Innate Immune Pathway Activation To Modulate Mesenchymal Stromal Cell (MSC) Interaction With Synovium And Cartilage

Osteoarthritis (OA) is a progressive degenerative condition prevalent in aged animals and humans. Regenerative cellular therapies have emerged as a treatment option for OA with mixed results in efficacy which have been attributed in part to heterogeneity within stromal cell populations and between donors. Activation of mesenchymal stromal cells (MSC) by priming their ligands prior to administration has been demonstrated as a means to generate more homogenous cell populations thereby potentially achieving more consistent outcomes with cell therapy administration. We have demonstrated improved functional and histological outcomes in a murine destabilization of the medial meniscus model of OA with primed MSC therapy. Therefore, the goal of this study was to evaluate and compare the immunomodulatory properties of two ligands on cytokine and gene expression of MSC themselves and the effect of primed MSC conditioned media on key joint cells to further understand that observed effect mechanistically in the context of OA. Overall design: MSC were activated with either Toll-like receptor (TLR)-3 agonist polyinosinic:polycytidylic acid (pIC) or Stimulator of INterferon Genes (STING) agonist (2'3'cGAMP) at 10 ug/mL for 2 hours, washed and cultured an additional 24 hours to generate MSC conditioned media (MSC-CM). Key effector cells relevant to OA (peripheral blood derived macrophages, chondrocytes and synoviocytes) were stimulated with IL-1ß and TNF-a and treated with activated MSC supernatants for 24 hours. The cells were washed and cultured an additional 24 hours and supernatants were collected. Secreted cytokines in conditioned media from MSC themselves and MSC-treated joint cells were measured by multiplex immunoassay and ELISA. Bulk RNA sequencing was performed on activated MSC and MSC-CM treated joint cells to determine transcriptomic responses to treatment via an Illumina based platform. Data analysis included comparison of differentially expressed genes and pathway analysis in treated vs. untreated MSC, macrophages, synoviocytes, and chondrocytes, and between treatment groups themselves.