Inflammation is a biological response to tissue injury or infection, mediated by signalling cascades and triggered by endogenous and exogenous danger signals. The nuclear protein High Mobility Group Box 1 (HMGB1), when released extracellularly, activates inflammatory pathways (e.g., nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway) via pattern recognition receptors (PRRs) such as the Receptor for Advanced Glycation Endproducts (RAGE). Its activity is shaped by the positively charged A- and B-boxes and negatively charged acidic C-terminal tail, influencing receptor binding. Here, the anti-inflammatory properties of dendritic polyglycerol sulfate (dPGS), a synthetic, Heparin-mimetic polyanion, were investigated. Surface plasmon resonance (SPR) revealed that commercial HMGB1 from mammalian cells binds dPGS with nanomolar affinity (KD = 94.3 ± 1.7 nM). Alongside, recombinant full-length HMGB1 and subdomains expressed in E. coli and purified by a two-step affinity chromatography, showed that the A-box (KD = 83.5 nM) and A-+B-box (KD = 82.3 nM) exhibited similar affinities, while full-length HMGB1 displayed biphasic kinetics. Fitting with the heterogenous ligand binding model resulted in KD1 = 5 nM and KD2 = 0.009 nM, while dPGS also bound soluble (s)RAGE (KD1 = 6 nM, KD2 = 0.006 nM). HMGB1 bound sRAGE with KD1 = 70 nM, KD2 = 52 nM. Competitive binding studies demonstrated that dPGS impaired the HMGB1/sRAGE complex (IC50 = 3.3 nM). At higher dPGS concentrations biophysical data indicated a ternary complex of HMGB1/sRAGE/dPGS. Further, imaging showed that dPGS reduced lipopolysaccharide (LPS)-induced HMGB1/RAGE complex formation by 18 % in RAW 264.7 macrophages and 24 % in human microglia. NF-κB nuclear translocation induced by either HMGB1 or LPS was reduced to approximately 13 % of the control. As a result, RT-PCR showed that TNF-α mRNA levels were lowered owing to dPGS by 91 % (HMGB1 stimulus) and 81% (LPS stimulus), IL-6 by 71 % and 26 %, and MCP-1 by 34 % and 48 %, respectively. Protein quantification via ELISA confirmed reduced secretion levels of TNF-α upon co-stimulation with HMGB1 (50 %) or LPS (25 %). To prove that dPGS is capable to act intracellularly, uptake studies were performed with a cyanine 5 (Cy5) fluorescent label. Flow cytometry analyses showed time-dependent dPGS-Cy5 uptake peaking at 16–24 h. HMGB1 and LPS reduced intracellular accumulation by up to 30-40 % from 6 h onwards. Summarising, these results show that dPGS inhibits HMGB1-mediated inflammation at multiple structural and signalling checkpoints, supporting its potential as a therapeutic agent.
