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

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.

Grant Number: 5R01HL150658-03
NIH Institute/Center: NIH
Principal Investigator: Joshua Chandler