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, Dorward David, Edward F McKone, Gordon Cooke, Seamus C. Donnelly, Pradeep K Singh, David A Stoltz, Christopher Haslett, Paul B McCray, Moira K B WhyteAdriano G Rossi, Donald J Davidson

Research output: Contribution to journalArticlepeer-review

Abstract

Background: 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 hypothesized 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 CF patients, CF pigs and appropriate controls. Neutrophils were also obtained from CF patients 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 by cytokine generation by ELISA and qRT-PCR.

Results: CF neutrophils live longer due to decreased apoptosis. This was observed in both CFTR null piglets and CF patients, 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 (CDKi) exposure. NETs provided a pro-inflammatory stimulus to macrophages, which was enhanced in CF.

Conclusions: CF neutrophils have a pro-survival 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.
Original languageEnglish
Pages (from-to)134-144
Number of pages11
JournalThorax
Volume73
Issue number2
Early online date15 Sep 2017
DOIs
Publication statusPublished - Feb 2018

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