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Cystic fibrosis
Original Article
Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis
  1. Robert D Gray1,
  2. Gareth Hardisty1,
  3. Kate H Regan1,
  4. Maeve Smith1,
  5. Calum T Robb1,
  6. Rodger Duffin1,
  7. Annie Mackellar1,
  8. Jennifer M Felton1,
  9. Lily Paemka2,
  10. Brian N McCullagh2,
  11. Christopher D Lucas1,
  12. David A Dorward1,
  13. Edward F McKone3,
  14. Gordon Cooke4,
  15. Seamas C Donnelly4,
  16. Pradeep K Singh5,
  17. David A Stoltz2,
  18. Christopher Haslett1,
  19. Paul B McCray2,
  20. Moira K B Whyte1,
  21. Adriano G Rossi1,
  22. Donald J Davidson1
  1. 1UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
  2. 2Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
  3. 3Department of Respiratory Medicine, St Vincent’s Hospital, Dublin, Ireland
  4. 4Department of Medicine, Trinity College Dublin and Tallaght Hospital, Dublin, Ireland
  5. 5Department of Microbiology, Washington University Medical School, Seattle, Washington, USA
  1. Correspondence to Dr Robert D Gray, UoE/MRC Centre for Inflammation Research, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK; r.d.gray{at}ed.ac.uk

Abstract

Background Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation.

Methods Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR.

Results CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF.

Conclusions CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

  • Cystic Fibrosis
  • Neutrophil Biology

This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/

https://doi.org/10.1136/thoraxjnl-2017-210134

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    Footnotes

    • GH and KHR contributed equally.

    • Contributors RDG, GH, KHR, MS, CTR, RD, AM, JMF, LP, BNM, GC, CDL and DAD performed research. RDG, AGR, DJD, CH, MKBW, PKS, SCD, PBM, DAS and EFM designed the research. RDG, MKBW, AGR and DJD wrote the manuscript.

    • Funding This work was supported by the Wellcome Trust WT093767 (RDG), WT094415 (CDL) and WT096497 (DAD); the UK Medical Research Council G1002046 (DJD) and MR/K013386/1 (AGR, RD, and CH). The studies in CF pigs were supported by NIH grants: P01 HL-51670, P01 HL-091842, and the Cystic Fibrosis Foundation RDP. Specimen collection from the patients receiving Ivacaftor was supported by an unrestricted investigator-initiated grant from Vertex Pharmaceuticals (Boston, MA, USA).

    • Competing interests None declared.

    • Ethics approval East of Scotland Research Ethics Committee, 15/ES/0094; West of Scotland Research Ethics Committee, 11/WS/0074; Lothian Research Ethics Committee, 08/S1103/38.

    • Provenance and peer review Not commissioned; externally peer reviewed.