Objectives: In recent years, it has become clear that mesenchymal stem cells (MSCs) do not only have the ability to differentiate into different cell types but also possess immunomodulatory properties. However, the underlying mechanism of MSCs-mediated immunoregulation is not fully understood so far. Proinflammatory (M1) and alternatively activated (M2) macrophages exhibit different functions and properties. In this study, we have explored the immunosuppressive effects of bone marrow-derived MSCs in respect to their ability to promote a shift in macrophage phenotype in dependence of their activation state. We hypothesize that pre-activation of MSCs might represent a better strategy for their future application in clinic by improving immunosuppressive responsiveness.
Methods: Murine bone-marrow derived MSCs and cytokine pre-activated MSCs were co-cultured with macrophages under M1 and M2 polarizing conditions. Both M1 macrophages as well as three different M2 macrophage subtypes (M2a, M2b and M2c) were further characterized by real time PCR, flow cytometry, ELISA and Griess assay.
Results: Pre-activation of MSCs resulted in a strong increase of inducible nitric oxide (iNOS) expression accompanied by increased amounts of secreted NO and proinflammatory IL-6 in the culture supernatants. Co-culture experiments under M1 polarizing conditions revealed significant downregulation of CD86, intracellular iNOS expression and secretion of pro-inflammatory tumor necrosis factor α (TNF-α) in macrophages in the presence of pre-stimulated MSCs independent of direct cell contact. Additionally, under M2 polarizing conditions, macrophages were found to upregulate CD86, iNOS and IL-10 indicating M2b polarization in the presence of activated, but not in the presence of unstimulated MSCs. Indeed, MSC-mediated polarization toward a M2b phenotype in M1- as well M2-like cells was confirmed by strong upregulation of the M2b markers sphingosine kinase 1 (SPHK1) and LIGHT, whereas only weak changes in M2a-specific chitin-like 3 (Ym1) and M2c-typical Mer tyrosine Kinase (MertK) could be observed.
Conclusion: Pre-activated MSCs suppress M1 polarization and rather promote a polarization shift toward a regulatory M2b phenotype which is known to reduce inflammation. The soluble factors involved in MSCs-mediated M2b polarization are not fully identified and warrant further investigation in the future.
