Datasheet regarding Figs 1–5.

Objective The airway epithelium provides a first line of defense against pathogens by release of antimicrobial factors and neutrophil-attracting chemokines. Pseudomonas (P.) aeruginosa, a Gram-negative bacterium that expresses flagellin as an important virulence factor, is a common cause of injurious airway inflammation. The aim of our study was to determine the contribution of flagellin to the inflammatory, antimicrobial, and metabolic responses of the airway epithelium to P. aeruginosa. Furthermore, as we previously showed that targeting mTOR limited the glycolytic and inflammatory response induced by flagellin, we assessed the effect of rapamycin on human bronchial epithelial (HBE) cells stimulated with flagellated and non-flagellated P. aeruginosa. Methods Primary pseudostratified HBE cells, cultured on an air-liquid-interface, were treated on the basolateral side with medium, vehicle or rapamycin, exposed on the apical side with flagellated or flagellin-deficient P. aeruginosa, and analyzed for their inflammatory, antimicrobial, and glycolytic responses. Results Flagellin augmented the P. aeruginosa-induced expression of antimicrobial factors and secretion of chemokines by HBE cells but did not further increase the glycolytic response. Treatment of HBE cells with rapamycin inhibited mTOR activation in general and flagellin-augmented mTOR activation in particular, but did not affect the glycolytic response. Rapamycin, however, diminished the flagellin-augmented inflammatory and antimicrobial response induced by Pseudomonas. Conclusions These results demonstrate that flagellin is a significant factor that augments the inflammatory and antimicrobial response of human airway epithelial cells upon exposure to P. aeruginosa and suggest that mTOR inhibition by rapamycin in the airway epithelium diminishes these exaggerated responses.研究目的 气道上皮（airway epithelium）通过释放抗菌因子（antimicrobial factors）与趋化中性粒细胞的趋化因子（neutrophil-attracting chemokines），构成抵御病原体的第一道免疫防线。铜绿假单胞菌（Pseudomonas aeruginosa，简称P. aeruginosa）是一类以鞭毛蛋白（flagellin）作为核心毒力因子的革兰氏阴性菌（Gram-negative bacterium），亦是引发损伤性气道炎症的常见致病菌。本研究旨在明确鞭毛蛋白在气道上皮针对铜绿假单胞菌产生的炎症应答、抗菌应答及代谢应答中的作用。此外，鉴于本团队前期研究证实，靶向雷帕霉素靶蛋白（mTOR）可抑制鞭毛蛋白诱导的糖酵解（glycolytic）与炎症应答，本研究进一步评估了雷帕霉素（rapamycin）对分别经携带鞭毛蛋白与鞭毛蛋白缺陷型铜绿假单胞菌刺激的人支气管上皮（HBE）细胞的干预效果。 研究方法 将在气液界面（air-liquid interface）培养的原代假复层人支气管上皮（HBE）细胞，于其基底外侧侧（basolateral side）分别施加培养基、溶媒（vehicle）或雷帕霉素处理，随后在顶侧（apical side）分别给予携带鞭毛蛋白或鞭毛蛋白缺陷型的铜绿假单胞菌刺激，最终对细胞的炎症应答、抗菌应答及糖酵解应答进行检测分析。 研究结果 鞭毛蛋白可增强HBE细胞中铜绿假单胞菌诱导的抗菌因子表达与趋化因子分泌，但未进一步提升糖酵解应答水平。雷帕霉素处理HBE细胞后，可普遍抑制mTOR活化，尤其能削弱鞭毛蛋白介导的mTOR活化，但未对糖酵解应答产生显著影响。然而，雷帕霉素可显著降低铜绿假单胞菌诱导的、经鞭毛蛋白增强的炎症与抗菌应答水平。 研究结论 本研究结果证实，鞭毛蛋白是人体气道上皮细胞接触铜绿假单胞菌后，增强其炎症与抗菌应答的关键调控因子；同时表明，气道上皮中经雷帕霉素介导的mTOR抑制，可有效削弱这类过度活化的免疫应答反应。