Regulation of macrophage migration and activity by high-mobility group box 1 protein released from periodontal ligament cells during orthodontically induced periodontal repair: an in vitro and in vivo experimental study

Abstract

Recent studies have shown that periodontal ligament (PDL) cells interact with macrophages from the immune system during orthodontically induced repair of periodontal tissue. Hypothesizing that high-mobility group box1 (HMGB1) protein is released by mechanically stressed PDL cells into the extracellular space and has a role in mediating the local immune response by acting as an "alarmin", this study was performed to further elucidate these cellular interactions, with a special focus on the impact of proinflammatory mediators secreted by PDL cells on macrophage physiology. The study included an in vivo part in which orthodontic stress was induced in rats and their PDL analyzed for expression of HMGB1 by immunohistochemistry after 5days of tooth movement. In the in vitro part, human PDL cells were subjected to compressive loading, followed by stimulating human macrophages with conditioned supernatants of these stressed PDL cells and analyzing how mediators that had been released by these cells into the medium would impact macrophage physiology. Assays for macrophage migration and osteoclast differentiation were used in addition to immunohistochemistry, enzyme-linked immunosorbent assays, and western blotting. Induction of mechanical stress was found to upregulate HMGB1 expression both in vivo and in vitro. At the same time, translocation HMGB1 from nuclei into cytoplasm was observed. Culturing macrophages in conditioned PDL cell medium was associated with enhanced chemotactic migration and osteoclast differentiation. Addition of anti-HMGB1 antibodies to inhibit HMGB1 in the conditioned medium was found to significantly attenuate these effects. A less marked increase of migration and osteoclast differentiation by macrophages was observed after isolated addition of HMGB1, at its observed pathological concentration, to nonconditioned medium. This study clearly indicates an immunomodulatory potential of human PDL cells via release of mediators, including HMGB1 protein. Our finding that these mediators modify the migration and differentiation of macrophages as a function of periodontal repair during orthodontic treatment broadens the theoretical basis toward developing interventional strategies to avoid orthodontically induced root resorption.