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.
Vamadevan
Arunan S
AS
Division of Gastroenterology, Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida 33101, USA.
Fukata
Masayuki
M
Arnold
Elizabeth T
ET
Thomas
Lisa S
LS
Hsu
David
D
Abreu
Maria T
MT
D001374:Azacitidine / Q000494:pharmacology; D018938:Caco-2 Cells; D016764:Cell Polarity; D019175:DNA Methylation; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D015212:Inflammatory Bowel Diseases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D007413:Intestinal Mucosa / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects
D006877:Hydroxamic Acids; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D051197:Toll-Like Receptor 4; C012589:trichostatin A; D001374:Azacitidine