Extracellular vesicles in sputum of children with cystic fibrosis pulmonary exacerbations.

BACKGROUND

The aim of this study was to quantify mediators of neutrophilic inflammation within airway extracellular vesicles (EVs) of children treated for a cystic fibrosis (CF) pulmonary exacerbation (PEx).

METHODS

EVs were isolated from stored sputum samples collected before and after antibiotic therapy for PEx between 2011 and 2013, and characterised by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Western blot analysis of EV protein extracts was used for EV canonical protein markers CD63, CD9 and flotillin-1 (FLOT1), as well as neutrophil elastase (NE), myeloperoxidase (MPO) and interleukin-8. The EV content of NE and MPO were expressed as ratios of NE/FLOT1 and MPO/FLOT1 protein band densities.

RESULTS

Sputum samples from 21 children aged 13.3 (range 8.0-17.0) years were analysed. NTA showed high concentrations of particles at the size of small EVs (50-200 nm), and typical EV morphology was confirmed by TEM. CD63, CD9 and FLOT1 were detectable in all samples. Median (interquartile range (IQR)) NE/FLOT1 increased from 2.46 (1.68-5.25) before to 6.83 (3.89-8.89, p<0.001) after PEx therapy, and median (IQR) MPO/FLOT1 increased from 2.30 (1.38-4.44) before to 5.76 (3.45-6.94, p<0.01) after, while EV size remained unchanged. Improvement in lung function (percent predicted forced expiratory volume in 1 s (ppFEV)) with PEx therapy correlated with NE EV content (r=0.657, p=0.001).

CONCLUSIONS

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, compared with after therapy, and the correlation with pulmonary function suggest both a functional role of EVs in CF airway inflammation and the potential of EVs as a biomarker to monitor CF lung disease.