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

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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.