotoxigenic Bacteroides fragilis (BFT) has been associated with mucosal inflammation and diarrhoeal diseases. In this study, the anti-inflammatory molecular mechanism of 5,7-dihydroxy-3,4,6-trimethoxyflavone (eupatilin) was characterized in an HT-29 intestinal epithelial cell line stimulated with BFT. Pre-treatment of HT-29 cells with eupatilin decreased the production significantly of both interleukin (IL)-8 and prostaglandin E2 induced by BFT in a dose-dependent manner. BFT-activated nuclear factor-kappaB (NF-κB) signals in HT-29 cells and pretreatment with eupatilin suppressed NF-κB activation that resulted in the significant inhibition of IL-8 and cyclo-oxygenase-2 expression. BFT-induced phosphorylation of both IκBα and IκB kinase (IKK) signals was prevented in eupatilin-pretreated HT-29 cells. Transfection of siRNA for IKK-α and IKK-β decreased the production of IL-8 and prostaglandin E2; however, the transfection of IKK-β siRNA showed a more significant reduction of BFT-induced IκBα phosphorylation compared with that of IKK-α siRNA. In addition, herbimycin A, a specific inhibitor of heat shock protein 90 (Hsp90), decreased the BFT-induced activation of IKK and NF-κB, suggesting that Hsp90 is associated with a pathway of IKK-NF-κB-IL-8/cyclo-oxygenase-2 gene signalling. Furthermore, eupatilin dissociated the complex between Hsp90 and IKK-γ in BFT-stimulated HT-29 cells. These results suggest that eupatilin can suppress the NF-κB signalling pathway by targeting the Hsp90-IKK-γ complex in intestinal epithelial cells and may attenuate BFT-induced inflammatory responses. [ABSTRACT FROM AUTHOR]

Escherichia coliis associated with inflammation in the brain. To investigate whether astrocytes are involved inE. coil-induced inflammation, we assessed the levels of expression of proinflammatory mediators produced byE. coli-infected astrocytes.E. coliinfection in primary human astrocytes and cell lines increased expression of the CXC chemokine IL-8/GRO-α, the CC chemokine MCP-1, TNF-α, and iNOS.E. coliinfection activated p65/p50 heterodimeric NF-κB and concurrently decreased the signals of IκBα. Blocking the NF-κB signals by IκBα-superrepressor-containing retrovirus or antisense p50 oligonucleotide transfection resulted in down-regulation of expression of the proinflammatory mediators. Furthermore, superrepressors of IκBα, IκB kinase (IKK) or NF-κB inducing kinase (NIK) inhibited the up-regulated expression of the downstream target genes of NF-κB such as IL-8 and MCP-1, and superrepressors of TNF receptor-associated factor (TRAF)2 and TRAF5 also inhibited expression of theE. coli-induced target genes of NF-κB. These results indicate that proinflammatory mediators such as the CXC chemokine IL-8/GRO-α, the CC chemokine MCP-1, TNF-α, and iNOS can be expressed inE. coli-infected astrocytes via an NF-κB pathway, suggesting that these mediators may contribute to inflammation in the brain, including infiltration of inflammatory cells. [ABSTRACT FROM AUTHOR]

Umbilical cord blood has emerged as an alternative source of haematopoietic CD34+ cells for allogeneic stem cell transplantation. Although bacteraemia induced by Escherichia coli is considered one of the complications of transplantation, expression of proinflammatory cytokines is poorly understood. In this study, we report the altered expression of proinflammatory cytokines in CD34+ cells and their in vitro cultured cells following E. coli infection. CD34+ stem cells and their cultured cells up-regulated expression of proinflammatory cytokines such as interleukin (IL)-1 α, IL-6, IL-8 and tumour necrosis factor (TNF)- α after infection with E. coli. Expression of the proinflammatory cytokines was generated mainly by the granulocyte-macrophage lineages. E. coli infection activated the signals of p50/p50 nuclear factor-kappaB (NF- κB) homodimers and I κB kinase. Furthermore, inhibition of NF- κB activation lowered the up-regulated expression of the proinflammatory cytokines. These results suggest that CD34+ cells and their cultured cells infected with E. coli induce the expression of proinflammatory cytokines via the NF- κB pathway. [ABSTRACT FROM AUTHOR]

SUMMARY Although intestinal epithelial cells are known to up-regulate the expression of several chemokine genes in response to the stimulation with B. fragilis enterotoxin (BFT), there has been little understanding on the cellular mechanisms of BFT-induced mucosal inflammation. To test whether nuclear transcriptional factor-kappa B (NF-κB) is involved in the process, we stimulated intestinal epithelial cells with BFT, and evaluated the signalling NF-κB pathways. BFT increased signals of NF-κB in HT-29 and T84 epithelial cell lines as well as primary human colon epithelial cells. NF-κB molecules activated by BFT stimulation were composed of p65 and p50 heterodimers. In contrast, BFT decreased the signals of IκBα and IκBℇ, as assessed by immunoblot. Super-repressors of IκBα, IκB kinase (IKK)β, and NF-κB inducing kinase (NIK) inhibited an up-regulated transcription of downstream target gene (CXCL8) of NF-κB. Moreover, blocking the activation of NF-κB by MG-132 or antisense p50 oligonucleotide transfection resulted in down-regulated expression of chemokines such as CXCL1, CXCL8, and CCL2 in BFT-stimulated HT-29 cells. In addition, NF-κB inhibition suppressed the BFT-induced neutrophil transepithelial migration in T84 cells. These results indicate that NF-κB can be a central regulator of chemokine gene expression in BFT-stimulated intestinal epithelial cells and may be an important regulator of neutrophil migration. [ABSTRACT FROM AUTHOR]