Optimal interventions for lung inflammation in early cystic fibrosis (CF) may improve patient lifespan and healthspan. This project seeks to identify mechanisms of damaging luminal hypochlorous acid (HOCl) evolution by myeloperoxidase (MPO) in early CF, with an ultimate goal of creating new scientific knowledge that may aid in development of new or improved therapeutics for early CF lung inflammation. Previously published research from our group showed that MPO is actively released by neutrophils (PMNs) in early CF and is associated with early stage lung damage and methionine sulfoxide, a product of HOCl reaction with methionine. To identify mechanisms by which this occurs, we will use a translational model of PMN transmigration toward CF airway fluid supernatant (CFASN), which faithfully reproduces hallmarks of CF airway PMNs observed in early and adult CF, including hyperexocytosis of PMN primary granules which contain MPO. In preliminary studies, we observed increased MPO protein secretion by PMNs, increased MPO specific activity and increased methionine oxidation in apical fluid after PMNs transmigrated to CFASN, compared to that of paired PMNs transmigrated to leukotriene B4 (LTB4). This was accompanied by less cellular production of superoxide and hypochlorous acid by CFASN-transmigrated PMNs. We observed large increases in de novo extracellular vesicles (EVs) in apical fluid after PMN transmigration to CFASN compared to LTB4 and retention of MPO in the >300 kDa fraction of apical fluid, suggesting EV complexation. EV- associated MPO from either condition was fully active, unlike free MPO in LTB4 which was inhibited. We hypothesize that hyperexocytosis is a critical mechanism of MPO secretion in early CF and that PMN- derived EVs protect MPO from inactivation, enabling lung damage by luminal hypochlorous acid. To test this hypothesis, we have designed the following Aims:
Aim 1 : Determine mechanisms of hypochlorous acid evolution by CF airway PMNs. Sub-Aim 1A will quantify the impact of hyperexocytic PMNs on fluid and epithelial cell MPO exposure, MPO specific activity, oxidation of methionine and thiols and fluid metabolomics. Sub-Aim 1B will determine the capacity of transmigrated PMNs to generate oxidants (superoxide, hypochlorous acid) de novo and kill early CF pathogens.
Aim 2 : Characterize oxidative mechanisms of CF airway PMN-derived EVs. Sub-Aim 2A will determine the capacity of PMN-derived EVs to generate oxidants, quantify proteins and metabolites in EVs that can support oxidant production and determine capacity of EVs to injure epithelial cells and kill bacteria. Sub-Aim 2B will use a panel of physiological substrate conditions to compare the resistance of free and EV-bound MPO to endogenous and pharmacological inhibitors. Expected outcomes include new mechanistic information about MPO activity, localization and mechanisms of inhibition relevant to early CF airways. Ongoing prospective clinical studies of early CF in the immediate environment will provide excellent opportunities to validate our findings.
This project aims to determine the impact of cystic fibrosis (CF) airway neutrophil hyperexocytosis on the activity of myeloperoxidase (MPO), a heme protein that generates hypochlorous acid (i.e., chlorine bleach) and has been associated with lung injury in early CF. To this end, we will leverage a highly translational model of neutrophil transmigration to CF airway fluid supernatant that faithfully recapitulates profound changes in neutrophil phenotype observed in early CF clinical airway samples, including hyperexocytosis of MPO- containing primary granules. Expected outcomes include mechanistic knowledge of MPO activity and localization in early CF, associated molecular markers, and new scientific knowledge to aid in MPO-targeted therapeutic development.
