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. 2018 Feb;73(2):134-144.
doi: 10.1136/thoraxjnl-2017-210134. Epub 2017 Sep 15.

Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis

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"V体育平台登录" Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis

VSports手机版 - Robert D Gray et al. Thorax. 2018 Feb.

V体育2025版 - 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. VSports手机版.

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. V体育安卓版.

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 V体育ios版. 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 VSports最新版本. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation. .

Keywords: Cystic Fibrosis; Neutrophil Biology V体育平台登录. .

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Conflict of interest statement (VSports在线直播)

Competing interests: None declared.

Figures

Figure 1
Figure 1
CF neutrophils have increased survival due to less apoptosis. (A) Cultured CF neutrophils are more viable at 24 hours than healthy controls when assessed by morphology (n=6 CF, 5 controls). (B) Freshly isolated viable neutrophils in ex vivo culture demonstrating multilobed nuclei (×100 objective). (C) Apoptotic neutrophils following prolonged ex vivo culture demonstrating characteristic nuclear condensation (×100 objective). (D) Flow plot of control neutrophils at 24 hours ex vivo culture demonstrating small numbers of viable cells (V=annexin V−ve/PI−ve cells) with the majority of cells having entered apoptosis (A=annexin V+ve/PI−ve) or completed apoptosis (CA=annexin V+ve/PI+ve). (E) Control neutrophils have a higher rate of completed apoptosis than CF (n=19 CF and 20 controls, for patient details see online supplementary table E1). (F) More viable neutrophils are present in CF culture at 24 hours (n=19 CF and 20 controls). (G) Addition of the pan-caspase inhibitor Q-VD-OPh hydrate to CF neutrophil culture increases survival by inhibiting constitutive apoptosis. For patient details, see online supplementary table E1. Data presented as mean±SEM. Analysis with two-way analysis of variance and Bonferroni (A, E, G), unpaired t-test (F). **p<0.01; ***p<0.001. CF, cystic fibrosis; PI, propidium iodide; QVD, Q-VD-OPh hydrate.
Figure 2
Figure 2
Delayed neutrophil apoptosis in CF is related to a loss of cystic fibrosis transmembrane conductance regulator (CFTR) function. Neutrophils were harvested from 12 patients with at least one G551D mutation before and after starting the CFTR potentiator ivacaftor (for patient details, see online supplementary table E2), and cultured ex vivo for 24 hours. (A) and (B) demonstrate example flow plots (following 24 hours ex vivo culture) from an individual patient before (A) and 2 days after (B) starting ivacaftor, showing that neutrophil survival decreased with treatment. (C) Combined data for 12 patients receiving ivacaftor, neutrophil viability decreased significantly with treatment. (D) Neutrophils were harvested from 2-week-old CF piglets and wild-type (WT) controls. Flow cytometry following 24 hours ex vivo culture demonstrated increased survival in CF (5 CF pigs and 5 WT controls). (E) Representative cytocentrifuge preparations of CF and WT neutrophils at 0 and 24 hours (×100 objective) demonstrating increased numbers of apoptotic neutrophils in WTs at 24 hours. Data presented as mean±SEM. Analysis with paired t-test (C), Mann-Whitney U test (D). *p<0.05. CF, cystic fibrosis; PI, propidium iodide.
Figure 3
Figure 3
Prolonged CF neutrophil lifespan is not caused by inflammation. CF neutrophils have normal baseline apoptotic signalling of (A) Mcl-1, a 37 kDa antiapoptotic protein, and (B) BAX, a 21 kDa proapoptotic protein when freshly isolated (n=8 CF and 8 healthy controls, for patient details see online supplementary table E3). (C) Mcl-1 and BAX densitometry. (D) CF neutrophils retain sensitivity to delayed apoptosis with prosurvival stimuli GM-CSF (20 ng/mL) and LPS (10 ng/mL) for 24 hours (n=19 CF and 20 controls, for patient details see online supplementary table E1). (E) The primary apoptosis defect in CF is not due to a circulating CF serum factor. Healthy control neutrophils were cultured in media containing 