Extracellular vesicles derived from DFO-preconditioned canine AT-MSCs reprogram macrophages into M2 phase

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) are effective therapeutic agents that ameliorate inflammation through paracrine effect; in this regard, extracellular vesicles (EVs) have been frequently studied. To improve the secretion of anti-inflammatory factors from MSCs, preconditioning with hypoxia or hypoxia-mimetic agents has been attempted and the molecular changes in preconditioned MSC-derived EVs explored. In this study, we aimed to investigate the increase of hypoxia-inducible factor 1-alpha (HIF-1α)/cyclooxygenase-2 (COX-2) in deferoxamine (DFO)-preconditioned canine MSC (MSC(DFO)) and whether these molecular changes were reflected on EVs. Furthermore, we focused on MSC(DFO) derived EVs (EV(DFO)) could affect macrophage polarization via the transfer function of EVs. RESULTS: In MSC(DFO), accumulation of HIF-1α were increased and production of COX-2 were activated. Also, Inside of EV(DFO) were enriched with COX-2 protein. To evaluate the transferring effect of EVs to macrophage, the canine macrophage cell line, DH82, was treated with EVs after lipopolysaccharide (LPS) stimulation. Polarization changes of DH82 were evaluated with quantitative real-time PCR and immunofluorescence analyses. When LPS-induced DH82 was treated with EV(DFO), phosphorylation of signal transducer and transcription3 (p-STAT3), which is one of key factor of inducing M2 phase, expression was increased in DH82. Furthermore, treated with EV(DFO) in LPS-induced DH82, the expression of M1 markers were reduced, otherwise, M2 surface markers were enhanced. Comparing with EV(DFO) and EV(non). CONCLUSION: DFO preconditioning in MSCs activated the HIF-1α/COX-2 signaling pathway; Transferring COX-2 through EV(DFO) could effectively reprogram macrophage into M2 phase by promoting the phosphorylation of STAT3.