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

Abstract

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 TLR4/MD-2 complex results in proinflammatory gene expression and cytokine secretion in intestinal epithelial cells (IEC). We have shown that IEC 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 IEC 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-κB transcriptional activation was used as a measure of LPS responsiveness. Intestinal epithelial cellsin patients with IBD exhibited increased expression of MD-2 and TLR4 mRNA. Lipopolysaccharide responsiveness in IEC 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 deactylation by trichostatin A, two forms of epigenetic silencing, resulted in increased mRNA expression of MD-2 in IEC. These results demonstrate various molecular mechanisms by which IEC down-regulate MD-2 and thereby protect against dysregulated inflammation to commensal bacteria in the intestinal lumen.

Interferon-γ-induced MD-2 Protein Expression and Lipopolysaccharide (LPS) Responsiveness in Corneal Epithelial Cells Is Mediated by Janus Tyrosine Kinase-2 Activation and Direct Binding of STAT1 Protein to the MD-2 Promoter

“…Cytosine methylation plays an important role in gene regulation and occurs in the MD-2 promoter region of human colonic and intestinal epithelial cells (38). In that study, IFN-␥ dependent activation was found by deletion studies to be dependent on a region of the promoter between Ϫ728 and Ϫ1000.…”

Epigenetic Regulation of TLR4 Gene Expression in Intestinal Epithelial Cells for the Maintenance of Intestinal Homeostasis

“…It has been reported that MD-2 expression in healthy, normal intestinal mucosa is minimal, while it is increased in active inflammatory bowel disease colitis (33,34). Moreover, inhibition of CpG methylation and histone deacetylation was shown to result in increased mRNA expression of MD-2 gene in IECs just recently (35). TLR4 and MD-2 expression may be down-regulated in part by common or related mechanisms in IECs.…”

Epigenetic Control of the Host Gene by Commensal Bacteria in Large Intestinal Epithelial Cells

“…Increased expression of TLR4 and an elevated response to commensal organisms are often observed in patients with inflammatory bowel disease (12,13). We have previously reported using human IEC lines that low responsiveness of IECs to LPS is mainly brought about by down-regulation of the TLR4 gene and that transcriptional repression of this gene in IECs is mediated by epigenetic mechanisms including DNA methylation and histone deacetylation (14).…”