Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium
Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium
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DOI：
10.1111/j.1365-2249.2005.02848.x
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发表时间：
2005-08-01
影响因子：
4.6
通讯作者：
Brynskov, J
中科院分区：
文献类型：
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作者：
Pedersen, G;Andresen, L;Brynskov, J
通过Toll样受体(TLR)9识别重复的CpG基序是先天免疫系统的组成部分，这种重复基序在细菌中很常见，但在哺乳动物中不存在。由于TLR9在人类肠道中的作用尚不清楚，我们测定了TLR9在正常和炎症结肠中的表达谱，并检测了上皮细胞对特定TLR9配体刺激的反应。用逆转录聚合酶链式反应或Western blotting检测TLR9在人结肠黏膜活检组织、新鲜分离的人结肠上皮细胞和HT-29细胞中的表达。用合成的CpG-寡脱氧核苷酸(ODN)刺激培养的结肠上皮细胞，对B细胞有很强的免疫刺激作用。用酶联免疫吸附试验检测白细胞介素8(IL-8)的分泌，用凝胶电泳法测定核因子-kB(NF-kB)的活性，用Western blotting检测IKB的磷酸化。TLR9mRNA在正常对照组(n=6)和溃疡性结肠炎或克罗恩病(n=13)患者的结肠黏膜中表达相同。HT-29细胞表达TLR9mRNA和蛋白，并对CpG-ODN产生反应(P<；0.01)，但不对非CpG-ODN刺激产生IL-8，显然是在不激活NF-kB的情况下。原代培养的人结肠上皮细胞表达TLR9mRNA，但对CpG-ODN刺激完全无反应。总之，分化的人结肠上皮细胞在体外对TLR9配体刺激没有反应，尽管TLR9基因自发表达。这表明，人类上皮细胞能够通过调节TLR9通路来避免对管腔细菌产物的不适当免疫反应。
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 expresssion 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.