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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s10020-018-0021-x) contains supplementary material, which is available to authorized users.