Neutrophil elastase and myeloperoxidase in airway extracellular vesicles of cystic fibrosis children during pulmonary exacerbations

<bold>Background:</bold> Extracellular vesicles (EVs) play critical roles in disease pathogenesis. The aim of this study was to quantify mediators of neutrophilic inflammation within airway EVs during cystic fibrosis(CF) pulmonary exacerbation(PEx). <bold>Methods:</bold> EVs were isolated from sputum samples collected before and after antibiotic therapy for PEx and characterized according to international guidelines. Western blot analysis of EV protein extracts was used for EV canonical markers CD63, CD9 and flotillin-1(FLOT1), as well as neutrophil elastase(NE) and myeloperoxidase(MPO). EV content of NE and MPO was expressed as NE/FLOT1 and MPO/FLOT1 protein band densities. <bold>Results:</bold> Sputum samples from 21 CF children were analysed. Nanoparticle tracking analysis showed high concentrations of particles at the size of small EVs, and transmission electron microscopy confirmed typical EV morphology. Median (IQR) NE/FLOT1 increased from 2.46(1.68-5.25) to 6.83(3.89-8.89, p<0.001) and MPO/FLOT1 from 2.30(1.38-4.44) before to 5.76(3.45-6.94, p<0.01) after PEx therapy; EV concentration and size remained unchanged. There was a correlation between the changes in lung function (ppFEV1) and NE/FLOT1 with therapy in all patients (r=0.66, p<0.001) and in those receiving intravenous antibiotics (r=0.94, p<0.0001). Changes in the lung clearance index (LCI) correlated with changes in MPO/FLOT1 (r=0.68, p=0.05). <bold>Conclusions:</bold> Airways of children with CF contain EVs that carry NE and MPO as cargo. The lower NE and MPO content at the time of PEx and the correlation with pulmonary function suggest both a functional role of EVs in CF airway inflammation and potential as a biomarker to monitor CF lung disease.