Disulfide and Fully Reduced HMGB1 Induce Different Macrophage Polarization and Migration Patterns.

Macrophage plasticity enables cells to obtain different functions over a broad proinflammatory and repairing spectrum. In different conditions, macrophages can be induced by high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein that activates innate immunity, to polarize towards a pro- (M1) or anti-inflammatory (M2) phenotype. In this study, we investigated the phenotypes of murine bone-marrow-derived macrophages (BMDMs) induced by different HMGB1 redox isoforms in depth. Our results demonstrate that disulfide HMGB1 (dsHMGB1) induces a unique macrophage phenotype that secretes pro-inflammatory cytokines, rather than inducing metabolic changes leading to nitric oxide production. Fully reduced HMGB1 (frHMGB1) did not induce macrophage polarization. The migrating function of BMDMs was measured by scratch assay after the stimulation with dsHMGB1 and frHMGB1. Both dsHMGB1 and frHMGB1 induced cell migration. We found that dsHMGB1 mediates cytokine secretion and cellular motility, mainly through toll-like receptor 4 (TLR4). Importantly, our data shows that dsHMGB1 and frHMGB1 induce distinct BMDM polarization phenotypes, and that dsHMGB1 induces a unique phenotype differing from the classical proinflammatory macrophage phenotype.

Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain.

BACKGROUND: High mobility group box 1 (HMGB1) protein is a central endogenous inflammatory mediator contributing to the pathogenesis of several inflammatory disorders. HMGB1 interacts with toll-like receptors (TLRs) but contradictory evidence regarding its identity as a TLR2 ligand persists. The aim of this study was to investigate if highly purified HMGB1 interacts with TLR2 and if so, to determine the functional outcome. METHODS: Full length or C-terminal truncated (Δ30) HMGB1 was purified from E.coli. Binding to TLR2-Fc was investigated by direct-ELISA. For the functional studies, proteins alone or in complex with peptidoglycan (PGN) were added to human embryonic kidney (HEK) cells transfected with functional TLR2, TLR 1/2 or TLR 2/6 dimers, macrophages, whole blood or peripheral blood mononuclear cells (PBMCs). Cytokine levels were determined by ELISA. RESULTS: In vitro binding experiments revealed that Δ30 HMGB1, lacking the acidic tail domain, but not full length HMGB1 binds dose dependently to TLR2. Control experiments confirmed that the interaction was specific to TLR2 and could be inhibited by enzymatic digestion. Δ30 HMGB1 alone was unable to induce cytokine production via TLR2. However, full length HMGB1 and Δ30 HMGB1 formed complexes with PGN, a known TLR2 ligand, and synergistically potentiated the inflammatory response in PBMCs. CONCLUSIONS: We have demonstrated that TLR2 is a receptor for HMGB1 and this binding is negatively regulated by the C-terminal tail. HMGB1 did not induce functional activation of TLR2 while both full length HMGB1 and Δ30 HMGB1 potentiated the inflammatory activities of the TLR2 ligand PGN. We hypothesize that Δ30 HMGB1 generated in vivo by enzymatic cleavage could act as an enhancer of TLR2-mediated inflammatory activities.

Neuroinflammation in Response to Intracerebral Injections of Different HMGB1 Redox Isoforms.

BACKGROUND: Neuroinflammation triggered by infection or trauma is the cause of central nervous system dysfunction. High-mobility group box 1 protein (HMGB1), released from stressed and dying brain cells, is a potent neuroinflammatory mediator. The proinflammatory functions of HMGB1 are tightly regulated by post-translational redox modifications, and we here investigated detailed neuroinflammatory responses induced by the individual redox isoforms. METHODS: Male Dark Agouti rats received a stereotactic injection of saline, lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1, and were accessed for blood-brain barrier modifications using magnetic resonance imaging (MRI) and inflammatory responses by immunohistochemistry. RESULTS AND CONCLUSIONS: Significant blood-brain barrier disruption appeared 24 h after injection of lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1 compared to controls, as assessed in post-gadolinium T1-weighted MRI images and confirmed by increased uptake of FITC-conjugated dextran. Immunohistochemistry revealed that both HMGB1 isoforms also induced a local production of IL-1ß. Additionally, disulfide HMGB1 increased major histocompatibility complex class II expression and apoptosis. Together, the results demonstrate that extracellular, cerebral HMGB1 causes significant blood-brain barrier disruption in a redox-independent manner and activates several components of neuroinflammation. Blocking HMGB1 might potentially improve clinical outcome in conditions such as stroke and traumatic brain injury.