Dendritic Polyglycerol Sulfate Reduces Inflammation Through Inhibition of the HMGB1/RAGE Axis in RAW 264.7 Macrophages

Abstract

High Mobility Group Box 1 (HMGB1) is a central pro-inflammatory mediator released from damaged or stressed cells, where it activates receptors such as the Receptor for Advanced Glycation Endproducts (RAGE). Dendritic polyglycerol sulfate (dPGS), a hyperbranched polyanionic polymer, is known for its anti-inflammatory activity. In this study, we examined how dPGS modulates HMGB1-driven signaling in RAW 264.7 macrophages and human microglia. Recombinant human HMGB1 expressed in Escherichia coli (E. coli) was purified by nickel-nitrilotriacetic acid (Ni-NTA) and heparin chromatography. Proximity ligation assays (PLA) revealed that dPGS significantly disrupted HMGB1/RAGE interactions, particularly under lipopolysaccharide (LPS) stimulation, thereby reducing inflammatory signaling complex formation. This correlated with reduced activation of the nuclear factor kappa B (NF-κB) pathway, demonstrated by decreased nuclear translocation and transcriptional activity. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (RT-qPCR) showed that dPGS suppressed HMGB1- and LPS-induced transcription of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) and Griess assays confirmed reduced TNF-α secretion and nitric oxide production. Electron paramagnetic resonance (EPR) spectroscopy further showed that dPGS altered HMGB1/soluble RAGE (sRAGE) complex dynamics, providing mechanistic insight into its receptor-disruptive action.

Keywords: cytokines; dendritic polyglycerol sulfate; high mobility group box 1; inflammation; macrophages; receptor for advanced glycation end products.

Figure 1

dPGS reduces HMGB1/RAGE interactions in LPS-stimulated RAW 264.7 macrophages. (A) RAW 264.7 macrophages were seeded onto glass coverslips at 10,000 cells/coverslip. Cells were then treated with 100 ng/mL LPS, 50 nM dPGS, and 100 ng/mL LPS + 50 nM dPGS under serum-free conditions for 24 h. PLA visualized the interaction of HMGB1 and RAGE, as indicated by orange dots and white arrows. Phalloidin-iFluor 488 (green) and DAPI (blue) were used to visualize actin and nuclei, respectively. The scale bars represent 10 µm, and images were taken with a Zeiss Axio Observer. Z1 microscope and the ZEN blue software (version: 3.7.97.04000). (B) Evaluation of data obtained by PLA. The mean fluorescence intensity (MFI) per image of the PLA signal was normalized to that of the untreated control and set to 1. Bar graphs show the mean values ± SEM. Statistical analysis was assessed using two-way ANOVA, followed by Tukey’s multiple comparison test. At least 40 cells in three independent experiments were analyzed (*** p ≤ 0.001, * p ≤ 0.05, ns = not significant). The diagram was generated with GraphPad Prism 10 (version 10.5.0).

Figure 2

Shuttling of NF-κB is inhibited by dPGS. (A) RAW 264.7 macrophages were seeded onto glass coverslips at 10,000 cells/coverslip. Cells were then treated with 1 µg/mL HMGB1, 100 ng/mL LPS, 50 nM dPGS, and combinations under serum-free conditions for 90 min. For visualization of NF-κB, cells were incubated with an NF-κB p65 rabbit mAb followed by an anti-rabbit FITC-conjugated antibody. The nucleus was labeled with Hoechst 33342, and images were taken with a Zeiss Axio Observer. Z1 microscope and the ZEN blue (version: 3.7.97.04000) software. (B) The nuclear MFI was calculated for each sample and normalized to the untreated control (set to 1). At least 70 cells in three independent experiments were analyzed. Statistical analysis was performed by using two-way ANOVA followed by Tukey’s multiple comparisons test. Mean ± SEM are shown (**** p ≤ 0.0001, ** p ≤ 0.01, * p ≤ 0.05, ns = not significant). Scale bar 30 µm. The diagram was generated with GraphPad Prism (version 10.5.0).

