Molecular Medicine

, Volume 20, Issue 1, pp 639–648 | Cite as

Toll-like Receptor 4 (TLR4) Antagonist Eritoran Tetrasodium Attenuates Liver Ischemia and Reperfusion Injury through Inhibition of High-Mobility Group Box Protein B1 (HMGB1) Signaling

  • Kerry-Ann McDonald
  • Hai Huang
  • Samer Tohme
  • Patricia Loughran
  • Kimberly Ferrero
  • Timothy Billiar
  • Allan Tsung
Research Article


Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1-TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepatocytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury.



This work was supported by a Howard Hughes Medical Institute Physician-Scientist Award (to A Tsung), R01-GM95566 (to A Tsung) and R01-GM50441 (to T Billiar). The authors would like to thank Eisai for the generous gift of eritroran and Xinghua Liao for technical assistance in preparing the manuscript.

Supplementary material

10020_2014_2001639_MOESM1_ESM.pdf (1 mb)
Supplementary material, approximately 1.04 MB.


  1. 1.
    Tsung A, et al. (2005) Hepatic ischemia/reperfusion injury involves functional TLR4 signaling in non-parenchymal cells. J. Immunol. 175:7661–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Abu-Amara M, et al. (2010) Liver ischemia/reperfusion injury: processes in inflammatory networks: a review. Liver Transpl. 16:1016–32.CrossRefPubMedGoogle Scholar
  3. 3.
    Evankovich J, Billiar T, Tsung A. (2010) Toll-like receptors in hepatic ischemia/reperfusion and transplantation. Gastroenterol. Res. Pract. 2010:537263.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wu HS, et al. (2004) Toll-like receptor 4 involvement in hepatic ischemia/reperfusion injury in mice. Hepatobiliary Pancreat. Dis. Int. 3:250–3.PubMedGoogle Scholar
  5. 5.
    Zhai Y, et al. (2004) Cutting edge: TLR4 activation mediates liver ischemia/reperfusion inflammatory response via IFN regulatory factor 3-dependent MyD88-independent pathway. J. Immunol. 173:7115–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Gill R, Tsung A, Billiar T. (2010) Linking oxidative stress to inflammation: Toll-like receptors. Free Radic. Biol. Med. 48:1121–32.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Shen XD, et al. (2005) Toll-like receptor and heme oxygenase-1 signaling in hepatic ischemia/reperfusion injury. Am. J. Transplant. 5:1793–800.CrossRefGoogle Scholar
  8. 8.
    Kim HM, et al. (2007) Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130:906–17.CrossRefGoogle Scholar
  9. 9.
    Barochia A, Solomon S, Cui X, Natanson C, Eichacker PQ. (2011) Eritoran tetrasodium (E5564) treatment for sepsis: review of preclinical and clinical studies. Expert Opin. Drug Metab. Toxicol. 7:479–94.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Mullarkey M, et al. (2003) Inhibition of endotoxin response by e5564, a novel Toll-like receptor 4-di-rected endotoxin antagonist. J. Pharmacol. Exp. Ther. 304:1093–102.CrossRefPubMedGoogle Scholar
  11. 11.
    Rossignol DP, et al. (2004) Safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (E5564) during continuous intravenous infusion into healthy volunteers. Antimicrob. Agents Chemother. 48:3233–40.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Czeslick E, Struppert A, Simm A, Sablotzki A. (2006) E5564 (Eritoran) inhibits lipopolysaccharide-induced cytokine production in human blood monocytes. Inflamm. Res. 55:511–5.CrossRefPubMedGoogle Scholar
  13. 13.
    Ehrentraut S, et al. (2011) In vivo Toll-like receptor 4 antagonism restores cardiac function during endotoxemia. Shock. 36:613–20.CrossRefPubMedGoogle Scholar
  14. 14.
    Figueiredo MD, Moore JN, Vandenplas ML, Sun WC, Murray TF. (2008) Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes. Am. J. Vet. Res. 69:796–803.CrossRefPubMedGoogle Scholar
  15. 15.
    Rossignol DP, Lynn M. (2002) Antagonism of in vivo and ex vivo response to endotoxin by E5564, a synthetic lipid A analogue. J. Endotoxin. Res. 8:483–8.CrossRefPubMedGoogle Scholar
  16. 16.
    Opal SM, et al. (2013) Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial. JAMA. 309:1154–62.CrossRefPubMedGoogle Scholar
  17. 17.
    Korff S, et al. (2013) Eritoran attenuates tissue damage and inflammation in hemorrhagic shock/trauma. J. Surg. Res. 184:e17–25.CrossRefPubMedGoogle Scholar
  18. 18.
    Liu M, et al. (2010) Protective effects of Toll-like receptor 4 inhibitor eritoran on renal ischemia-reperfusion injury. Transplant Proc. 42:1539–44.CrossRefPubMedGoogle Scholar
  19. 19.
    Shimamoto A, et al. (2006) Inhibition of Toll-like receptor 4 with eritoran attenuates myocardial ischemia-reperfusion injury. Circulation. 114 (1 Suppl): 1270–4.Google Scholar
  20. 20.
    Shirey KA, et al. (2013) The TLR4 antagonist Eritoran protects mice from lethal influenza infection. Nature. 497:498–502.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Sun Y, Pearlman E. (2009) Inhibition of corneal inflammation by the TLR4 antagonist Eritoran tetrasodium (E5564). Invest. Ophthalmol. Vis. Sci. 50:1247–54.CrossRefPubMedGoogle Scholar
  22. 22.
    Tsung A, et al. (2005) The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J. Exp. Med. 201:1135–43.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Park JS, et al. (2004) Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein. J. Biol. Chem. 279:7370–7.CrossRefPubMedGoogle Scholar
  24. 24.
