Plasma extracellular vesicles dampen acute inflammator y responses in neutrophils stimulated with bacterial PAMPS

FABIANO, MARTINA; ADAMCZYK, ALAN; LEICAJ, LUZ; PEREZ, PAULA; MAZZITELLI, IGNACIO; ERRA DIAZ, FERNANDO; SABBIONE, FLORENCIA; TREVANI, ANALIA; OSTROWSKI, MATÍAS

Seattle

Congreso; SEV2023 Annual Meeting | Seattle, Washington | 17-21 May; 2023

ISEV

Introduction: Neutrophils are key players at inflammatory foci, where they exert defensive functions by producing reactive oxygen species and cytokines, phagocytosing microbes and releasing NETs. However, to avoid tissue damage neutrophil activity has to be controlled. We have previously shown that healthy donors’ plasma EVs (pEVs) inhibit macrophage inflammatory response 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. Western blot 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 degranu- lation (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 (Annexin V 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 both oxidative burst and degranulation following fMLP stimulation. Likewise, phagocytosis of Candida sp. was impaired in pEV- treatedneutrophils.Incontrast,pEVsboostedIL-8productioninresponsetoLPSstimulation,ascomparedtocellsnotexposed to pEVs.Summar y/Conclusion: In conclusion, pEVs modulate neutrophils activity at the inflammator y foci, contributing to control acute inflammation by diminishing neutrophils’ respiratory burst, degranulation and phagocytosis. Concurrently, pEVs could contribute to tissue repair by promoting the secretion of the angiogenic cytokine IL-8. Together with previous observations from our group, these results suggest that pEVs are homeostatic regulators of acute inflammation.Funding: Argentinean National Agency for Science and Technology Promotion (ANPCYT) under Grants No. PICT 2015-0658, PICT-2018-02202 and PICT-2019-02506.