TLR signaling at the intestinal epithelial interface.

The intestinal epithelium provides a critical interface between lumenal bacteria and the mucosal immune system. Whereas normal commensal flora do not trigger acute inflammation, pathogenic bacteria trigger a potent inflammatory response. Our studies emanate from the hypothesis that the intestinal epithelium is normally hyporesponsive to commensal pathogen-associated molecular patterns (PAMPs) such as LPS. Our data demonstrate that normal human colonic epithelial cells and lamina propria cells express low levels of TLR4 and its co-receptor MD-2. This expression pattern is mirrored by intestinal epithelial cell (IEC) lines. Co-expression of TLR4 and MD-2 is necessary and sufficient for LPS responsiveness in IEC. Moreover, LPS sensing occurs along the basolateral membrane of polarized IEC in culture. Expression of MD-2 is regulated by IFN-gamma. Cloning of the MD-2 promoter demonstrates that promoter activity is increased by IFN-gamma and blocked by the STAT inhibitor SOCS3. We conclude from our studies that the intestinal epithelium down-regulates expression of TLR4 and MD-2 and is LPS unresponsive. The Th1 cytokine IFN-gamma up-regulates expression of MD-2 in a STAT-dependent fashion. The results of our studies have important implications for understanding human inflammatory bowel diseases.

Regulation of Toll-like receptor 4-associated MD-2 in intestinal epithelial cells: a comprehensive analysis.

The intestinal epithelium maintains a state of controlled inflammation despite continuous contact with Gram-negative commensal bacteria and lipopolysaccharide (LPS) on its luminal surface. Recognition of LPS by the Toll-like receptor (TLR) 4/MD-2 complex results in pro-inflammatory gene expression and cytokine secretion in intestinal epithelial cells (IECs). We have shown that IECs express low levels of MD-2 and TLR4 and are poorly responsive to LPS. In this study, we did a comprehensive analysis to understand the immune-mediated and epigenetic mechanisms by which IECs down-regulate MD-2 expression. Expression of MD-2 and TLR4 mRNA was examined in human inflammatory bowel disease and intestinal epithelial cell lines (T84, HT-29, Caco-2). Nuclear factor-kappaB transcriptional activation was used as a measure of LPS responsiveness. Intestinal epithelial cells in patients with inflammatory bowel disease exhibited increased expression of MD-2 and TLR4 mRNA. Lipopolysaccharide responsiveness in IECs was polarized to the basolateral membrane. Bisulfite sequencing of the MD-2 promoter demonstrated methylation of CpG dinucleotides. Inhibition of methylation by 5-azacytidine and histone de-actylation by trichostatin A, two forms of epigenetic silencing, resulted in increased mRNA expression of MD-2 in IECs. These results demonstrate various molecular mechanisms by which IECs down-regulate MD-2 and, thereby, protect against dysregulated inflammation to commensal bacteria in the intestinal lumen.

TLR4 and MD-2 expression is regulated by immune-mediated signals in human intestinal epithelial cells.

The normal intestinal epithelium is not inflamed despite contact with a high density of commensal bacteria. Intestinal epithelial cells (IEC) express low levels of TLR4 and MD-2 and are lipopolysaccharide (LPS)-unresponsive. We hypothesized that immune-mediated signals regulate the expression of TLR4 and MD-2 in IEC. Expression of TLR4 and MD-2 was examined in normal colonic epithelial cells or intestinal epithelial cell lines. The effect of the cytokines interferon (IFN)-gamma, IFN-alpha, and tumor necrosis factor-alpha (TNF-alpha) on TLR4 and MD-2 expression was examined by reverse transcription-PCR and Western blot. NF-kappaB transcriptional activation and interleukin-8 secretion were used as measures of LPS responsiveness. Native colonic epithelial cells and IEC lines express a low level of TLR4 and MD-2 mRNA. IFN-gamma regulates MD-2 expression in both IEC lines, whereas IFN-gamma and TNF-alpha regulate TLR4 mRNA expression in IEC lines. Pre-incubation with IFN-gamma and/or TNF-alpha sensitizes IEC to LPS-dependent interleukin-8 secretion. To examine MD-2 transcriptional regulation, we cloned a 1-kb sequence proximal to the MD-2 gene translational start site. This promoter directed expression of a reporter gene in endothelial cells and IEC. IFN-gamma positively regulated MD-2 promoter activity in IEC. Co-expression of a STAT inhibitor, SOCS3, blocked IFN-gamma-mediated MD-2 promoter activation. T cell-derived cytokines lead to increased expression of TLR4 and MD-2 and LPS-dependent pro-inflammatory cytokine secretion in IEC. IFN-gamma regulates expression of the critical TLR4 co-receptor MD-2 through the Janus tyrosine kinase-STAT pathway. Th1 cytokines may initiate or perpetuate intestinal inflammation by altering toll-like receptor expression and bacterial reactivity.