LTB4 and 5-oxo-ETE from extracellular vesicles stimulate neutrophils in granulomatosis with polyangiitis.

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)B4 and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) as compared with EVs from healthy controls. Moreover, neutrophils stimulated with LTB4 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.