The CF airway environment affects neutrophil antibacterial effector mechanisms: phagocytosis and NET formation

Abstract Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the epithelial chloride channel CFTR. Lung disease is the main cause of mortality and morbidity in CF. CF airways are characterized by chronic, mainly neutrophil-mediated inflammation and persistent bacterial infections. Despite their increased presence in the CF lung, airway neutrophils are unable to clear respiratory pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. We hypothesized that the CF airway environment affects two antibacterial effector mechanisms of neutrophils: phagocytosis and neutrophil extracellular trap (NETs) formation. While neutrophils are known to kill P. aeruginosa and S. aureus mainly by phagocytosis-mediated intracellular killing, NET-mediated extracellular killing remains relatively inefficient against these pathogens. To examine this in CF, blood PMNs from healthy donors were incubated in sputum supernatants from CF patients to mimic the CF airway environment. Using this in vitro model, different markers and functions of neutrophils were measured by flow cytometry. Phagocytosis of fluorescently-labelled P. aeruginosa was significantly inhibited by CF sputum treatment. Surface expression of CD33, a myeloid-derived suppressor cell marker known to inhibit phagocytosis, was increased following CF sputum treatment. CF sputum exposure slightly increased both of the NET-specific markers studied: intracellular levels of citrullinated histone H3 and surface expression of gasdermin-D. These results suggest that the CF airway environment contributes to the inability of neutrophils to eliminate bacteria while promoting the release of dangerous, tissue-damaging neutrophil cargo via NET formation.