Annexin a5(An5)-bound extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) show enhanced and specific antiinflammatory effects

Background: An5 is a physiological molecule with tolerogenic properties. Since An5 can bind to negatively charged phospholipids like phosphatidylserine on the surface of EVs, we reasoned that this molecule could affect the well-recognized immune regulatory properties of MSCEVs. Therefore, we compared the immunological effects of MSC-EVs and of the AnV-MSC-EVs complex both in vitro and in vivo. Methods: An5 was bound to human Wharton Jelly-derived MSC-EVs using a commercial kit. Our in vitro assay consisted of a mixed lymphocyte reaction with splenocytes from two different mice strains (C57BL/6 and BALB/c). Mice with dextran sulphate sodium (DSS)- induced colitis (3% in drinking water for 5 days) were used to test the anti-inflammatory effect in vivo of EVs given via enema. Results: In vitro, both MSC-EVs and An5-MSC-EVs exhibited a suppressive effect both on B and T lymphocytes, although the latter showed a more pronounced effect. In vivo, MSC-EV administration via enema had no effect on body weight, disease activity index, colon length and histological parameters. In contrast, enema administration of AnVMSC- EVs dramatically improved both clinical and morphometric/histological scores. Free (unbound) An5 administration had no effect on colitis severity. MSC-EVs and An5-MSC-EVs induced a different pattern of cytokine expression in colon mucosa. Summary/conclusion: In conclusion, An5 binding enhanced the antiinflammatory activity of MSC-EVs both in vitro and in vivo. The dissimilar pattern of cytokine expression observed in vivo following treatment with MSC-EVs or An5-MSC-EVs suggests that An5 binding affects the mechanisms of action of MSC-EVs.