Plasma extracelular vesicles dampen acute inflammatory responses in neutrophils stimulated with bacterial PAMPs

MARTINA P FABIANO; MARIA L LEICAJ; ALAN M ADAMCZYK; IGNACIO G MAZZITELLI; FERNANDO ERRA DIAZ; PAULA S PEREZ; MATIAS OSTROWSKI

Mar del Plata

Congreso; SAIC.SAI.SAFIS, 2022; 2022

Extracellular vesicles are heterogeneous membrane structures thatmediate intercellular communication both in physio and pathologicalconditions. We have previously shown that healthy donors’ plasmaEVs (pEVs) have anti-inflammatory and pro-resolution effectson monocyte derived macrophages simultaneously treated with aPAMP. Herein we aimed at studying the pEV-mediated modulationof neutrophil activation by diverse PAMPs. We analyzed neutrophils’oxidative burst, degranulation, cytokine production and cell viability.pEVs were purified from healthy donor plasma by size-exclusionchromatography followed by centrifugation, and characterized bywestern blotting (WB). Neutrophils were isolated from blood fromhealthy donors by centrifugation on Ficoll-Paque followed by dextransedimentation and hypotonic lysis of erythrocytes. N-Formyl-Met-Leu-Phe (fMLP)-induced oxidative burst was analyzed bydetermining dihydrorhodamine 123 (DHR) oxidation; neutrophils’degranulation was generated also with fMLP and assessed byCD11b and CD66b surface staining; cell viability was evaluated withAnnexin V and propidium iodide. Measurements were obtained byflow cytometry (FC). Cytokines in cell culture supernatants wereevaluated by ELISA. We observed that pEV treatment induced adose-dependent reduction of both oxidative burst and degranulationof neutrophils following fMLP stimulation. However, IL-8 concentrationswere higher in simultaneously LPS and pEVs treated neutrophils.Neutrophils stimulated with LPS 150 ng/mL and pEVs for18 hs showed similar live cells percentages than only LPS treatedones. In conclusion, pEVs may contribute to controlling inflammationby diminishing neutrophils’ respiratory burst and degranulation whilecontributing to wound healing by promoting the secretion of the angiogeniccytokine IL-8.