Flagellin-induced corneal antimicrobial peptide production and wound repair involve a novel NF-kappaB-independent and EGFR-dependent pathway.
Flagellin-induced corneal antimicrobial peptide production and wound repair involve a novel NF-kappaB-independent and EGFR-dependent pathway.
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DOI：
10.1371/journal.pone.0009351
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发表时间：
2010-02-26
期刊：
影响因子：
3.7
通讯作者：
Yu FS
中科院分区：
文献类型：
--
作者：
Gao N;Kumar A;Jyot J;Yu FS
细菌鞭毛蛋白在刺激粘膜表面对细菌感染的先天免疫反应中起主要作用，并独特地诱导对病原体、化学物质和辐射的深刻细胞保护。本研究试图确定鞭毛蛋白对人角膜上皮细胞(HCECs)的炎症和细胞保护作用的信号通路。用从铜绿假单胞菌(PAK)或活细菌中提纯的鞭毛蛋白攻击培养的人脐静脉内皮细胞。用Western印迹法检测细胞信号通路的激活程度，用ELISA法和斑点印迹法检测细胞因子/趋化因子的分泌和抗菌肽(AMPs)的产生。在培养的猪角膜上评价了鞭毛蛋白对伤口愈合的影响。L94A(TLR5结合区突变)鞭毛蛋白和表达L94A鞭毛蛋白的PAK不能刺激NF-κB的激活，但能在转化生长因子-α相关途径中诱导EGFR信号转导。L94A鞭毛蛋白不能诱导人脐静脉内皮细胞产生IL-6和IL-8，同时缺乏对NF-κB的激活作用。令人惊讶的是，两个可诱导的AMP，LL-37和hBD2的分泌不受L94A突变的影响。与野生型鞭毛蛋白相似，L94A通过维持EGFR介导的信号转导，诱导培养的猪角膜上皮伤口闭合。我们的研究结果表明，由NF-κB介导的炎症反应可以从上皮固有防御机制(即腺苷脱氢酶的表达)和主要的上皮细胞增殖/修复途径中解离出来，鞭毛蛋白及其衍生物在细胞保护和控制角膜和其他粘膜组织的感染方面可能具有广泛的治疗应用。
The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs). Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-κB activation, but were potent in eliciting EGFR signaling in a TGF-α–related pathway in HCECs. Concomitant with the lack of NF-κB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling. Our data suggest that inflammatory response mediated by NF-κB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues.
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