Article Text
Abstract
Rationale Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene form the basis of cystic fibrosis (CF). There remains an important knowledge gap in CF as to how diminished CFTR activity leads to the dominant inflammatory response within CF airways.
Objectives To investigate if extracellular vesicles (EVs) contribute to inflammatory signalling in CF.
Methods EVs released from CFBE41o-, CuFi-5, 16HBE14o- and NuLi-1 cells were characterised by nanoparticle tracking analysis (NTA). EVs isolated from bronchoalveolar lavage fluid (BALF) from 30 people with CF (PWCF) were analysed by NTA and mass spectrometry and compared with controls. Neutrophils were isolated from the blood of 8 PWCF to examine neutrophil migration in the presence of CFBE41o- EVs.
Results A significantly higher level of EVs were released from CFBE41o- (p<0.0001) and CuFi-5 (p=0.0209) relative to control cell lines. A significantly higher level of EVs were detected in BALF of PWCF, in three different age groups relative to controls (p=0.01, 0.001, 0.002). A significantly lower level of EVs were released from CFBE41o- (p<0.001) and CuFi-5 (p=0.0002) cell lines treated with CFTR modulators. Significant changes in the protein expression of 126 unique proteins was determined in EVs obtained from the BALF of PWCF of different age groups (p<0.001–0.05). A significant increase in chemotaxis of neutrophils derived from PWCF was observed in the presence of CFBE41o EVs (p=0.0024) compared with controls.
Conclusion This study demonstrates that EVs are produced in CF airway cells, have differential protein expression at different ages and drive neutrophil recruitment in CF.
- cystic fibrosis
- paediatric lung disaese
- neutrophil biology
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Footnotes
ZU and MPW are joint first authors.
Correction notice This article has been corrected since it was published Online First. Figure 3 has been corrected due to an incorrect actin included in figure 3D and the sentence relating to this has been amended from, ‘Validation of expression of EPCAM, VCAM and S100 A12, and exosomal markers (CD63, CD9, Hps90) was confirmed by immunoblotting in both CFBE41o- and CuFi-5 EV fractions (figure 3D).’ to ‘Validation of expression of EPCAM, VCAM and S100 A12, was confirmed by immunoblotting in both CFBE41o- and CuFi-5 EV fractions (figure 3D) to complement a number of exosomal markers already validated and shown in figure 1C and figure 1F (CD63, CD9, Hps90).’
Contributors Conception and design: JC, PM, ZU and MW. Experiments: ZU, MW, RR, ED, HC, KB and AT. Clinical data collection and support: RR, JL, HD, SCD, EM, SC, PM and BL. Analysis and interpretation: ZU, MW, HC, DM, KB, JC, SD, PM and AT. Manuscript preparation: JC, PM, ZU, MW and AT.
Funding This publication has emanated from research conducted with the financial support of The National Children’s Research Centre under Project Grant No C/17/3. HC and DF are funded by the European Union’s Horizon 2020 research and innovation under grant agreement no. 686098.
Patient consent for publication Not required.
Ethics approval The study was approved by the research ethics committee of Children’s Health Ireland at Crumlin, St. Vincent’s University Hospital and Tallaght University Hospital where informed consent was obtained from each participant.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.
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