ABSTRACT
A 20 kDa heat-labile toxin (BFT) produced by enterotoxigenic Bacteroides fragilis (B. fragilis) is associated with diarrhea and mucosal inflammation. Although intestinal epithelial cells are known to activate mitogen-activated protein kinase (MAPK) in response to bacterial infection, there has been little understanding on the association between MAPK activation and BFT-induced enteritis. This study was performed to investigate the role of MAPK in enteritis induced by BFT. In human colon epithelial cells, BFT increased IL-8 secretion in a dose-dependent manner. BFT activated the three main MAPK cascades, including extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). BFT stimulation also activated AP-1 activation signals. Overexpression of dominant-negative plasmid of the c-Jun decreased the activated AP-1 signals and the up-regulated IL-8 expression induced by BFT stimulation. In addition, SB203580 and ERK inhibitor U0126 significantly reduced IL-8 secretion in colon epithelial cells stimulated with BFT. Furthermore, SB203580 significantly prevented BFT-induced severity of enteritis and fluid secretion in mouse ileum. These results suggest that MAPK activation may be required for IL-8 transcription in intestinal epithelial cells exposed to BFT and that the activated MAPK can mediate intestinal inflammation and mucosal damage induced by BFT.
Subject(s)
Animals , Humans , Mice , Bacterial Infections , Bacteroides fragilis , Bacteroides , Colon , Diarrhea , Enteritis , Enterotoxins , Epithelial Cells , Ileum , Inflammation , Interleukin-8 , Phosphotransferases , Plasmids , Protein Kinases , Transcription Factor AP-1ABSTRACT
A ~20 kDa heat-labile toxin (BFT) produced by enterotoxigenic B. fragilis induces chemokine responses that are associated with mucosal inflammation. In the present study, we assessed whether the activation of mitogen-activated protein kinase (MAPK) affects the levels of IL-8 and MCP-1 produced by BFT stimulation in human epithelial HT-29 cells. Human intestinal epithelial HT-29 cell lines were incubated with purified BFT. MAPK and AP-1 in HT-29 cells were measured by Western blot and luciferase assay, respectively. The expression of chemokines such as IL-8 and MCP-1 were determined by quantitative RT-PCR, ELISA, and luciferase assay. BFT stimulation activated MAPK such as ERK1/2 and p38 in HT-29 cells. Treatment with MAPK inhibitors attenuated BFT-induced expression of IL-8 and MCP-1. Transfection with mutant genes for Ras or c-Jun did not only suppressed AP-1 reporter genes, but also inhibited BFT-induced expression of IL-8 and MCP-1 reporter genes. These results suggest that Ras and MAPK cascade may act as the upstream signaling for the activation of AP-1, which induce chemokine expression in BFT-stimulated intestinal epithelial cells.
Subject(s)
Humans , Bacteroides fragilis , Bacteroides , Blotting, Western , Chemokines , Enterotoxins , Enzyme-Linked Immunosorbent Assay , Epithelial Cells , Genes, Reporter , HT29 Cells , Inflammation , Interleukin-8 , Luciferases , Phosphotransferases , Protein Kinases , Transcription Factor AP-1 , TransfectionABSTRACT
A ~20 kDa heat-labile toxin (BFT) produced by enterotoxigenic B. fragilis induces chemokine responses that may be associated with mucosal inflammation. To determine whether epithelial cells can contribute to BFT-induced inflammation, we assessed the expression of cyclooxygenase (COX)-2 in BFT-stimulated human intestinal epithelial cells. Human intestinal epithelial cell lines, HT-29 and Caco-2, were incubated with purified BFT. COX-2 mRNA and protein expression were measured by quantitative RT-PCR and Western blot analysis, respectively. BFT increased expression of COX-2, not that of COX-1, in human intestinal epithelial cell lines. Up-regulated COX-2 expression was paralleled by increased prostaglandin E2 (PGE2) production measured by the radioimmunoassay. PGE2 was predominantly secreted from the basolateral surface of BFT-treated epithelial cells, whereas lactate dehydrogenase was released predominantly from the apical surface. Moreover, the COX-2 expression and PGE2 production were significantly suppressed when NF-kappaB activity was inhibited. The basolateral secretion of PGE2 by up-regulated COX-2 in the BFT-stimulated colon epithelial cells seems to contribute to the inflammatory response in the underlying intestinal mucosa.