Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium.
Recognition of repeat CpG motifs, which are common in bacterial, but not in mammalian, DNA, through Toll-like receptor (TLR)9 is an integral part of the innate immune system. As the role of TLR9 in the human gut is unknown, we determined the spectrum of TLR9 expression in normal and inflamed colon and examined how epithelial cells respond to specific TLR9 ligand stimulation. TLR9 expression was measured in human colonic mucosal biopsies, freshly isolated human colonic epithelial cells and HT-29 cells by reverse transcriptase-polymerase chain reaction or Western blotting. Colonic epithelial cell cultures were stimulated with a synthetic CpG-oligodeoxynucleotide (ODN), exhibiting strong immunostimulatory effects in B cells. Interleukin (IL)-8 secretion was determined by enzyme-linked immunosorbent assay, nuclear factor-kappaB (NF-kB) activity by electrophoretic mobility shift assay and IkB phosphorylation by Western blotting. TLR9 mRNA was equally expressed in colonic mucosa from controls (n = 6) and patients with ulcerative colitis or Crohn's disease disease (n = 13). HT-29 cells expressed TLR9 mRNA and protein and responded to CpG-ODN (P < 0.01), but not to non-CpG-ODN stimulation, by secreting IL-8, apparently in the absence of NF-kB activation. Primary epithelial cells isolated from normal human colon expressed TLR9 mRNA, but were completely unresponsive to CpG-ODN stimulation in vitro. In conclusion, differentiated human colonic epithelial cells are unresponsive to TLR9 ligand stimulation in vitro despite spontaneous TLR9 gene expression. This suggests that the human epithelium is able to avoid inappropriate immune responses to luminal bacterial products through modulation of the TLR9 pathway.
Department of Gastroenterology, Herlev University Hospital, DK-2730 Herlev, Denmark. giped@dadlnet.dk
G
G
Pedersen
L
L
Andresen
M W
MW
Matthiessen
J
J
Rask-Madsen
J
J
Brynskov
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003093:Colitis, Ulcerative / Q000378:metabolism; D003106:Colon / Q000276:immunology / Q000378:metabolism; D003424:Crohn Disease / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007413:Intestinal Mucosa / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors
C408982:CPG-oligonucleotide; D020672:I-kappa B Proteins; D016209:Interleukin-8; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors