Human plasma extracelular vesicles induce an alternative/growth promoting phenotype in M-CSF macrophages exposed to resiquimod via PGE-2 signalling

FABIANO MARTINA; LEICAJ M LUZ; ADAMCZYK ALAN; CABRERIZO GONZALO; REMES LENICOV FEDERICO; OSTROWSKI MATIAS; PEREZ PAULA S

Congreso; SAIC.SAI.AAFE.NANOMED.AR 2021; 2021

Extracellular Vesicles are cell-derived membranous structures important in cell communication, capable of modifying recipient cells’responses due to their different cargoes. We previously showedthat plasma EVs (pEVs) dampen inflammatory cytokine responseof M-CSF macrophages exposed to a viral PAMP, having a possiblerole in tissue homeostasis after an immune response.Herein, we aim to characterize the phenotype of pEV-treated M-CSFmacrophages exposed to a viral PAMP and to investigate whether PGE2, a known regulatory mediator, could be implicated in thisphenotype.pEVs were purified from healthy donor plasma by size-exclusionchromatography followed by ultracentrifugation, and characterizedby western blotting (WB). Human monocytes were isolated fromhealthy donors’ buffy coats via density gradient centrifugation followed by positive isolation with CD14 magnetic beads, and differentiated with M-CSF for 7 days. Resiquimod (RSQ), a TLR-7/8 agonist,was used as a viral PAMP. Apoptotic-cell phagocytosis was evaluated by flow cytometry. Expression of selected genes was assessedby qPCR. Phosphorylated signaling proteins were detected by WB.PGE2 was detected by a competitive assay.Macrophages exposed to RSQ in the presence of pEVs showedincreased apoptotic-cell phagocytosis and expression of VEGFa,CD300e, RGS2 and THBS1 transcripts at 4h and 24h. They alsopresented higher levels of PGE2 in 24h-supernatants, and augmented pCREB at 20 min post-stimuli.To conclude, pEVs promote a growth-promoting and wound-healingphenotype on M- CSF macrophages exposed to RSQ, which maycontribute to inflammation resolution. Although further studies areneeded, we propose that by stimulating PGE2 production and CREBmediated transactivation, pEVs dampen macrophage activation following an encounter with a viral PAMP.