Fig. 1

Study design overview. MSCs were primed with polyinosinic:polycytidylic acid (poly I:C) to stimulate TLR3 receptors or interferon gamma (IFN-γ) to induce inflammation. MSCs on co-culture inserts were exposed to proinflammatory macrophages in the bottom of transwells. After co-culture, MSCs were removed, placed in a new well, and lymphocytes were added to allow direct MSC–lymphocyte contact. Gene expression of immunogenicity (MHC-I, MHC-II), immunomodulation (IDO, PTGS2, TGF-β1), and inflammatory (IL-6, IL-8, CCL2, CXCL10) mediators was analyzed. T-cell proliferation assays with mitogen concavalinA (ConA) characterized functional changes in MSC immunomodulation. Macrophage gene expression of cytokines (IL-6, IL-10, TNF-α, IFN-γ) and chemokines (CCL2, CXCL10) assessed to characterize effect of inflammatory primed MSCs on macrophages. To analyze the functional significance of MSC secretome after priming, MSC conditioned media was generated over a period of 48 h. This primed MSC secretome was then placed on IL-1β-stimulated or untreated chondrocytes, and effect of conditioned media was assessed by measuring inflammatory (IL-6, TNF-α, CCL2, CXCL10, MMP-13, PTGS2) and matrix (ACAN, COL2A1) gene expression changes in chondrocytes. MSC mesenchymal stem cell, MHC major histocompatibility complex, IDO indoleamine 2,3-dioxygenase, NOS2 inducible nitric oxide synthase, PTGS2 prostaglandin-endoperoxide synthase 2, TGF-β1 transforming growth factor beta-1, IL interleukin, CCL2 C-C motif chemokine 2, CXCL10 C-X-C motif chemokine 10, TNF-α tumor necrosis factor-alpha, ACAN aggrecan, COL2A1 collagen type 2