MRP8/14 Enhances Corneal Susceptibility to Pseudomonas aeruginosa Infection by Amplifying Inflammatory Responses
MRP8/14 Enhances Corneal Susceptibility to Pseudomonas aeruginosa Infection by Amplifying Inflammatory Responses
复制标题
MRP8/14 通过增强炎症反应来增强角膜对铜绿假单胞菌感染的敏感性。
DOI：
10.1167/iovs.12-10172
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发表时间：
2013-02-01
影响因子：
4.4
通讯作者：
Huang, Xi
中科院分区：
文献类型：
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作者：
Deng, Qiuchan;Sun, Mingxia;Huang, Xi
目的。我们探讨了骨髓相关蛋白8和14 （MRP8/14）在铜绿假单胞菌（PA）角膜炎中的作用。采用实时荧光定量PCR检测PA感染的人角膜擦伤和小鼠角膜中MRP8/14 mRNA的表达水平。Western blot和免疫组化检测C57BL/6 （B6）角膜中MRP8/14蛋白的表达。在B6小鼠结膜下注射MRP8/14 siRNA，然后感染PA。用细菌平板计数和髓过氧化物酶测定感染B6角膜的细菌载量和多形核中性粒细胞（PMN）浸润。采用PCR和ELISA检测小鼠体内和体外促炎细胞因子水平。在小鼠巨噬细胞样RAW264.7细胞中，采用平板计数法检测其吞噬能力和细菌杀伤能力，采用流式细胞术和Griess法检测其活性氧（ROS）和一氧化氮（NO）水平。PA感染后，人角膜擦伤和B6角膜中MRP8/14表达水平显著升高。在B6角膜中，MRP8/14的沉默显著降低了PA感染后角膜疾病的严重程度、细菌清除、PMN浸润和促炎细胞因子的表达。体外研究进一步表明，MRP8/14的沉默抑制了促炎细胞因子的产生、细菌杀灭和ROS的产生，但不抑制吞噬或NO的产生。我们的研究证明了MRP8/14在细菌性角膜炎中的双重作用。虽然MRP8/14通过增强ROS的产生来促进细菌清除，但它更重要的功能是通过增强促炎细胞因子的表达，在眼表充当炎症放大器，从而促进角膜的易感性。（Invest Ophthalmol Vis Sci. 2013;54:1227-1234） DOI:10.1167/iovs.12-10172
PURPOSE. We explored the role of myeloid-related protein 8 and 14 (MRP8/14) in Pseudomonas aeruginosa (PA) keratitis.METHODS. MRP8/14 mRNA levels in human corneal scrapes and mouse corneas infected by PA were tested using real-time PCR. MRP8/14 protein expression in C57BL/6 (B6) corneas was confirmed using Western blot assay and immunohistochemistry. B6 mice were injected subconjunctivally with siRNA for MRP8/14, and then infected with PA. Bacterial plate counts and myeloperoxidase assays were used to determine the bacterial load and polymorphonuclear neutrophil (PMN) infiltration in infected B6 corneas. Pro-inflammatory cytokine levels in vivo and in vitro were examined with PCR and ELISA. In murine macrophage-like RAW264.7 cells, phagocytosis and bacterial killing were assessed using plate count assays, and reactive oxygen species (ROS) and nitric oxide (NO) levels were tested with flow cytometry and Griess assay, respectively.RESULTS. MRP8/14 expression levels were increased significantly in human corneal scrapes and B6 corneas after PA infection. Silencing of MRP8/14 in B6 corneas significantly reduced the severity of corneal disease, bacterial clearance, PMN infiltration, and pro-inflammatory cytokine expression after PA infection. In vitro studies demonstrated further that silencing of MRP8/14 suppressed pro-inflammatory cytokine production, bacterial killing, and ROS production, but not phagocytosis or NO production.CONCLUSIONS. Our study demonstrated a dual role for MRP8/14 in bacterial keratitis. Although MRP8/14 promotes bacterial clearance by enhancing ROS production, it functions more importantly as an inflammatory amplifier at the ocular surface by enhancing pro-inflammatory cytokine expression, thus contributing to the corneal susceptibility. (Invest Ophthalmol Vis Sci. 2013;54:1227-1234) DOI:10.1167/iovs.12-10172