The Function and Potential Application of Extracellular Vesicle Derived Clara Cell Protein 16 in Gram-negative Bacterial Pneumonia

项目摘要/摘要 革兰氏阴性(G-)细菌是美国医院获得性肺炎(HAP)的主要原因。 美国医院每年发生近30万起HAP病例。HAP平均增加了 住院时间从7天到9天，每名患者的额外费用超过40,000美元；它负责一项- 占所有ICU感染的第四位，占所有抗生素使用的一半。G-细菌造成的毁灭性破坏导致 从杀菌作用和压倒性炎症的失衡。尽管进行了数十年的研究，但 导致炎症失控的潜在机制仍不完全清楚。 因此，迫切需要开发针对这种毁灭性疾病的新的治疗策略。 Clara细胞蛋白16(CC16)是Clara细胞分泌的主要蛋白质，也是 支气管肺泡灌洗液(BALF)。据报道，CC16的调节失调与多种 肺部疾病和多项研究表明，CC16可作为肺的潜在生物标志物 多种疾病状态下的上皮损伤，包括特发性肺纤维化、结节病、慢性阻塞性肺病、 哮喘、急性肺损伤等。从功能上讲，CC16对暴露于 臭氧、过敏原、病毒和香烟烟雾。这一保护作用已被使用基因的研究证实 改良的小鼠，表明Cc16缺乏与肺部对感染的易感性增加有关 刺激和氧化应激。然而，CC16在急性肺损伤中的作用尚不清楚。 我们的初步研究表明，CC16可以以细胞外小泡(EV)依赖的方式分泌。 为了研究EV来源的CC16(EV-CC16)的功能，我们建立了一个稳定表达CC16的细胞系 从细胞中收集分泌的EV-CC16。我们观察到EV-CC16抑制了NF-κB信号转导 对脂多糖和克雷伯氏菌有较强的抗炎作用 肺炎克雷伯菌(Kpneu)引起炎症。值得注意的是，考虑到CC16的剂量及其保护作用 EV-CC16对脂多糖诱导的肺损伤的保护作用是重组CC16的5万多倍 (RCC16)。此外，我们的数据还表明，EV-CC16比rCC16具有更强的抗冻融能力，使 EV-CC16是一种潜在的治疗肺损伤的新药物。利用基因芯片，我们发现了EV- CC16显著激活了多条DNA修复信号通路。一以贯之，我们观察到了更严重的DNA Cc16基因敲除小鼠的肺损伤程度高于野生型小鼠。因此，我们打算进一步调查 CC16保护肺组织免受损伤的机制。我们的最终目标是测试是否交付了 雾化吸入EV-CC16对G-细菌所致肺炎具有保护作用。 成功完成本申请中提出的目标不仅可能为该领域提供新的见解 通过填补我们对CC16在G-细菌性肺炎中的作用的了解的空白， 可能为肺部感染的治疗提供一种新的治疗策略。

PROJECT SUMMARY/ABSTRACT Gram-negative (G-) bacteria are the leading cause of hospital-acquired pneumonia (HAP) in the United States. Nearly 300,000 episodes of HAP occur in U.S. hospitals each year. HAP on average increases the length of hospital stay from 7 to 9 days, at an additional cost of more than $40,000 per patient; it is responsible for one- fourth of all ICU infections and half of all antibiotic use. The devastating damage caused by G- bacteria results from the imbalance of bactericidal effects and overwhelming inflammation. Despite decades of research, the underlying mechanisms by which the runaway inflammation is developed remain incompletely understood. Thus, there is an urgent need to develop novel therapeutic strategies against this devastating disease. Clara Cell Protein 16 (CC16) is the major protein secreted by Clara cells and the most abundant protein in bronchoalveolar lavage fluid (BALF). Dysregulation of CC16 has been reported to be associated with various lung disorders and several studies have demonstrated that CC16 could serve as a potential biomarker of lung epithelial injury in numerous disease states including idiopathic pulmonary fibrosis, sarcoidosis, COPD, asthma, acute lung injury, etc. Functionally, CC16 has anti-inflammatory activities in lung tissues exposed to ozone, allergens, viruses and cigarette smoke. This protective role is confirmed by studies using genetically modified mice, showing that Cc16 deficiency is associated with increased susceptibility of the lung to infectious stimuli and oxidative stress. However, the role of CC16 in acute lung injury is still not clear. Our preliminary study showed that CC16 can be secreted in an extracellular vesicle (EV)-dependent manner. To explore the function of EV-derived CC16 (EV-CC16), we generated a stable cell line overexpressing CC16 and collected secreted EV-CC16 from cells. We observed that EV-CC16 suppresses NF-κB signaling activation and has a strong anti-inflammatory effect against lipopolysaccharide (LPS) and Klebsiella pneumoniae (K. pneu) induced inflammation. Notably, considering the dose of CC16 and its protective effect against LPS-induced lung injury, EV-CC16 is over 50,000 times more efficient than recombinant CC16 (rCC16). Besides, our data also indicated EV-CC16 is much more resistant to freeze-thaw than rCC16, making EV-CC16 a potential novel therapeutic agent for the treatment of lung injury. Using microarray, we found EV- CC16 strikingly activated multiple DNA repair signaling pathways. Consistently, we observed more severe DNA damage in lungs from Cc16 knockout mice than wild-type mice. Thus, we aim to further investigate the mechanism by which CC16 protects lung tissues from injury. Our ultimate goal is to test whether the delivery of aerosolized EV-CC16 via nebulization can provide protective effects against G- bacterial-induced pneumonia. Successful completion of the aims proposed in this application may not only provide new insights into the field of innate immunity, by filling gaps in our knowledge about the role of CC16 in G- bacterial pneumonia but also might provide a novel therapeutic strategy for the treatment of pulmonary infection.