5% pooled CF serum and this did not lead to increased neutrophil survival (n=3 separate healthy donors). (F) The survival defect in CF could be corrected by culture with AT7519 (1 µM) for 24 hours to augment neutrophil apoptosis and effectively reduce survival to healthy control levels (n=19 CF and 20 controls, for patient details see online supplementary table E1). Data presented as mean±SEM. Analysis with unpaired t-test (C), one-way analysis of variance with Newman-Keuls post-test (D, F). *p<0.05; **p<0.01; ***p<0.001. CF, cystic fibrosis; GMCSF, granulocyte-macrophage colony-stimulating factor; LPS, lipopolysaccharides; PI, propidium iodide.
Figure 4
Figure 4
CF neutrophils form more NETs than healthy controls under conditions of ageing in culture. (A) Freshly isolated CF neutrophils form similar amounts of NETs as healthy controls (11 CF vs 6 controls, for patient details see online supplementary table E3) following stimulation with 10 nM phorbol myristate acetate (PMA). (B) DNA release as surrogate marker of NET production yielded similar results from PMA-treated freshly isolated neutrophils (7 CF vs 6 controls), although we observed a non-statistically significant increase in DNA release at later time points by CF neutrophils. (C) Neutrophils aged in culture for 6 hours prior to stimulation with PMA (10 nM) demonstrated increased NET production by CF neutrophils versus controls (n=6 CF vs 6 controls). (D) Increased NET production in aged neutrophils was confirmed by DNA release assay (n=6 CF vs 6 controls). (E) Representative fluorescent microscopy following addition of SYTOX green in non-fixed cells, demonstrating increased PMA-induced NETs in CF following neutrophil ageing compared with controls (and also non-aged CF). Both diffuse morphology and spread morphology NETs are seen (scale bar=10 µm). Data presented as mean±SEM. Analysis with unpaired t-test (A, C), two-way analysis of variance with Bonferroni (D). **p<0.01; ***p<0.001. CF, cystic fibrosis; NET, neutrophil extracellular trap; OD, optical density.
Figure 5
Figure 5
CF neutrophils form more NETs due to their prosurvival phenotype. (A) Healthy control and CF neutrophils were cultured for 6 hours in the presence of GM-CSF (2.5 ng/mL)±AT7519 (1 µM) and then stimulated with PMA (10 nM). The addition of GM-CSF to control neutrophils increased NET formation to that of CF levels. This effect was reversed by AT7519, suggesting that inducing apoptosis stopped NET formation (n=6 CF and 6 healthy controls). (B) Representative fluorescent microscopic images showing SYTOX positive diffuse NETs in culture, enhanced by GM-CSF and inhibited by AT7519 in a patient with CF (scale bar=10 µm). Data presented as mean±SEM. Analysis with one-way analysis of variance with Newman-Keuls post-test (A). **p<0.01; ***p<0.001. CF, cystic fibrosis; GMCSF, granulocyte-macrophage colony-stimulating factor; NET, neutrophil extracellular trap; PMA, phorbol myristate acetate; AT, AT7519.
Figure 6
Figure 6
NETs stimulate an inflammatory response from macrophages and this is exaggerated in CF. (A) Scanning electron microscopy of bronchus from patient with explanted CF lung showing characteristic strands of NET-like material associated with neutrophils and bacteria. (B) Outline of novel NET/MDM coculture system. (C) NETs are proinflammatory to MDMs, causing an increase in IL-8 production after 24 hours of coculture and this effect is more pronounced with CF MDMs (n=8 CF and 7 healthy MDM donors, for patient details see online supplementary table E3). (D) NETs induce TNF production from MDMs. (E) NETs induce CCL17 expression in healthy and CF MDMs. (F) NETs induce a non-significant increase in CXCL-9 expression in healthy controls, whereas CXCL-9 is overexpressed at baseline in CF. Data presented as mean±SEM. Analysis by one-way analysis of variance with Newman-Keuls post-test (C–F). **p<0.01; ***p<0.001. CCL, chemokine (C-C motif) ligand; CF, cystic fibrosis; CXCL, chemokine (C-X-C motif) ligand; IL, interleukin; MDM, monocyte-derived macrophage; NET, neutrophil extracellular trap; PMA, phorbol myristate acetate; TNF, tumour necrosis factor; WT, wild type.

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