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

细胞外囊泡衍生的 Clara 细胞蛋白 16 在革兰氏阴性细菌性肺炎中的功能和潜在应用

• 批准号: 10905165
• 负责人: Duo Zhang
• 金额: $ 50.15万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10905165
• 关键词: Acute Lung Injury; Allergens; Alveolar; Alveolar wall; Anti-Inflammatory Agents; Antibiotics; Antiinflammatory Effect; Asthma; Bacterial Infections; Bacterial Pneumonia; Biogenesis; Biological; Blood capillaries; Bronchoalveolar Lavage Fluid; Cells; Chronic Obstructive Pulmonary Disease; Clara cell; Clinical; Communicable Diseases; DNA Damage; DNA Repair; DNA Repair Pathway; Data; Disease; Dose; Edema; Engineering; Epithelial Cells; Exposure to; Freezing; Goals; Gram-Negative Bacteria; Hospitals; Human; Hypoxemia; Infection; Inflammation; Inflammatory; Inflammatory Response; Inhalation; Injury; Klebsiella pneumoniae; Knockout Mice; Knowledge; Length of Stay; Lipopolysaccharides; Lung; Lung Compliance; Lung diseases; Lung infections; Measures; Mediating; Micro Array Data; Mus; N-terminal; Natural Immunity; Nebulizer; Nosocomial pneumonia; Oxidative Stress; Ozone; Pathogenesis; Patients; Peptide Signal Sequences; Permeability; Pharmaceutical Preparations; Play; Pneumonia; Predisposition; Property; Protein Secretion; Proteins; RNA; Recombinants; Regulation; Reporting; Research; Research Project Grants; Resistance; Role; Runaway; Sarcoidosis; Signal Pathway; Signal Transduction; Stimulus; Structure of parenchyma of lung; System; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Toxic effect; Ultrasonics; United States; Virus; Wild Type Mouse; aerosolized; bactericide; cell regeneration; cell type; cigarette smoke; comparative; cost; epithelial injury; exosome; extracellular vesicles; idiopathic pulmonary fibrosis; immunogenicity; insight; lung injury; novel; novel therapeutic intervention; novel therapeutics; overexpression; potential biomarker; protective effect; response; single-cell RNA sequencing; stable cell line; treatment strategy; ventilator-associated pneumonia

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.

项目概要/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。 美国医院每年平均发生近 300,000 起 HAP，这延长了 HAP 的持续时间。 住院 7 至 9 天，每位患者的额外费用超过 40,000 美元； G 细菌造成的破坏性损害占所有 ICU 感染的四分之一和一半。 尽管经过数十年的研究，细菌作用的不平衡和压倒性的炎症仍然存在。 失控炎症发生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，也是细胞中最丰富的蛋白质。 据报道，支气管肺泡灌洗液 (BALF) 的失调与多种疾病有关。 肺部疾病和多项研究表明 CC16 可以作为肺部疾病的潜在生物标志物 许多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。从功能上来说，CC16 在暴露于以下环境的肺组织中具有抗炎活性 臭氧、过敏原、病毒和香烟烟雾的这种保护作用已通过基因研究得到证实。 改良小鼠，表明 Cc16 缺乏与肺部对感染的易感性增加有关 然而，CC16 在急性肺损伤中的作用仍不清楚。 我们的初步研究表明，CC16 可以以细胞外囊泡 (EV) 依赖性方式分泌。 为了探索 EV 衍生的 CC16 (EV-CC16) 的功能，我们生成了过表达 CC16 的稳定细胞系 并从细胞中收集分泌的 EV-CC16 我们观察到 EV-CC16 抑制 NF-κB 信号传导。 激活并对脂多糖（LPS）和克雷伯氏菌有很强的抗炎作用 值得注意的是，考虑到 CC16 的剂量及其保护作用。 针对 LPS 引起的肺损伤，EV-CC16 的效率比重组 CC16 高 50,000 倍以上 (rCC16) 此外，我们的数据还表明 EV-CC16 比 rCC16 更耐冻融，使得 EV-CC16 是一种潜在的治疗肺损伤的新型治疗剂，我们使用微阵列发现了 EV-CC16。 CC16 惊人地激活了多种 DNA 修复信号通路，我们观察到更严重的 DNA。 Cc16 敲除小鼠的肺部损伤程度高于野生型小鼠因此，我们的目标是进一步研究 Cc16 敲除小鼠的肺部损伤。 CC16 保护肺组织免受损伤的机制我们的最终目标是测试 CC16 是否能够传递。 通过雾化雾化 EV-CC16 可提供针对 G 细菌诱导的肺炎的保护作用。 成功完成本申请中提出的目标不仅可以为该领域提供新的见解 通过填补我们关于 CC16 在 G 细菌肺炎中的作用的知识空白， 可能为肺部感染的治疗提供新的治疗策略。