Analysis of the immunomodulatory potential of mesenchymal stem cell-derived extracellular vesicles in an model of Alzheimer’s disease

Aims: Human mesenchymal stem cells (hMSCs) possess potent immunosuppressive capacity that mainly depends on paracrine mechanisms. Here we have optimized ex vivo strategies to obtain MSCs endowed with improved capabilities of modulating the innate immunity. With the aim to develop a cell-free system to target inflammatory processes in AD we are investigating the immunomodulatory potential of MSC-derived extracellular veiscles (MSC EVs) on microglia polarization in in vitro model of inflammation and in vivo AD animal model. Method: To induce an immunosuppressive phenotype, bone marrow hMSCs were subjected to different preconditioning protocols [(oxygen deprivation (OD) or pro-inflammatory cytokines (PC)] and characterized by WB for COX2 and IDO (indoleamine 2,3-dioxygenase) protein levels, known to be responsible for MSC immunomodulatory competence. Preconditioned MSC EVs were isolated from MSC culture medium by ultracentrifugation. In in vitro experiments microglia - extracted from cortices and hippocampi of postnatal 1-2 day old mice - were treated with EVs 2 h after inflammatory challenge and their immunomodulatory effects were assessed after 48 h by evaluating cytokine release. 7-month-old 3xTg mice used in in vivo experiments were intranasally injected with 15 x10 9 EVs and sacrificed after 3 weeks for the analysis of cortical and hippocampal microglial phenotype. Golgi Cox was used for the staining of neuronal dendritic branching. Results: PC preconditioning turned out to be the best strategy to switch MSC phenotype towards a more immunosuppressive one as indicated by the upregulation of COX2 and IDO markers. MSC EVs seem to foster microglia M2 phenotype as evidenced by the increased release of anti-inflammatory cytokines (IL-10, IL-4) and negative modulation on the pro-inflammatory cytokine IL-6. Conclusion: Preconditioned-MSC EVs may represent therapeutic tools to dampen neuroinflammation in AD by influencing the behaviour of microglia. The analysis of EV content and their potential effects on glial and neuronal cells in 3xTg models is under investigation