Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis

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.

NETs in pneumonia: is just enough the right amount?

“…The observation that corticosteroids lead to a prolonged lower level of NETs in recovering patients who have a better outcome does perhaps suggest a beneficial role for NETosis during the process of recovering from pneumonia, and would be in keeping with other recent data suggesting that patients who survive sepsis may indeed have a greater ability to make NETs than non-survivors [24]. Recent data from CF patients has suggested that a delay in neutrophil apoptosis in these subjects leads to an increased ability to form NETs [21], suggesting that the activation of alternative routes of disposal for neutrophils may have consequences in terms of promoting inflammation. Indeed the fact that corticosteroids are known to delay neutrophil apoptosis may be of direct consequence in the present study by preferentially allowing NETosis in those treated with prednisolone.…”

“…NETs have been associated with inflammation in a number of sterile inflammatory conditions such as systemic lupus erythematosus, rheumatoid arthritis, small vessel vasculitis, gout and cardiovascular disease [19], underlining a role for NETs in inflammation, although the role of NETs in the inflammatory response to infection is less well defined. Crucially the mechanisms by which NETs can promote inflammation are now being revealed and recent evidence suggests that the interaction of NETs with macrophages may be key to this [20,21]. Therefore, NETs may have a crucial role to play in the crosstalk between cells of the innate (and adaptive) immune system, promoting inflammation, whereas other forms of neutrophil disposal such as apoptosis have a defined role in driving the resolution of inflammation [22].…”

Inflammation in cystic fibrosis: An update

“…In this later situation, NETs are more likely to be harmful and contribute to the immunopathology in the CF airway. Gray et al demonstrated that CF neutrophils formed more NETs and lived longer due to decreased apoptosis than did non‐CF neutrophils . The opposite was found in people with G551D who were being treated with ivacaftor.…”

“…The opposite was found in people with G551D who were being treated with ivacaftor. The investigators also found that NETs provided a pro‐inflammatory stimulus to macrophages, which was enhanced in CF . The CF lung is an especially conducive environment for NETosis.…”

Mechanisms and disease relevance of neutrophil extracellular trap formation

“…During the release of NETs, the plasma membrane ruptures in a programmed manner. This suggests that the formation of NETs involves a suicide programme, morphologically different from other types of cell death, such as apoptosis and necrosis . One form of programmed cell death, necroptosis, can result in cytolysis.…”