    El GM. (2007) HMGB1 modulates inflammatory responses in LPS-activated macrophages. Inflamm. Res. 56:162–7.CrossRefGoogle Scholar
  25. 25.
    Yu M, et al. (2006) HMGB1 signals through tolllike receptor (TLR) 4 and TLR2. Shock. 26:174–9.CrossRefGoogle Scholar
  26. 26.
    Yang H, Antoine DJ, Andersson U, Tracey KJ. (2013) The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis. J. Leukoc. Biol. 93:865–73.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research, Division on Earth and Life Studies, National Research Council of the National Academies. (2011) Guide for the Care and Use of Laboratory Animals. 8th edition. Washington (DC): National Academies Press.Google Scholar
  28. 28.
    Tsung A, et al. (2006) The transcription factor interferon regulatory factor-1 mediates liver damage during ischemia-reperfusion injury. Am. J. Physiol. Gastrointest. Liver Physiol. 290:G1261–8.CrossRefPubMedGoogle Scholar
  29. 29.
    West MA, Billiar TR, Curran RD, Hyland BJ, Simmons RL. (1989) Evidence that rat Kupffer cells stimulate and inhibit hepatocyte protein synthesis in vitro by different mechanisms. Gastroenterology. 96:1572–82.CrossRefPubMedGoogle Scholar
  30. 30.
    Huang H, et al. (2011) Hepatic arterial perfusion is essential for the spontaneous recovery from focal hepatic venous outflow obstruction in rats. Am. J. Transplant. 11:2342–52.CrossRefPubMedGoogle Scholar
  31. 31.
    Sun Q, et al. (2013) Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress. J. Biol. Chem. 288:15947–58.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    McCloskey CA, Kameneva MV, Uryash A, Gallo DJ, Billiar TR. (2004) Tissue hypoxia activates JNK in the liver during hemorrhagic shock. Shock. 22:380–6.CrossRefPubMedGoogle Scholar
  33. 33.
    Colletti LM, et al. (1996) The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. Hepatology. 23:506–14.CrossRefPubMedGoogle Scholar
  34. 34.
    Wanner GA, et al. (1999) Differential effect of anti-TNF-alpha antibody on proinflammatory cytokine release by Kupffer cells following liver ischemia and reperfusion. Shock. 11:391–5.CrossRefPubMedGoogle Scholar
  35. 35.
    Nace GW, et al. (2013) Cellular-specific role of toll-like receptor 4 in hepatic ischemia-reperfusion injury in mice. Hepatology. 58:374–87.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Tsung A, et al. (2007) HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling. J. Exp. Med. 204:2913–23.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Bhogal RH, Sutaria R, Afford SC. (2011) Hepatic liver ischemia/reperfusion injury: processes in inflammatory networks: a review. Liver Transpl. 17:95.CrossRefPubMedGoogle Scholar
  38. 38.
    Evankovich J, et al. (2010) High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity. J. Biol. Chem. 285:39888–97.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Gero D, et al. (2013) Identification of pharmacological modulators of HMGB1-induced inflammatory response by cell-based screening. PLoS One. 8:e65994.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Li J, et al. (2003) Structural basis for the proin-flammatory cytokine activity of high mobility group box 1. Mol. Med. 9:37–45.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Wang H, Yang H, Czura CJ, Sama AE, Tracey KJ. (2001) HMGB1 as a late mediator of lethal systemic inflammation. Am. J. Respir. Crit. Care Med. 164:1768–73.CrossRefPubMedGoogle Scholar
  42. 42.
    Kim S, et al. (2013) Signaling of high mobility group box 1 (HMGB1) through toll-like receptor 4 in macrophages requires CD14. Mol. Med. 19:88–98.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Li J, et al. (2004) Recombinant HMGB1 with cytokine-stimulating activity. J. Immunol. Methods. 289:211–23.CrossRefPubMedGoogle Scholar
  44. 44.
    Andersson U, Tracey KJ. (2011) HMGB1 is a therapeutic target for sterile inflammation and infection. Annu. Rev. Immunol. 29:139–62.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Harris HE, Andersson U, Pisetsky DS. (2012) HMGB1: a multifunctional alarmin driving autoimmune and inflammatory disease. Nat. Rev. Rheumatol. 8:195–202.CrossRefPubMedGoogle Scholar
  46. 46.
    Klune JR, Dhupar R, Cardinal J, Billiar TR, Tsung A. (2008) HMGB1: endogenous danger signaling. Mol. Med. 14:476–84.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Park JS, et al. (2006) High mobility group box 1 protein interacts with multiple Toll-like receptors. Am. J. Physiol. Cell Physiol. 290:C917–24.CrossRefGoogle Scholar
  48. 48.
    Hoppe G, Talcott KE, Bhattacharya SK, Crabb JW, Sears JE. (2006) Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1. Exp. Cell Res. 312:3526–38.CrossRefPubMedGoogle Scholar
  49. 49.
    Venereau E, et al. (2012) Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release. J. Exp. Med. 209:1519–28.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Author(s) 2014

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • Kerry-Ann McDonald
    • 1
  • Hai Huang
    • 1
  • Samer Tohme
    • 1
  • Patricia Loughran
    • 2
  • Kimberly Ferrero
    • 1
  • Timothy Billiar
    • 1
  • Allan Tsung
    • 1
    • 3
  1. 1.Department of SurgeryUniversity of Pittsburgh Medical CenterPittsburghUSA
  2. 2.Center for Biologic Imaging, Department of Cell BiologyUniversity of Pittsburgh Medical CenterPittsburghUSA
  3. 3.South PittsburghUSA

Personalised recommendations