Single dose of anti-high mobility group box 1 protein (HMGB-1) neutralizing antibody ameliorates dysregulated inflammation and restores fracture healing in a polytrauma rat (Rattus norvegicus) model

Abstract

Abstract Polytrauma (PT) is associated with dysregulated inflammation (DI) and complicated/delayed fracture healing. We recently reported delayed fracture healing and increased circulating levels of HMGB-1 in a rat model of PT compared to osteotomy (OST). Since HMGB-1 binds to and activates TLR2, TLR4 and RAGE surface receptors to induce pro-inflammation, we investigated this protein’s role in delayed healing. We hypothesized that neutralization of cell-free HMGB-1 immediately after PT would resolve DI and restore fracture healing in PT. Anti-HMGB-1 polyclonal antibody (2mg/kg) was administered via IP injection following PT (PT-Ab) and bone/tissue volume (BV/TV) of fractures was evaluated by micro-CT imaging at 5 weeks post PT. As hypothesized, by neutralizing HMGB-1, fracture healing was improved with significantly higher BV/TV ratio in PT-Ab compared to PT rats. Changes to circulating CD68+ CD86+ (M1) and CD68+CD163+ (M2) monocyte/macrophage populations, as well as alterations to CD4+ and CD8+ T cells and their RAGE and TLR4 surface expression, were assessed on 1 and 3 days post-injury (d). While circulating M1 and M2 populations are scarce in naïve rats, these populations elevated post trauma. M1 was significantly higher in PT than in OST and PT-Ab at 1d but not at 3d. M2 was significantly high in only OST at 1d. In contrast to OST, we observed decreased RAGE and TLR4 expression on circulating T cells at 1d in PT and PT-Ab and at 3d in PT but not in OST and PT-Ab. Our observations suggest that elevated levels of HMGB-1 following severe trauma initiate immune dysregulation causing delayed fracture repair and the data presented herein indicates that HMGB-1 may be a therapeutic target to restore normal immune response and normal fracture healing in PT.