TY - JOUR

T1 - Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium

AU - Pedersen, G

AU - Andresen, Lars

AU - Matthiessen, M W

AU - Rask-Madsen, J

AU - Brynskov, J

PY - 2005

Y1 - 2005

N2 - 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.

AB - 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.

KW - Adult

KW - Aged

KW - Aged, 80 and over

KW - Blotting, Western

KW - Cell Line, Transformed

KW - Cells, Cultured

KW - Colitis, Ulcerative

KW - Colon

KW - Crohn Disease

KW - Epithelial Cells

KW - Female

KW - Gene Expression Regulation

KW - Humans

KW - I-kappa B Proteins

KW - Interleukin-8

KW - Intestinal Mucosa

KW - Male

KW - Membrane Glycoproteins

KW - Middle Aged

KW - NF-kappa B

KW - Oligodeoxyribonucleotides

KW - RNA, Messenger

KW - Receptors, Cell Surface

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Toll-Like Receptor 9

KW - Toll-Like Receptors

U2 - 10.1111/j.1365-2249.2005.02848.x

DO - 10.1111/j.1365-2249.2005.02848.x

M3 - Journal article

C2 - 15996194

VL - 141

SP - 298

EP - 306

JO - Clinical and Experimental Immunology, Supplement

JF - Clinical and Experimental Immunology, Supplement

SN - 0964-2536

IS - 2

ER -