Neutrophil hyperexocytosis and hypochlorous acid exposure in early cystic fibrosis lung disease

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PROJECT SUMMARY / ABSTRACT Current standard of care for cystic fibrosis (CF) does not include drugs unequivocally effective at curbing airway inflammation, in contrast to crucial gains made in correcting defects in the CF transmembrane conductance regulator (CFTR) via highly effective modulator therapy (HEMT) and an array of antimicrobial drugs. Effectively treating exuberant neutrophil-dominated inflammation in CF, particularly in the earliest stages of the disease, is a critical goal because doing so would increase patient lifespan and health span. This project is designed to test the hypothesis that neutrophil hyperexocytosis, which occurs in CF from a very young age, is a critical event that releases active myeloperoxidase (MPO) from neutrophil granules and enables the generation of extracellular hypochlorous acid (HOCl), a strong and promiscuous oxidant that can damage cells. We further hypothesize that the released MPO becomes associated with extracellular vesicles (EVs), providing MPO with sustained metabolism to fuel its HOCl-generating activity and conferring resistance to inhibitors. Ultimately, we hypothesize that increased airway HOCl injures airway epithelial cells (AECs) and promotes maladaptive cellular responses that contribute to bronchiectasis. Research from our group has shown that MPO is active in CF airways from the earliest stages of disease and is associated with initial manifestations of bronchiectasis. Furthermore, we have identified molecular products of HOCl exposure, such as methionine sulfoxide, that can be monitored by LC-MS across a broad range of biological specimens, including basic and translational models and clinical samples. Using state-of-the-art LC-MS, rigorous cell-, protein-, and EV- detection methods, highly translational models of neutrophils and EVs, and clinical sample validation, this project sets out a series of parallel experiments designed to identify the sequence of events leading from neutrophil transmigration into CF airways, to active MPO release and extracellular HOCl generation, to CF AEC injury and maladaptation to the potent oxidative insult. These experiments are enumerated in the following Specific Aims: (1) Determine the impact of hyperexocytosis on extracellular HOCl and innate defense; (2) Determine mechanisms of granule-releasing, immunomodulatory, and metabolically active (GRIM) neutrophil EV-based HOCl production; and (3) Determine mechanisms of CF airway epithelial cytotoxicity mediated by MPO and HOCl. Furthermore, these experiments account for potential impacts of novel HEMT therapy elexacaftor-tezacaftor-ivacaftor (ETI) in inflammatory pathways by comparing CF sputum samples with HEMT to the same samples from donors not receiving ETI. The ultimate goal of the proposed studies is to generate knowledge informing clinical and drug development research and forestall CF lung disease progression at the earliest stages and before end organ damage. Furthermore, owing to the prevalence of GRIM neutrophils in a number of lung diseases beyond CF, information pertinent to other lung diseases including neutrophil inflammation will also be gained by the completion of this project.