The TLR4 antagonist Eritoran protects mice from lethal influenza infection



There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation1. Subsequently, we reported that Tlr4−/− mice are highly refractory to influenza-induced lethality2, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)—a potent, well-tolerated, synthetic TLR4 antagonist3,4—blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

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Figure 1: Eritoran treatment protects mice from lethal influenza challenge.
Figure 2: Eritoran treatment inhibits influenza-induced lung pathology and lung function.
Figure 3: Treatment with Eritoran reduces lung viral titres.
Figure 4: Eritoran treatment suppresses influenza-induced cytokine gene expression.
Figure 5: Molecular requirements of Eritoran-induced protection.

Change history

  • 22 May 2013

    A minor change was made to the Fig. 4 legend.


  1. 1

    Imai, Y. et al. Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell 133, 235–249 (2008)

  2. 2

    Nhu, Q. M. et al. Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo. Mucosal Immunol. 3, 29–39 (2010)

  3. 3

    Kalil, A. C. et al. Influence of severity of illness on the effects of eritoran tetrasodium (E5564) and on other therapies for severe sepsis. Shock 36, 327–331 (2011)

  4. 4

    Mullarkey, M. et al. Inhibition of endotoxin response by E5564, a novel Toll-like receptor 4-directed endotoxin antagonist. J. Pharmacol. Exp. Ther. 304, 1093–1102 (2003)

  5. 5

    Thompson, W. W. et al. Mortality associated with influenza and respiratory syncytial virus in the United States. J. Am. Med. Assoc. 289, 179–186 (2003)

  6. 6

    Thompson, W. W. et al. Influenza-associated hospitalizations in the United States. J. Am. Med. Assoc. 292, 1333–1340 (2004)

  7. 7

    Reid, A. H., Taubenberger, J. K. & Fanning, T. G. The 1918 Spanish influenza: integrating history and biology. Microbes Infect. 3, 81–87 (2001)

  8. 8

    Taubenberger, J. K., Reid, A. H., Janczewski, T. A. & Fanning, T. G. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Phil. Trans. R. Soc. Lond. B 356, 1829–1839 (2001)

  9. 9

    Hurt, A. C., Holien, J. K., Parker, M., Kelso, A. & Barr, I. G. Zanamivir-resistant influenza viruses with a novel neuraminidase mutation. J. Virol. 83, 10366–10373 (2009)

  10. 10

    McKimm-Breschkin, J. L. et al. Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors. J. Virol. 72, 2456–2462 (1998)

  11. 11

    Mishin, V. P., Hayden, F. G. & Gubareva, L. V. Susceptibilities of antiviral-resistant influenza viruses to novel neuraminidase inhibitors. Antimicrob. Agents Chemother. 49, 4515–4520 (2005)

  12. 12

    Goldblum, S. E., Ding, X., Brann, T. W. & Campbell-Washington, J. Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: concurrent F-actin depolymerization and new actin synthesis. J. Cell. Physiol. 157, 13–23 (1993)

  13. 13

    Verhoeven, D., Teijaro, J. T. & Farber, D. L. Pulse-oximetry accurately predicts lung pathology and the immune response during influenza infection. Virology 390, 151–156 (2009)

  14. 14

    Blanco, J. C. G. et al. Receptor characterization and suceptibility of cotton rats to avian and 2009 pandemic influenza virus strains. J. Virol. 87, 2036–2045 (2013)

  15. 15

    Ottolini, M. G. et al. The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis. J. Gen. Virol. 86, 2823–2830 (2005)

  16. 16

    Polakos, N. K. et al. Kupffer cell-dependent hepatitis occurs during influenza infection. Am. J. Pathol. 168, 1169–1178 (2006)

  17. 17

    Taubenberger, J. K. & Morens, D. M. The pathology of influenza virus infections. Annu. Rev. Pathol. 3, 499–522 (2008)

  18. 18

    Shirey, K. A. et al. The anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), induces IFN-β-mediated antiviral activity in vitro and in vivo. J. Leukoc. Biol. 89, 351–357 (2011)

  19. 19

    Thomas, K. E., Galligan, C. L., Newman, R. D., Fish, E. N. & Vogel, S. N. Contribution of interferon-β to the murine macrophage response to the toll-like receptor 4 agonist, lipopolysaccharide. J. Biol. Chem. 281, 31119–31130 (2006)

  20. 20

    Kim, H. M. et al. Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist eritoran. Cell 130, 906–917 (2007)

  21. 21

    Yoon, S. I., Hong, M., Han, G. W. & Wilson, I. A. Crystal structure of soluble MD-1 and its interaction with lipid IVa. Proc. Natl Acad. Sci. USA 107, 10990–10995 (2010)

  22. 22

    Esparza, G. A., Teghanemt, A., Zhang, D., Gioannini, T. L. & Weiss, J. P. Endotoxin-albumin complexes transfer endotoxin monomers to MD-2 resulting in activation of TLR4. Innate Immun. 18, 478–491 (2012)

  23. 23

    Gioannini, T. L., Zhang, D., Teghanemt, A. & Weiss, J. P. An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation. J. Biol. Chem. 277, 47818–47825 (2002)

  24. 24

    Means, T. K. et al. The CD14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors. J. Immunol. 163, 6748–6755 (1999)

  25. 25

    Pauligk, C., Nain, M., Reiling, N., Gemsa, D. & Kaufmann, A. CD14 is required for influenza A virus-induced cytokine and chemokine production. Immunobiology 209, 3–10 (2004)

  26. 26

    Lee, R. M., White, M. R. & Hartshorn, K. L. Influenza A viruses upregulate neutrophil Toll-like receptor 2 expression and function. Scand. J. Immunol. 63, 81–89 (2006)

  27. 27

    Shinya, K. et al. Toll-like receptor pre-stimulation protects mice against lethal infection with high pathogenic influenza viruses. Virol. J. 8, 97–101 (2011)

  28. 28

    Wong, Y. N. et al. Safety, pharmacokinetics, and pharmacodynamics of E5564, a lipid A antagonist, during an ascending single-dose clinical study. J. Clin. Pharmacol. 43, 735–742 (2003)

  29. 29

    Tidswell, M. et al. Phase 2 trial of eritoran tetrasodium (E5564), a Toll-like receptor 4 antagonist, in patients with severe sepsis. Crit. Care Med. 38, 72–83 (2010)

  30. 30

    Cohen, J., Opal, S. & Calandra, T. Sepsis studies need new direction. Lancet Infect. Dis. 12, 503–505 (2012)

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This work was supported by National Institutes of Health grants AI057575 (J.C.G.B.), AI018797 (S.N.V.), AI059372 (J.W.) and NCRR K12-RR-023250 (W.H.C.), VA Merit Award 1I01BX0000949-01A1 (T.L.G.) and Cystic Fibrosis Foundation RDP Center Grant (C.L.K.).

Author information

K.A.S. and S.N.V. carried out the study design (with advice from J.C.G.B., D.P.R., J.W., R.K.E. and C.L.K.). K.A.S., W.L., J.C.G.B., L.M.P., A.J.S., T.L.G., J.M. and P.M. performed experiments. M.L. performed histological analysis. F.G., W.H.C. and D.P.R. provided crucial reagents and advice. K.A.S. and S.N.V. prepared the manuscript, with input and approval from all other co-authors.

Correspondence to Stefanie N. Vogel.

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With the exception of F.G., none of the authors have a competing interest. F.G. is an employee of Eisai Inc.

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Shirey, K., Lai, W., Scott, A. et al. The TLR4 antagonist Eritoran protects mice from lethal influenza infection. Nature 497, 498–502 (2013).

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