Departments of Internal Medicine Jagiellonian University Medical College, Krakow, Poland.
Institute of Pharmacology, University of Bern, Bern, Switzerland.
J Lipid Res. 2020 Jan;61(1):1-9. doi: 10.1194/jlr.M092072. Epub 2019 Nov 18.
Activation of neutrophils is an important mechanism in the pathology of granulomatosis with polyangiitis (GPA). In this study, we evaluated whether extracellular vesicles (EVs) circulating in the plasma of GPA patients could contribute to this process. EVs from the plasma of GPA patients in the active stage of the disease (n = 10) and healthy controls (n = 10) were isolated by ultracentrifugation and characterized by flow cytometry (CD63, CD8) and nanoparticle tracking analysis. Targeted oxylipin lipidomics of EVs was performed by HPLC-MS/MS. EV/oxylipin-induced neutrophil extracellular traps (NETs) were analyzed by confocal microscopy, and released double-stranded DNA (dsDNA) was quantified by PicoGreen fluorescent dye. Reactive oxygen species (ROS) production and neutrophils' EV binding/uptake were evaluated by flow cytometry. Brief priming with granulocyte-macrophage colony-stimulating factor was required for EV-mediated ROS production and dsDNA release. It was observed that priming also increased EV binding/uptake by neutrophils only for EVs from GPA patients. EVs from GPA patients had higher concentrations of leukotriene (LT)B and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) as compared with EVs from healthy controls. Moreover, neutrophils stimulated with LTB or 5-oxo-ETE produced ROS and released dsDNA in a concentration-dependent manner. These results reveal the potential role of EVs containing oxylipin cargo on ROS production and NET formation by activated neutrophils.
中性粒细胞的激活是肉芽肿性多血管炎（GPA）病理生理学中的一个重要机制。在本研究中，我们评估了 GPA 患者循环血浆中的细胞外囊泡（EVs）是否有助于这一过程。通过超速离心从处于疾病活动期的 GPA 患者（n=10）和健康对照者（n=10）的血浆中分离 EVs，并通过流式细胞术（CD63、CD8）和纳米颗粒跟踪分析进行特征鉴定。通过 HPLC-MS/MS 进行 EV/氧化脂质组学靶向分析。通过共聚焦显微镜分析 EV/氧化脂质诱导的中性粒细胞细胞外陷阱（NETs），并通过 PicoGreen 荧光染料定量释放的双链 DNA（dsDNA）。通过流式细胞术评估活性氧（ROS）的产生和中性粒细胞 EV 结合/摄取。EV 介导的 ROS 产生和 dsDNA 释放需要用粒细胞-巨噬细胞集落刺激因子进行短暂预刺激。观察到，仅针对来自 GPA 患者的 EV，预刺激还增加了 EV 结合/摄取中性粒细胞的作用。与健康对照组相比，来自 GPA 患者的 EV 具有更高浓度的白三烯（LT）B 和 5-氧代二十碳四烯酸（5-oxo-ETE）。此外，ROS 和 dsDNA 以浓度依赖的方式由 LTB 或 5-oxo-ETE 刺激的中性粒细胞释放。这些结果揭示了含有氧化脂质货物的 EV 在激活的中性粒细胞中 ROS 产生和 NET 形成中的潜在作用。
