Mesenchymal stem cell therapy modulates synovial macrophages in a murine model of osteoarthritis

Abstract

Purpose: Inflammation is one of the major hallmarks of osteoarthritis (OA), with participation of synovial macrophages and infiltrated lymphocytes. Mesenchymal stem cells (MSCs) are known to modulate the response of all cells of the immune system, with a central role for apoptosis becoming evident in many disease models. Intra-articular (IA) injections of MSCs are known to protect from cartilage degeneration and modulate local inflammation, although the lack of engraftment suggests that they exert their action via paracrine mechanisms. It is also hypothesised that locally-delivered MSCs undergo apoptosis or necrosis.The aim of this study was to examine the mechanism of action of IA-delivered MSCs in an animal model of OA. Macrophages were investigated as recipients of the MSC action through ex vivo cultures. Also, the role of apoptosis was assessed with an in vitro model of cell death. Methods: Collagenase-induced OA (CIOA) was performed on C57BL/6 mice, with a saline-treated group (SHAM) used as a control. 2×10ˆ5 GFP+ MSCs were IA-injected in the animals’ knees after one week. Three days later, knee joints were digested into a single-cell suspension and MSCs retrieved by cell sorting, based on cell fluorescence intensity, and expanded for two passages in standard culture conditions. Capacity to form colonies in vitro was assessed by CFU-F assay and expression of surface markers characteristic of MSCs was measured by flow cytometry. Conditioned medium (CM) of retrieved cells was tested on syngeneic bone marrow-derived macrophages (BMDMs) activated with lipopolysaccharides (LPS) for 4 hours. Expression of major histocompatibility complex class II (MHC-II) and co-stimulatory molecule CD86 were measured by flow cytometry, while secretion of cytokines interleukin (IL)-10 and tumor necrosis factor (TNF)-α was quantified by enzyme-linked immunosorbent assay (ELISA). Apoptosis of MSCs was induced in vitro with staurosporine (STS) and confirmed by pre-treatment with the pan-caspase inhibitor Q-VD-OPh, while necrosis was induced by heat treatment at 90°C. Cell death was evaluated by Annexin V/Sytox Blue staining, analysed by flow cytometry. CM were collected and tested on BMDMs as previously described. Results: 1.63% of injected cells were retrieved and proliferated in culture. Compared to naïve (CTRL) MSCs, retrieved cells maintained morphology, proliferation rates, clonogeneicity and surface marker expression profile. CM of CIOA-retrieved MSCs modulated activation of macrophages, significantly downregulating expression of MHC-II and CD86 and upregulating secretion of IL-10, compared to both SHAM-retrieved MSCs and CTRL MSCs. TNF-α was downregulated in a similar fashion, but not significantly. STS induced apoptosis, which was completely prevented by pre-treatment with Q-VD-OPh, thus confirming initiation of caspase pathway. Necrosis was also confirmed by Annexin V/Sytox staining. Apoptotic but not necrotic MSC-CM prevented activation of BMDMs, with suppressed secretion of TNF-α and significantly increased production of IL-10. Conclusions: MSCs can be administered and retrieved from murine knees by cell sorting. Retrieval yield was low, consistently with previous in vivo cell tracking studies, confirming that most therapeutic action is performed via a hit-and-run mechanism. MSCs were licensed from the OA joint to produce an immunosuppressive milieu that modulated macrophages ex vivo, inducing an M2-like (anti-inflammatory) switch. In vitro, apoptotic MSCs showed an increased immunomodulatory potential in a way that reflected the mechanism observed in OA-challenged MSCs. This set of data suggests that the OA synovial environment licensed the MSCs to secrete immunomodulatory factors, but also that apoptosis is a contributing factor to the effects of cell therapy in OA. As expected, macrophages have a crucial role in propagating the MSC-mediated immunomodulation, by acquiring an M2-like phenotype, with inhibition of stimulatory and co-stimulatory molecules and further release of anti-inflammatory cytokines. These findings are key to the understanding of OA and the development of future cell therapies, which could be focused on apoptosis rather than integration in the degenerated tissues.