Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis

Robert D Gray; Gareth Hardisty; Kate H Regan; Maeve Smith; Calum T Robb; Rodger Duffin; Annie Mackellar; Jennifer M Felton; Lily Paemka; Brian N McCullagh; Christopher D Lucas; David A Dorward; Edward F McKone; Gordon Cooke; Seamas C Donnelly; Pradeep K Singh; David A Stoltz; Christopher Haslett; Paul B McCray; Moira K B Whyte; Adriano G Rossi; Donald J Davidson

doi:10.1136/thoraxjnl-2017-210134

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Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis

Journal article

Open access

Peer reviewed

Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis

Robert D Gray, Gareth Hardisty, Kate H Regan, Maeve Smith, Calum T Robb, Rodger Duffin, Annie Mackellar, Jennifer M Felton, Lily Paemka, Brian N McCullagh, …

Thorax, Vol.73(2), pp.134-144

02/2018

DOI: 10.1136/thoraxjnl-2017-210134

PMCID: PMC5771859

PMID: 28916704

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Abstract

BackgroundCystic 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.MethodsBlood 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.ResultsCF 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.ConclusionsCF 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.

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Title: Subtitle: Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis
Creators: Robert D Gray - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Gareth Hardisty - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Kate H Regan - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Maeve Smith - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Calum T Robb - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Rodger Duffin - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Annie Mackellar - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Jennifer M Felton - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Lily Paemka - Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
Brian N McCullagh - Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
Christopher D Lucas - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
David A Dorward - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Edward F McKone - Department of Respiratory Medicine, St Vincent’s Hospital, Dublin, Ireland
Gordon Cooke - Department of Medicine, Trinity College Dublin and Tallaght Hospital, Dublin, Ireland
Seamas C Donnelly - Department of Medicine, Trinity College Dublin and Tallaght Hospital, Dublin, Ireland
Pradeep K Singh - Department of Microbiology, Washington University Medical School, Seattle, Washington, USA
David A Stoltz - Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
Christopher Haslett - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Paul B McCray - Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
Moira K B Whyte - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Adriano G Rossi - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Donald J Davidson - UoE/MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK
Resource Type: Journal article
Publication Details: Thorax, Vol.73(2), pp.134-144
DOI: 10.1136/thoraxjnl-2017-210134
PMID: 28916704
PMCID: PMC5771859
NLM abbreviation: Thorax
ISSN: 0040-6376
eISSN: 1468-3296
Grant note: DOI: 10.13039/100004440, name: Wellcome Trust
Language: English
Date published: 02/2018
Academic Unit: Roy J. Carver Department of Biomedical Engineering; Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Microbiology and Immunology; Pulmonary Medicine; Stead Family Department of Pediatrics; Surgery; Internal Medicine
Record Identifier: 9984025416602771

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