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.

项目摘要/摘要 革兰氏阴性（G-）细菌是美国医院获得肺炎（HAP）的主要原因。 每年在美国医院发生近300,000集HAP。 HAP平均增加 住院时间从7到9天，每位患者的额外费用超过40,000美元；它负责一个 所有ICU感染中的四分之一和所有抗生素使用的一半。由G-细菌结果造成的破坏性损害 造成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 发育失控感染的基本机制仍未完全理解。 这是迫切需要制定针对这种毁灭性疾病的新型治疗策略。 克拉拉细胞蛋白16（CC16）是克拉拉细胞分泌的主要蛋白质，是最丰富的蛋白 支气管肺泡灌洗液（BALF）。据报道，CC16的失调与各种 肺部疾病和几项研究表明，CC16可以作为肺的潜在生物标志物 许多疾病状态的上皮损伤，包括特发性肺纤维化，结节病，COPD， 哮喘，急性肺损伤等。从功能上，CC16在暴露于肺组织中具有抗炎活性 臭氧，过敏原，病毒和香烟烟雾。通过一般研究证实了这种受保护的作用 改良的小鼠，表明CC16缺乏症与肺对感染性的敏感性增加有关 刺激和氧化应激。但是，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，我们生成了稳定的电池线过表达CC16 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，对脂多糖（LPS）和克雷伯氏菌具有强大的抗炎作用 肺炎（K。pneu）诱导炎症。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 （RCC16）。此外，我们的数据还表明，与RCC16相比，EV-CC16对冻结的抵抗力要高得多 EV-CC16一种潜在的新型热剂，用于治疗肺损伤。使用微阵列，我们发现 CC16惊人地激活了多个DNA修复信号通路。一致地，我们观察到更严重的DNA 与野生型小鼠相比，CC16敲除小鼠的肺损伤。这，我们旨在进一步调查 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 通过雾化的雾化EV-CC16可以为G-细菌诱导的肺炎提供受保护的作用。 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 天生的免疫力，通过填补我们对CC16在G-细菌性肺炎中的作用的了解 可能为治疗肺部感染提供了一种新颖的治疗策略。