Single-Step Purification of Sputum Extracellular vesicles from Cystic Fibrosis patients using aF4-MALS

Abstract Rationale Patients with cystic fibrosis (CF) present a large amount of respiratory damage caused in large part by chronic neutrophil influx into the lung. Neutrophils recruited to the lumen of the CF lung actively exocytose neutrophil elastase (NE) and other damaging molecules. Airway NE levels correlate strongly with disease severity in patients with CF. We previously showed that extracellular vesicles (EVs) from patients with other lung diseases carry NE and promote emphysema in a mouse model (Genschmer et al. 2019). Here, we hypothesized that airway NE also compartmentalizes within EVs in CF patients. Methods To avoid potential aggregation and purification biases, we gently sorted EVs from CF sputum samples using asymmetrical flow field flow fractionation (aF4). Sputum EV fractions were then subjected to analysis by multi-angle light scattering, nanoparticle tracking analysis and flow cytometry. Results Multi-angle light scattering and nanoparticle tracking analyses demonstrate the presence of EVs of different sizes ranging from 40–500 nm, with a concentration of ~5 × 107 EVs/mL. EVs were separated by size with aF4 and downstream flow cytometry analysis showed that discrete subfractions concentrate NE on their surface, but that NE burden was not proportional to size. Conclusions Our data show that there are discrete subtypes of EVs released by neutrophils carrying various amounts of NE on their surface. These findings provide evidence for a role of EVs in promoting inflammation in the lungs of patients with CF.