Using heparan sulfate octadecasaccharide (18-mer) as a multi-target agent to protect against sepsis

Abstract

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

Keywords: HDL; HMGB1; heparin; histone; sepsis.

Fig. 1.

18-mer inhibits inflammation in CLP-induced septic mice. (A) Chemical structures of 6-mer, 12-mer, and 18-mer. Short-hand structure for each oligosaccharide is also presented for clarity. Chemoenzymatic synthesis of the HS is shown in SI Appendix, Fig. S1A. (B–H) Mice underwent CLP surgery were administered (S.Q.) 20 mg/kg of 18-mer at 0, 6, 12 h after CLP, and euthanized 24 h after CLP to collect plasma for the following analysis. Concentrations of the biomarkers were individually presented for male and female groups in SI Appendix, Figs. S11 and S12. (B) Circulating H3 in mouse plasma was evaluated by Western analysis. A representative western analysis image is presented on top. The bar graph represents the H3 band intensities from the individual samples. The full image is presented in SI Appendix, Fig. S13. (C) Circulating HMGB1 in mouse plasma was evaluated by ELISA (n = 6 male and 3 female). (D–H) The levels of IL-6, MCP-1, soluble iCAM-1, creatinine, and BUN were tested in mice plasma. The Data was expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. n = 6 to 10 male and 3 to 5 female. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (I–K) Photomicrographs of proximal renal tubules. (I) Normal tubules of control mice. (J) 24 h post-CLP, proximal tubular epithelial cells have moderate vacuolation (blue arrowheads) and are less tall than in control mice. (K) 24 h post-CLP with 18-mer treatment, proximal tubular epithelial cells are similar to control mice.

Fig. 2.

18-mer reduces inflammation by targeting extracellular H3 and the LPS/HMGB1 complex. (A) The viability of endothelial cells EA.hy926 was analyzed by flow cytometry. Cells were treated with 30 μg/mL of H3 and the indicated concentration of HS for an hour before analysis (n = 3). The statistical difference in cell viability with the same HS treatment but the different concentrations is shown compared to the untreated group (group 3) (&P < 0.05 for 6-mer, #P < 0.05 for 12-mer, *P < 0.05 for 18-mer.) (B) The associate rate constant (ka), disassociation constant (kd), and binding affinity (KD) of oligosaccharides to H3 as measured by SPR. (C–F) Plasma and peritoneal lavage were collected from mice 2 h after biotinylated LPS (B*LPS) administration (i.p., 5 mg/kg) with or without the indicated concentration of HS (n = 4 to 7). (C, D) The protein level of IL-6 was evaluated by ELISA. (E, F) The complex of B*LPS/HMGB1 was isolated by affinity pull-down using streptavidin resin from mouse peritoneal lavage, shown as a representative image of immunoblot with HMGB1 (E) and bar graph for the intensity of the band (F). Samples before and after passing streptavidin resin are labeled “preload” and “elution,” respectively. Data expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. *P < 0.05; **P < 0.01.

Fig. 3.

18-mer enlists ApoA-I to reduce inflammation and exert a protective effect against sepsis. (A–D) Plasma and peritoneal lavage were collected from mice 2 h after B*LPS administration (i.p., 5 mg/kg) with or without the indicated concentration of HS (n = 4 to 7). (A) The SDS-PAGE image of the peritoneal lavage stained with Ponceau S. The membrane is the same as Fig. 2E. The protein migrated at 25 kDa was identified as ApoA-I by proteomic analysis. (B) Immunoblot of the level of B*LPS/HMGB1 (Top) and B*LPS/ApoA-I (Bottom) from peritoneal lavage. Samples before and after affinity purification by streptavidin resin are labeled “preload” and “elution,” respectively. The full image is presented in SI Appendix, Fig. S15. (C, D) The concentration of LPS in the plasma and peritoneal lavage (n = 4 to 7). Data expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. ***P < 0.001(E) The protein level of TNF-a from the cell supernatant of LPS-stimulated Raw264.7, with or without HDL pretreatment (n = 3). (F) The dissociation of human HDL by HS oligosaccharides. Human HDL (200 mg/mL) was incubated with 6-mer, 12-mer, and 18-mer (5 mg/mL) under mildly acidic conditions and centrifuged. The supernatant and precipitation from each reaction were analyzed by SDS-PAGE followed by Coomassie blue staining. (G) The 72-h survival in CLP mice was administered (S.Q.) with saline, 6-mer, and 18-mer at 0, 6, 12, 24, 36, and 52 h. (sham, n=8 male; CLP, n=22 male and 6 female; CLP + 18-mer, n = 20 male and 6 female, CLP + 6-mer, n = 20 male and 6 female). Data analyzed by log-rank test; overall, P = 0.0006; CLP vs. Sham, P = 0.0019; CLP vs. CLP + 18-mer, P = 0.0015; CLP vs. CLP + 6-mer, P = 0.2040. Survival data were individually presented for male and female groups in SI Appendix, Fig. S11.

Fig. 4.

Proposed the mechanism of action by 18-mer in sepsis. Sepsis causes systemic inflammation and releases extracellular H3 and HMGB1 and bacterial LPS. 18-mer displays the protection by directly neutralizing H3 and indirectly targeting HMGB1. 18-mer binds to H3 and neutralizes the cytotoxicity of H3 (Action 1). 18-mer causes the structural changes of HDL and releases ApoA-I (Action 2). ApoA-I binds to LPS to allow a clearance from the circulation to reduce the plasma concentration of LPS (Action 3). ApoA-I displaces HMGB1 from the LPS-HMGB1 complex, which is a pathway to deliver LPS into the cells to cause cell death (Action 4). [Illustration created with BioRender].