Figure 3

dPGS reduces mRNA levels of HMGB1-stimulated cytokines. (A) Gel-separated RT-PCR products received after indicated treatments of RAW 264.7 macrophages. (B–D) Quantification of cytokines was performed using ImageJ (version 1.54); values were normalized to the untreated control. Respective bands for MCP-1 (271 bp), IL-6 (474 bp), TNF-α (795 bp), and ACTB (150 bp) used as house-keeping gene. At least three independent experiments were performed. Statistical analysis was done by using one-way ANOVA followed by Tukey’s multiple comparison test. Mean ± SEM are shown (**** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, ns = not significant). The diagrams were generated with GraphPad Prism 10 (version 10.5.0).

Figure 4

Release of pro-inflammatory TNF-α from stimulated RAW 264.7 macrophages is reduced by dPGS. RAW 264.7 macrophages were seeded at 10,000 cells/well in a 96-well plate in complete medium and stimulated with respective treatments, (A) 1 µg/mL HMGB1 +/− 500 nM dPGS, 50 nM dPGS, 5 nM dPGS, 0.5 nM dPGS, 0.05 nM dPGS, and (B) 100 ng/mL LPS +/− 50 nM dPGS in serum-free medium for 16 h. Medium alone was used as an untreated control, and 500 nM dPGS as a polymer control. Secreted TNF-α was determined by ELISA and normalized to the untreated control. Data are shown as (A) fold change relative to 1 µg/mL HMGB1 and (B) relative to 100 ng/mL LPS treatment. At least three independent experiments were performed. The mean ± SEM are shown. Statistical analysis was done by using two-way ANOVA followed by Dunnett’s multiple comparison test (**** p ≤ 0.0001, ** p ≤ 0.01, * p ≤ 0.05). The diagrams were generated with GraphPad Prism 10 (version 10.5.0).

Figure 5

Co-incubation with LPS or HMGB1 reduces the uptake of dPGS-Cy5 into RAW 264.7 macrophages. In total, 1 × 10⁵ cells/well were seeded in 24-well plates and incubated with 50 nM dPGS-Cy5 in serum-free medium, without stimulus or in the presence of 1 μg/mL HMGB1 or 100 ng/mL for 1, 6, 16, and 24 h. The cellular uptake of dPGS-Cy5 was analyzed by flow cytometry and quantified as a fold change in Cy5 MFI relative to the unstimulated dPGS-Cy5 control (shown by a dotted line), after subtraction of the non-fluorescent background control. The data are shown as the mean ± SEM from three independent experiments, each performed with at least three technical replicates. For each time point, statistical significance was determined by one-way ANOVA, followed by Tukey-Kramer post hoc testing. **** p ≤ 0.0001, ** p ≤ 0.01, ns p > 0.05. bg-subtr. = background-subtracted; unstim. = unstimulated.

Figure 6

Binding analysis of HMGB1, sRAGE, and dPGS by electron paramagnetic resonance (EPR) spectroscopy and schematic illustration of potential complex formations. (A) EPR spectra of HMGB1 (black trace, 2A_zz = 61 G), HMGB1 + sRAGE premixed with 1 vol.% P20 (red trace, molar ratio 6:1, 2A_zz = 62.5 G), 11 µM HMGB1 + 2 µM sRAGE premixed with 1 vol.% P20 + 4 µM dPGS (blue trace, molar ratio 6:1:2, 2A_zz = 65.5 G), 20 µM HMGB1 + 20 µM dPGS (green trace, molar ratio 1:1, 2A_zz = 64.0 G). (B) Schematic representation of two different interaction scenarios upon addition of dPGS to the preformed HMGB1/sRAGE complex. Figure created with BioRender.com. (1) dPGS binds competitively to HMGB1, displacing sRAGE, occupying similar or overlapping binding sites, or (2) dPGS forms a ternary complex with both HMGB1 and sRAGE, hence, different binding sites.