(W0930-10-58) Modulating Host Response to Sepsis via Pure Anti-inflammatory Heparin-Like Compounds

Purpose: Sepsis, a lethal symptom caused by dysregulated host response, has no effective therapy and leads to millions of deaths annually (1). Heparin, an anticoagulant comprised of heparan sulfate (HS) mixtures, has been tested in clinical trials for sepsis, and the results are promising but inconclusive (2, 3). Previous studies indicated that heparin might benefit septic patients by targeting extracellular histone H3 or high mobility group box 1 (HMGB1), which are vital damage messengers that sometimes form a complex with bacterial fragment lipopolysaccharide (LPS) to trigger overwhelming inflammation and resulting severe sepsis (4,5). However, heparin’s heterogeneous structure hinders further investigation of the mechanism of actions and the structure-activity relationship (SAR) between H3/HMGB1. Also, its anticoagulant activity may cause bleeding at high doses, limiting the dose range for testing its anti-inflammatory effect (4). Therefore, improving the understanding of the heparin mechanism with appropriate probes is essential to advancing HS-based therapy in sepsis. This study aims to apply synthetic heparin-like compounds as potential treatments and probes for the mechanism of their anti-inflammatory effects in sepsis. In contrast to regular heparin, the synthetic heparin-like compounds are structurally homogenous HS without anticoagulant activity, enabling SAR and mechanistic studies without dose limitation. We hypothesize that the SAR between HS and key inflammatory molecules, H3 and HMGB1, can be elucidated by using synthesized heparin-like compounds. Furthermore, the optimized synthetic HS will better protect against sepsis-induced inflammation and lethality.

Methods: We used a novel technique, chemoenzymatic synthesis, to produce structurally defined HS with the same repeating disaccharide unit in different lengths, including 6-mer, 12-mer, and 18-mer. 6-mer is an analog of the shortest heparin-like compound on the market, and 18-mer is the longest HS that can be synthesized with biological function to date. All three HS are non-anticoagulants, and their structures were published previously (6,7). Mechanistic studies targeting H3 and HMGB1 were conducted in endothelial cell line EA.hy926 and macrophage cell line Raw264.7, respectively. Two murine sepsis models, cecal ligation and puncture (CLP) and lipopolysaccharide (LPS), were applied to evaluate the anti-inflammatory effect in septic mice (8). Various biochemical assays, including flow cytometry, ELISA, qPCR, and western blot, were applied to cell and animal model samples. Binding affinity between HS and H3/ HMGB1 was determined by surface plasmon resonance (SPR) and pull-down assays. In addition, proteomic analysis was included to identify unknown proteins purified from pull-down assays.

Results: Regarding extracellular H3, we found that the binding affinity of synthetic HS to H3 is size-dependent, with the longest synthetic HS 18-mer performing the best (Kd = 66 nM). Similarly, the neutralizing effect of HS to H3 is dose and size-dependent as tested in EA. Hy926. Regarding HMGB1, the inflammatory effect of the complex of LPS and HMGB1 can also be mitigated by HS in a size-dependent manner. Interestingly, the synthetic HS 18-mer effects on HMGB1/LPS complex are through enlisting apolipoprotein A-I (apoA-I) to replace HMGB1 complexation with LPS. ApoA-I is the main component of HDL, which has not been found associated with HS before. Unlike the HMGB1/LPS complex, ApoA-I/LPS complex is non-toxic. Based on the cell-based study, we chose 18-mer to evaluate the therapeutic effect in CLP mice. 18-mer significantly reduces the two key inflammatory targets, circulating H3 and HMGB1 in plasma. Moreover, 18-mer significantly reduces inflammation biomarkers, including IL-6, MCP-1, and iCAM-1 in plasma and kidney, suggesting a systemic anti-inflammatory effect with potential renoprotection. Finally, we show that 18-mer protects against CLP sepsis-induced lethality in a 72-hour study.

Conclusion: Our results show that the synthetic heparin-like compound effects on H3 and HMGB1 are size-dependent but with a different mechanism of action; HS directly neutralizes H3 but indirectly interrupts the HMGB1/LPS complex through recruiting apoA-I. The longest synthetic HS, 18-mer, shows the best protective effects for improving survival rate in septic mice with significant anti-inflammatory effects. Our study applied pure synthetic HS to elucidate its anti-inflammatory mechanism targeting H3/HMGB1 and identified a new target, apoA-I, which collectively contributes to its protective effect against sepsis. Overall, the study unveils the mechanism of the heparin-like compound as its ability to modulate the host response to sepsis, including reducing inflammatory H3/HMGB1 and increasing anti-inflammatory apoA-I to prevent infection (LPS) spreading. Moreover, the study advance carbohydrate-based therapy by providing a safer heparin-like compound with no bleeding risk and fewer possible side effects with its defined structure.

References: 1. Cavaillon, J. M., Singer, M. & Skirecki, T. Sepsis therapies: learning from 30 years of failure of translational research to propose new leads. EMBO Mol Med 12, e10128 (2020).
2. Jaimes, F. et al. Unfractioned heparin for treatment of sepsis: A randomized clinical trial (The HETRASE Study). Crit Care Med 37, 1185-1196 (2009).
3. Fan, Y., Jiang, M., Gong, D. & Zou, C. Efficacy and safety of low-molecular-weight heparin in patients with sepsis: a meta-analysis of randomized controlled trials. Sci Rep 6, 25984 (2016).
4. Wildhagen, K. C. et al. Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis. Blood 123, 1098-1101 (2014).
5. Tang, Y. et al. Heparin prevents caspase-11-dependent septic leathlity indepent of anticoagulant properties. Immunity 54, 454-467 (2021).
6. Xu, Y. et al. Synthetic oligosaccharides can replace animal-sourced low–molecular weight heparins. Sci Transl Med 9 (2017).
7. Arnold, K. et al. Design of anti-inflammatory heparan sulfate to protect against acetaminophen-induced acute liver failure. Sci Transl Med 12 (2020).
8. Seemann, Semjon, Franziska Zohles, and Amelie Lupp. "Comprehensive comparison of three different animal models for systemic inflammation." J Biomed Sci 24.1 (2017): 1-17.

Acknowledgements: UNC Michael Hooker Proteomics Center, UNC Flow Cytometry Core Facility, Dr. Nate Hathaway.

Funding: NIH (HL094463, HL144970, GM144019, HL157441, HL142604, and AR070179), Eshelman Innovation Institue Glycan Innovation Fund, the Government Scholarship to Study Abroad (GSSA) from Taiwan, NSF GlycoMIP (DMR-1933525).
Conflict of interest: Yongmei Xu and Jian Liu are founders of Glycan Therapeutics. Other authors declare no competing interests.


Synthetic HS directly neutralizes extracellular H3 and reduces H3 induced cytotoxicity


Anti-inflammatory synthetic HS remodels LPS complex by replacing HMGB1 with apoA-I


18-mer reduces local and systemic inflammation to protect against sepsis-induced kidney injury and lethality