Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin.
Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin.
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DOI：
10.1167/iovs.03-0219
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发表时间：
2003-10
影响因子：
4.4
通讯作者：
Jing Zhang;Keping Xu;B. Ambati;F. Yu
中科院分区：
文献类型：
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作者：
Jing Zhang;Keping Xu;B. Ambati;F. Yu
鞭毛蛋白是革兰氏阴性菌鞭毛的主要结构蛋白，是许多真核细胞和生物体中天然免疫应答的有效触发剂。在这项研究中，我们试图确定鞭毛蛋白是否诱导培养的人角膜上皮（HCE）细胞的炎症反应，并确定其潜在的机制。方法采用硫酸铵梯度沉淀法从铜绿假单胞菌（PA）PAO 1菌株中分离纯化鞭毛蛋白，离子交换层析法去除鞭毛蛋白中的脂多糖。纯化的鞭毛蛋白被用来挑战HUCL，端粒酶永生化的HCE细胞系，和原代培养的HCE细胞。通过Western印迹检测抑制性（I）kappaB-α磷酸化和降解。采用RT-PCR和酶联免疫吸附试验分别检测IL-6和IL-8的mRNA表达和分泌。使用抗TLR 5抗体通过免疫组织化学分析人角膜中的TLR 5定位。使用抗鞭毛抗血清和抗TLR 5抗体来功能性阻断鞭毛蛋白刺激和TLR 5活化。结果HUCL和原代HCE细胞暴露于纯化的PA鞭毛蛋白（250 ng/mL）导致IkappaB-α磷酸化和降解呈时间依赖性。伴随着NF-κ B活化，鞭毛蛋白也诱导这些细胞中IL-6和IL-8的转录表达和随后的分泌。Toll样受体（TLR）-5是鞭毛蛋白的先天性免疫受体，在人角膜上皮细胞中表达，位于角膜上皮基底层和翼层细胞表面，而在浅层细胞中不表达。培养基中存在鞭毛或TLR 5抗血清减弱鞭毛蛋白诱导的IkappaB-α磷酸化和降解以及IL-6和IL-8产生。结论PA等革兰阴性病原菌的鞭毛蛋白通过TLR 5-NF-κ B信号通路参与角膜上皮的炎症反应。
PURPOSE Flagellin is the major structural protein of the flagella of Gram-negative bacteria and is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms. In this study, we sought to determine whether flagellin induces an inflammation response in cultured human corneal epithelial (HCE) cells and to determine the underlying mechanisms. METHODS Flagellin was purified from Pseudomonas aeruginosa (PA) strain PAO1 with ammonium sulfate gradient precipitation and lipopolysaccharide in flagellin preparation was removed by ion exchange chromatography. Purified flagellin was used to challenge HUCL, a telomerase-immortalized HCE cell line, and primarily cultured HCE cells. Inhibitory (I)kappaB-alpha phosphorylation and degradation were detected by Western blot. Interleukin (IL)-6 and -8 expression in mRNA levels and secretion were assessed using RT-PCR and enzyme-linked immunosorbent assay, respectively. TLR5 localization in human cornea was analyzed by immunohistochemistry using anti-TLR5 antibody. Anti-flagellum antiserum and anti-TLR5 antibody were used for functional blocking of flagellin stimulation and TLR5 activation. RESULTS Exposure of both HUCL and primary HCE cells to purified PA flagellin (250 ng/mL) resulted in IkappaB-alpha phosphorylation and degradation in a time-dependent manner. Concomitant with NF-kappaB activation, transcriptional expression and subsequent secretion of IL-6 and -8 in these cells were also induced by flagellin. Toll-like receptor (TLR)-5, an innate immunity receptor for flagellin, was expressed in HUCL cells and located at the cell surface of the basal and wing, but not in superficial, cells of human corneal epithelium. Presence of flagellum- or TLR5-antisera in culture medium attenuated flagellin-induced IkappaB-alpha phosphorylation and degradation as well as IL-6 and -8 production. CONCLUSIONS Flagellin of Gram-negative pathogens such as PA contributes to the inflammatory responses of corneal epithelium in a TLR5-NF-kappaB signaling pathway-dependent manner.