(429) Myeloperoxidase in extracellular vesicles isolated from airways of children with cystic fibrosis

Background: Extracellular vesicles (EVs) are nano-sized particles released from different cell types and play critical roles in intercellular communication and disease pathogenesis (1). Neutrophil-derived EVs containing myeloperoxidase (MPO) have previously been identified in inflammatory conditions other than cystic fibrosis (CF). Since MPO is a key player in CF neutrophilic airways inflammation, the aims of this study were 1) to isolate and characterize EVs from sputum of children with CF, and 2) to analyze the expression of MPO in sputum-derived EVs during treatment for CF pulmonary exacerbation (PEx).

Methods: EVs were isolated from expectorated sputum of CF patients collected before and after 14 days of intravenous antibiotic therapy for PEx. EVs were characterized by nanoparticle tracking analysis (NTA) and electron microscopy (EM), following international guidelines (2). Western blot analysis of EV protein extracts was used to quantify expression of EV canonical proteins CD-63, CD-9, and flotillin-1 (FLOT1), as well as MPO and interleukin-8 (IL-8). MPO expression in EVs was quantified as MPO/FLOT1 ratio. IL-8 levels in sputum were measured by ELISA.

Results: Ten children (8 females) with a mean age of 13.5 (range 8.0-16.9) years were included. Mean (SD) % predicted (pp)FEV₁ was 59.8 (9.6) before and 71.8 (11.6) after treatment for PEx. There were no differences in sputum-derived EV concentration [1.7e*11 (1.1e*11) before vs. 2.0e*11 (1.4e*11) particles/ml after PEx therapy; p=0.44] and size [151.2 (26.6) nm before vs. 145.7 (31.8) nm after PEx therapy; p=0.47]. EV MPO/FLOT1 ratios were significantly lower before compared to after PEx (3.1±1.3 vs 6.0±1.12, p< 0.001, paired t-test). EV MPO/FLOT1 correlated with ppFEV₁ at all timepoints (r=0.64, p< 0.01, Pearson) and changes in EV MPO/FLOT1 with changes in ppFEV₁ (r=0.63, p< 0.05) with treatment. IL-8 protein was not detected in the EVs. Sputum IL-8 levels did not correlate with EV MPO/FLOT1. There was no difference in EV MPO content between children treated for Staphylococcus aureus (n=5) or Pseudomonas aeruginosa (n=5) infection.

Conclusions: CF airways contain EVs that carry MPO as cargo. The increase in EV MPO expression (MPO/FLOT1) following treatment for PEx suggests greater release of MPO from EVs before initiation of PEx therapy compared to after. The contribution of EV-derived MPO to CF lung disease and airways inflammation warrants further investigation.

Acknowledgements:

References: [1] Raghu K, Valerie SL. The biology, function, and biomedical applications of exosomes. Science 2020;367(6478):eaau6977. https://doi: 10.1126/science.aau6977.

[2] Thery CA, Witwer KA, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750. https://doi: 10.1080/20013078.2018.1535750.