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Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) deficiency, the cause of cystic fibrosis (CF), leads to unbalanced anion transport across the airway epithelium, limiting mucociliary clearance and resulting in chronic bacterial lung infections. The main CF pathogen, Pseudomonas aeruginosa stimulates neutrophil recruitment and activation in the lung. In CF, neutrophils are incapable of eliminating bacteria and undergo excessive activation, causing tissue damage and contributing to lung failure. Neutrophil extracellular traps (NETs) are one of the crucial innate immune, antimicrobial responses of neutrophil granulocytes. NET formation requires superoxide production and extrusion of DNA associated with citrullinated histones (citH4) and neutrophil granule proteins such as myeloperoxidase (MPO) and human neutrophil elastase (HNE). However, uncontrolled NET release offers a potential mechanism for excessive inflammatory response in CF airways. Thus, understanding the involvement of NET formation in CF airway pathophysiology is of clinical importance. We aimed to characterize P. aeruginosa-induced NET formation and its potential relevance in CF airways. Our data show that P. aeruginosa initiates robust release of extracellular DNA from human neutrophils. MPO, HNE and citH4 co-localize with extracellular DNA upon P. aeruginosa stimulation. The superoxide generating NADPH oxidase and MEK/ERK signaling contribute to NET formation. NET formation was quantified by our newly developed ELISA assays detecting MPO-DNA and HNE-DNA complexes. By comparing early and late bacterial isolates obtained from the same CF patient, we found that early isolates promoted more significant NET release than late isolates. This result suggested that release of P. aeruginosa-induced NETs is an important mechanism for release of neutrophil-derived CF inflammatory markers, and that decreased induction of NETs is required for adaptation and long-term survival of P. aeruginosa bacilli in CF airways. Correlation was performed between CF inflammatory markers and NET markers. We found that CF sputa contain significant amounts of NET markers whose levels correlate with lung disease severity. In summary, this work supports our hypothesis that P. aeruginosa-induced NET formation has deleterious effects to CF pulmonary airways.