Immunomodulatory abilities of licensed mesenchymal stem cells: a potential therapeutic strategy in osteoarthritis

Purpose: Synovial inflammation associated with production of pro-inflammatory mediators and active immune cells proliferation, plays an important role in the onset and progression of osteoarthritis (OA). Regenerative therapies based on mesenchymal stem/stromal cells (MSCs) hold great promise for the disease modulation of OA, however the precise therapeutic mechanisms and interactions with the inflammatory environment in the OA joint still needs to be elucidated. Our previous studies identified elevated production of pre- and anti-inflammatory cytokines in the synovial washouts from OA joints in a murine model. The aim of the present study was to investigate the in vitroimmunomodulatory effects of cytokine licensed MSCs on resting and activated bone-marrow derived macrophages (BMDMs). The proposed modulation strategy could provide a better understanding of the interaction between MSCs and immune cells, which gives valuable insight on the development of new therapies for the treatment of OA. Methods: MSCs isolated from bone marrow of C57/BL6 mice, were licensed singularly and simultaneously with IL - 6 (50ng/ml) and Monocyte Chemoattractant Protein-1 (MCP-1) (50ng/ml) for 72h. BMDMs of C57BL/6 mice were plated and differentiated for 6 days in macrophage media. Polarization was induced by stimulation with LPS (10ng/ml for 4h) and IFN-gamma (100ng/ml for 24 hours), to generate classical BMDMs. Alternate activated macrophages were obtained following an incubation with 10ng/ml IL-4 and IL-13 for 24h. Licensed MSCs were directly co-cultured respectively with resting (M0), classical activated (M1) and alternate activated (M2) macrophages for 72 hours, in ratio 2:1 (5x104MSCs: 2.5 x 104BMDMs). Co-cultured BMDMs were collected for flow cytometry analysis (FACS Canto cytometer). The release of pro- and anti-inflammatory cytokines in cell supernatants was measured with ELISA, and Griess assay was performed to quantify the production of nitrite. Results: Flow cytometric characterization of differentiated macrophages showed the expression of CD11b, a pan-macrophage marker. The successful generation of activated macrophages was confirmed with the positive expression of MHC-II in M1 and CD206 in M2 macrophages. Flow cytometrical analysis and ELISA showed a modulating effect of licensed MSCs on resting, classical and alternate activated macrophages. Co-cultured resting macrophages had an increased expression of MHC-II and CD206 expression compared to control BMDMs, associated with a higher production of PGE2 and MCP-1. Similarly, licensed MSCs increased the expression of CD206, while maintaining a high MHC-II expression on classical activated M1 macrophages, characterized with elevated production of PGE2 and MCP-1. M2 macrophages were characterized with a high expression of CD206 and MHC-II expression, and increased PGE2 and MCP-1 secretion. Griess assay showed that co-cultured M0, M1 and M2 macrophages had a higher production of NO, compared to control BMDMs. Conclusions: In summary, the present work demonstrates the effect of IL-6 and MCP-1 as licensing factors, on the immunomodulatory properties of MSCs on BMDMS in vitro. The effect is mediated mostly by an increased secretion of PGE2 and MCP-1, associated with MHC-II+CD206+ macrophages. In combination with the increased NO production, the licensing seems to induces an anti-inflammatory mediated BMDMs activity. We demonstrated that cytokine licensing could be a feasible strategy to enhance the therapeutic effects of MSCs for OA treatment. Our data give a valuable inside on the development of future in vivo validation studies and therapeutic strategies for the modulation of the inflammatory milieu of the OA joint.