Transient Exposure of Chondrocytes to Ccl2 in the Context of a Pro-Inflammatory Environment Enhances Chondrogenesis and Their Pro-Repair Response

Cartilage injury induces CCL2 expression in chondrocytes, which is associated with production of inflammatory cytokines such as IL1B and IL6. Recently, it has been reported in a cohort study that synovial fluid levels of CCL2 and IL6 are good indicators of acute cartilage injury. Previously, using a murine osteochondral defect model, we have shown that the recruitment of macrophages in damaged cartilage is key to its successful healing. However, CCL2's influence on cross-talk between macrophages and chondrocytes is yet to be investigated. Here we demonstrate the mechanism of how CCL2 could play a crucial role in acute injury and promote cartilage repair. Monocytes were isolated from human blood and polarized towards M1 and M2 macrophages. Pro-inflammatory M1 macrophages induced similar gene expression (CCL2, IL1B and IL6) in chondrocytes to that observed upon cartilage mechanical injury. Primary human chondrocytes cultured in vitro were primed with M1 macrophage conditioned medium (CM) for 24 hours, resulting in up-regulation of stem cell associated markers (CD44, CD105, CD73 and NOTCH1). Simultaneous 24 hours exposure of chondrocytes to CCL2 and M1 conditioned medium (CM) significantly increased (33%) their migratory activity in a scratch assay compared to M1 or CCL2 alone. Furthermore, in the presence of CCL2, fluorescently labelled chondrocytes exhibited enhanced adhesion to explants of human osteoarthritic cartilage. Chondrocyte co-stimulation with CCL2 and M1 CM for 24 hours subsequently resulted in their enhanced chondrogenic differentiation, with elevated expression of SOX9 and Decorin. Following direct or indirect co-culture of human bone marrow mesenchymal stromal cells with macrophages for 24 hours in the presence of CCL2, increased osteogenic differentiation was observed; RUNX2, OCN, ALP, OPN and SPARC expression were all markedly increased. Finally, using Western blot and immunofluorescence we show that, via the ERK, p38, and JNK pathways, CCL2 targets the chondrogenic progenitor cell (CPC) population developing from chondrocytes after M1 exposure. Here, we describe a mechanism through which CCL2 exerts a priming, pro-repair response in chondrocytes via the ERK, p38, and JNK pathways. Interestingly, the M1 macrophage secretome, together with CCL2, induces a more migratory, progenitor-like chondrocyte that subsequently exhibits stronger chondrogenic potential in vitro . CCL2 is a strong priming mediator of the early pro-regenerative pathway which can be exploited therapeutically.