The role of bioactive lipid mediators and extracellular vesicles in mesenchymal stromal cell immunomodulation

Human mesenchymal stromal cells (hMSCs) are fibroblast-like cells that have an exceptional ability to modulate immune cells. Due to their immuno-modulatory properties, hMSCs have been employed in various clinical trials in the treatment of autoimmune or inflammatory diseases. Even though hMSC therapy has yielded multiple promising results, not all trials have been successful. Due to the discrepancies in the therapeutic response, hMSC therapy requires further development and standardisation. While optimisation of the culture conditions provides one method to improve the therapeutic efficacy of hMSCs, fine-tuning the culture conditions requires deep understanding of the hMSC immunomodulatory mechanisms. It has been established that hMSCs mediate their therapeutic effect via cell-cell contact and especially by secreting several paracrine factors that include lipid mediators and extracellular vesicles (EVs). Intriguingly, hMSC-derived EVs (hMSC-EVs) are able to mediate the therapeutic response of MSCs, which has led to a growing interest in and research on cell-free hMSC-EV therapy. In addition to EVs, lipid metabolism and especially the lipid mediator prostaglandin E2 (PGE2) are vital to the hMSC immunomodulation. Interestingly, the membrane lipid composition correlates with the immunosuppressive capacity of hMSCs, suggesting that membrane lipids play a role in mediating the immunomodulatory response of hMSCs. Lipid mediators are derived from polyunsaturated fatty acids (PUFAs) that can be stored in membrane phospholipids. Over the past few decades, the significance of PUFA-derived lipid mediators in orchestrating the dampening phase of inflammation, i.e., resolution, has been unravelled. These novel specialized proresolving mediators (SPMs) have several essential roles in modulating immune cells, including macrophages. The role of these SPMs in hMSC immunomodulation has received only marginal interest, and whether hMSCs produce multiple SPMs has not been determined. Keeping in mind that hMSC-EVs mediate the therapeutic response of MSCs, we investigated the lipid metabolism of hMSCs and their EVs after supplementing the cells with lipid mediator precursor PUFAs: arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid (DHA). Due to the supplementation, the membrane phospholipid and fatty acid composition were notably modified and the downstream lipid mediator production was enhanced. Excitingly, we were able to demonstrate for the first time that hMSCs produce several SPMs, which may mediate the immunomodulatory response of hMSCs. Remarkably, we showed that the PUFA modifications in the phospholipid composition were transferred into the EVs, highlighting the importance of EVs as transporters of the immunomodulatory factors derived from hMSCs. Because of their importance in the resolution of inflammation, we investigated the effects of hMSCs and hMSC-EVs on macrophages with a focus on regulatory macrophages (Mregs). We elucidated that both hMSCs and hMSC-EVs enhanced the anti-inflammatory and proresolving properties of these less-studied macrophages, highlighting the importance of PGE2 in the function of hMSC-EVs. Moreover, the hMSC secretome increased the CD206 expression and Candida albicans phagocytosis activity of macrophages, demonstrating a functional change in the macrophage properties. Fascinatingly, the DHA-supplemented hMSCs even further skewed the macrophage phenotype in an anti-inflammatory direction. However, the change was limited, and the elucidation of the functional effects of PUFA supplementations on hMSCs require additional investigations. In conclusion, this thesis provides further evidence that the lipid metabolism has an essential role in hMSC functionality and that the SPM production may represent an additional mechanism in hMSC immunomodulation. For the first time, we have explored the impact of hMSCs and hMSC-EVs on Mregs and our results highlight the importance of EVs as the mediators of hMSC immunomodulation. Furthermore, we investigated the possibility of improving the hMSC immunomodulation with PUFA supplementations that would represent an easy and safe way to enhance the therapeutic potential of hMSCs or hMSC-EVs. A more detailed understanding of the complex immunomodulatory mechanisms of hMSCs is in key position when investigating new possibilities in the development of hMSC therapy.