Implication of 14-3-3epsilon as a potential alarmin in bone/cartilage communication in osteoarthritis

Abstract

Purpose: Osteoarthritis (OA) is the most frequent and disabling joint disease. OA is now considered as a whole-joint disease characterized by cartilage degradation, synovial inflammation and bone remodeling. Our team identified 14-3-3ε as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. This protein acts as a potent MMP-3 and MMP-13-stimulatory factor in chondrocytes leading to catabolic phenotype. 14-3-3ε seems to share common characteristics with alarmins, endogenous molecules with intracellular functions that are released in the extracellular media after infection or tissue damage. Alarmins (such as S100 and HMGB1 involved in cartilage degradation) bind to receptors expressed on the surface of cells engaged in host defense and tissue repair. These receptors include the Toll-like receptors, TLR2 and TLR4, as well as RAGE. Moreover, innate alarmins can stimulate the activation of resident immune cells, particularly macrophages. Our aim is to investigate the interactions between 14-3-3ε and its potential receptors, TLR2, TLR4 and RAGE and his role as an alarmin particularly as a new molecular mediator determining macrophage polarization in OA joint. Methods: Primary cultures of chondrocytes were performed from murine cartilage of the C57BL/6J mice (5 days old), and from human cartilage samples, obtained from OA patients undergoing total joint replacement surgery. The receptors TLR2, TLR4 and RAGE were blocked by monoclonal antibodies and pharmacologic inhibitors in chondrocytes (15mn) and then stimulated by recombinant 14-3-3ε during 24h. Immunocytochemistry was realized on murine chondrocytes with receptors antibodies. Chondrocyte gene expression and release of MMP-3, MMP-13 and IL-6 were evaluated by RT-PCR and ELISA. The direct interaction of 14-3-3ε and TLR2 was measured using SPR on a Biacore 3000 instrument. The potential 14-3-3ε-binding sites on receptors were investigated by in silico computational analysis PyMOL. Polarisation M1/M2 of marcrophages was performed by activating THP1 cells (ATCC) with PMA during 24h and stimulating them 24h hours with IL-4, LPS or 14-3-3ε. Different markers of the M1/M2 types were analysed by ELISA and RT-qPCR to identify the polarisation of macrophages (M1: TNF, IL-6, IL-1b, MCP1, Nos2, M2: IL-10, TGFb, Arg-1, FIZZ1, YM2). Results: TLR2, TLR4 and RAGE receptors were found on murine articular chondrocytes by immunocytochemistry using monoclonal antibodies. Invalidation of TLR2, TLR4 or RAGE receptors by blocking antibody in articular chondrocytes reduced significantly mRNA expression and protein release of MMP-3, MMP-13 and IL-6 induced by 14-3-3ε. For MMP3 mRNA expression, the inhibition rate reached 78% with blocking antibodies and 98% with OxPAPC, a TLR2/TLR4 inhibitor. The inhibition rate reached 65% (blocking antibodies), 98% (OxPAPC) for MMP13 and 74% (blocking antibodies) and 93% (OxPAPC) for IL-6. ELISA experiments showed an inhibition rate of 84% (blocking antibodies) and 97% (OxPAPC) for MMP3. Similar rates are observed for IL-6 secretion (83% and 97%). These results should be confirmed on cultures of human chondrocytes. Human recombinant TLR2 and TLR4 were covalently immobilized on CM5 chip. Results showed a direct interaction between recombinant TLR2 and 14-3-3ε (83 RU) and between TLR4 and 14-3-3ε (41 RU). Modeling experiments seems to show a direct interaction between 14-3-3ε and TLR2 receptor through a phosphorylated residue (TYR). Polarization of macrophages stimulated by recombinant 14-3-3ε is ongoing and expression of M1 type is expected. Conclusions: We have discovered 14-3-3ε, a new catabolic factor, produced by the subchondral bone and able to induce a catabolic phenotype of chondrocytes. Taking together, our results could designate 14-3-3ε as a novel alarmin to be explored in OA either for therapeutic or for prognostic purposes and its interaction with receptors could be a novel target in OA.