Extracellular vesicles in sputum of children with cystic fibrosis pulmonary exacerbations

E. Ben-Meir, Lina Antounians, Shafinaz Eisha, F. Ratjen, Augusto Zani, Hartmut Grasemann
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Abstract

The aim of this study was to quantify mediators of neutrophilic inflammation within airway extracellular vesicles (EVs) of children treated for a cystic fibrosis (CF) pulmonary exacerbation (PEx).EVs were isolated from stored sputum samples collected before and after antibiotic therapy for PEx between 2011–2013, and characterised by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Western blot analysis of EV protein extracts was used for EV canonical protein markers CD63, CD9, and flotillin-1 (FLOT1), as well as neutrophil elastase (NE), myeloperoxidase (MPO) and interleukin-8. EV content of NE and MPO was expressed as ratios of NE/FLOT1 and MPO/FLOT1 protein band densities.Sputum samples from 21 children aged 13.3 (range 8.0–17.0) years were analysed. NTA showed high concentrations of particles at the size of small EVs (50–200 nm), and typical EV morphology was confirmed by TEM. CD63, CD9 and FLOT1 was detectable in all samples. Median (IQR) NE/FLOT1 increased from 2.46 (1.68–5.25) before to 6.83 (3.89–8.89, p<0.001) after PEx therapy and MPO/FLOT1 from 2.30 (1.38–4.44) to 5.76 (3.45–6.94, p<0.01), while EV size remained unchanged. Improvement in lung function (ppFEV1) with PEx therapy correlated with NE EV content (r=0.657, p=0.001).Airways of children with CF contain EVs that carry NE and MPO as cargo. The lower NE and MPO content at the time of PEx compared to after therapy and the correlation with pulmonary function suggest both a functional role of EVs in CF airway inflammation and potential as a biomarker to monitor CF lung disease.
囊性纤维化肺恶化儿童痰中的胞外囊泡
本研究旨在量化因囊性纤维化(CF)肺部恶化(PEx)而接受治疗的儿童气道细胞外囊泡(EV)中的中性粒细胞炎症介质。EV是从2011-2013年间因PEx而接受抗生素治疗前后收集的贮存痰液样本中分离出来的,并通过纳米颗粒追踪分析(NTA)和透射电子显微镜(TEM)对其进行了表征。对 EV 蛋白提取物进行了 Western 印迹分析,以检测 EV 的典型蛋白标记物 CD63、CD9、flotillin-1 (FLOT1),以及中性粒细胞弹性蛋白酶 (NE)、髓过氧化物酶 (MPO) 和白细胞介素-8。NE和MPO的EV含量以NE/FLOT1和MPO/FLOT1蛋白条带密度的比率表示。NTA显示出高浓度的小EV颗粒(50-200 nm),TEM证实了典型的EV形态。所有样本均可检测到 CD63、CD9 和 FLOT1。PEx治疗后,NE/FLOT1的中位数(IQR)从治疗前的2.46(1.68-5.25)增加到6.83(3.89-8.89,p<0.001),MPO/FLOT1从2.30(1.38-4.44)增加到5.76(3.45-6.94,p<0.01),而EV的大小保持不变。PEx疗法对肺功能(ppFEV1)的改善与NE EV含量相关(r=0.657,p=0.001)。与治疗后相比,PEx治疗时的NE和MPO含量较低,且与肺功能相关,这表明EVs在CF气道炎症中发挥着功能性作用,并有可能成为监测CF肺部疾病的生物标记物。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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