Plasma extracellular vesicles dampen acute inflammatory responses in neutrophilsstimulated with bacterial PAMPS

MARTINA P FABIANO; ALAN ADAMCZYK; MARÍA LUZ LEICAJ; PAULA S.PEREZ; IGNACIO MAZZITELLI ; FERNANDO ERRA DIAZ; FLORENCIA SABBIONE; ANALÍA TREVANI; MATÍAS OSTROWSKI

Seattle

Congreso; ISEV2023 Annual Meeting; 2023

Introduction: Neutrophils are key players at inflammatory foci, where they exert defensive functions by producing reactiveoxygen species and cytokines, phagocytosing microbes and releasing NETs. However, to avoid tissue damage neutrophil activityhas to be controlled. We have previously shown that healthy donors’ plasma EVs (pEVs) inhibit macrophage inflammatoryresponse to a PAMP. Herein, we studied whether pEVs were also able to modulate neutrophil activation.Methods: pEVs were purified from healthy donor plasma by size-exclusion chromatography followed by centrifugation.Westernblot analysis of both EV and contaminant markers (ApoA1, ApoB1, IgG) revealed that our pEV preparations were highly pure.Neutrophils were isolated from healthy donors’ blood by a standard density gradient separation method. Neutrophil degranulation(CD11b/CD66b surface expression) and oxidative burst (dihydrorhodamine oxidation) were analyzed by flow cytometry(FC) following N-Formyl-Met-Leu-Phe (fMLP) stimulation. Phagocytosis of fluorescent Candida sp. and cell viability (AnnexinV and propidium iodide labeling) were also assessed by FC. Cytokine production was evaluated by ELISA.Results: Results showed that pEV exposure did not affect neutrophil viability but induced a dose-dependent reduction of bothoxidative burst and degranulation following fMLP stimulation. Likewise, phagocytosis of Candida sp. was impaired in pEVtreatedneutrophils. In contrast, pEVs boosted IL-8 production in response to LPS stimulation, as compared to cells not exposedto pEVs.Summary/Conclusion: In conclusion, pEVs modulate neutrophils activity at the inflammatory foci, contributing to controlacute inflammation by diminishing neutrophils’ respiratory burst, degranulation and phagocytosis. Concurrently, pEVs couldcontribute to tissue repair by promoting the secretion of the angiogenic cytokine IL-8. Together with previous observations fromour group, these results suggest that pEVs are homeostatic regulators of acute inflammation.