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.
Abreu
Maria T
MT
Inflammatory Bowel Disease Center, Division of Gastroenterology, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA. maria.abreu@cshs.org
Arnold
Elizabeth T
ET
Thomas
Lisa S
LS
Gonsky
Rivkah
R
Zhou
Yuehua
Y
Hu
Bing
B
Arditi
Moshe
M
D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D016207:Cytokines / Q000276:immunology; D017931:DNA Primers; D029721:Drosophila Proteins; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D000954:Antigens, Surface; D016207:Cytokines; D017931:DNA Primers; D029721:Drosophila Proteins; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors