Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation
Sepsis, a potentially fatal clinical syndrome, is mediated by an early (e.g., tumor necrosis factor and IL-1) and late [e.g., high mobility group B-1 (HMGB1)] proinflammatory cytokine response to infection. Specifically targeting early mediators has not been effective clinically, in part because peak mediator activity often has passed before therapy can be initiated. Late-acting downstream effectors, such as HMGB1, that mediate sepsis lethality may be more relevant therapeutic targets. Ethyl pyruvate (EP) recently was identified as an experimental therapeutic that significantly protects against lethal hemorrhagic shock. Here, we report that EP attenuates lethal systemic inflammation caused by either endotoxemia or sepsis even if treatment begins after the early tumor necrosis factor response. Treatment with EP initiated 24 h after cecal puncture significantly increased survival (vehicle survival = 30% vs. EP survival = 88%, P < 0.005). EP treatment significantly reduced circulating levels of HMGB1 in animals with established endotoxemia or sepsis. In macrophage cultures, EP specifically inhibited activation of p38 mitogen-activated protein kinase and NF-κB, two signaling pathways that are critical for cytokine release. This report describes a new strategy to pharmacologically inhibit HMGB1 release with a small molecule that is effective at clinically achievable concentrations. EP now warrants further evaluation as an experimental “rescue” therapeutic for sepsis and other potentially fatal systemic inflammatory disorders.
Sepsis, a lethal syndrome that develops in response to infection, occurs in 750,000 patients per year in the United States and is fatal in 20–40% of cases (1, 2). The pathological sequelae of sepsis are mediated by proinflammatory cytokines [e.g., tumor necrosis factor (TNF), IL-1, and high mobility group B-1 (HMGB1)] that are released from macrophages, neutrophils, and other cells of the innate immune system. The magnitude and duration of the systemic inflammatory response influence the development of tissue damage, hypotension, multiple organ failure, and death (3, 4). Significant advances have been made in understanding the role of proinflammatory mediators in the pathogenesis of sepsis, but effective therapies that target inflammatory mediators have not been clinically approved. A major difficulty in developing therapeutics that target cytokines (e.g., TNF and IL-1β) is that they are released early in the development of a systemic inflammatory response (3). This leaves a narrow therapeutic window for administration of antagonists, and inhibitors of TNF and IL-1β are not effective when delivered after the acute cytokine response has occurred (3). Unfortunately, in the typical clinical case, hours pass before sepsis is diagnosed and specific treatment is implemented. Thus, it is perhaps not surprising that agents directed against early proinflammatory cytokines are ineffective in large clinical trials (5, 6).
Recently, HMGB1 was implicated as a “late” mediator of lethal systemic inflammation in animal models of cytokine-mediated disease initiated by the Gram-negative bacterial product endotoxin [lipopolysaccharide (LPS)] (4). Known classically as an intracellular DNA-binding protein, HMGB1 is released by endotoxin-stimulated macrophages, but only after a delay of 12–18 h; a similar delay in HMGB1 appearance is observed in the serum of mice during endotoxemia (4). Anti-HMGB1 antibodies confer significant protection against delayed endotoxin lethality, even when antibody dosing is initiated at a time after the acute-phase cytokine responses have peaked and resolved (4). Cytokine activities of HMGB1 include stimulating the release of TNF, IL-1, and other inflammatory products from macrophages and pituicytes, inducing chemotaxis of smooth muscle cells, and mediating acute lung injury and lethality (7–9). These delayed kinetics, and the protective effects of anti-HMGB1 antibodies in vivo, indicate that HMGB1 is a late-acting mediator of lethal systemic inflammation that may provide a broader therapeutic window for treating sepsis and other systemic inflammatory disorders. To date, there has not been a report of an experimental therapeutic agent that inhibits HMGB1 release to modulate systemic inflammation.
Ethyl pyruvate (EP), a stable lipophilic pyruvate derivative identified recently by Fink and colleagues, is an experimental therapeutic that effectively protects animals from ischemia/reperfusion-induced tissue injury (10, 11). EP administration significantly improved survival in standard models of lethal hemorrhagic shock (12, 13). We reasoned that EP also might be protective in sepsis, because the pathogenesis of ischemia–reperfusion and hemorrhagic shock, like sepsis, depends on activation of early and late cytokine responses. Here, we show that EP rescued animals from lethal sepsis caused by peritonitis, even when dosing began 24 h after cecal puncture. EP inhibited the release of TNF and HMGB1 from endotoxin-stimulated RAW 264.7 murine macrophages and attenuated activation of both the p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. EP treatment of septic mice decreased circulating levels of HMGB1, indicating that delayed administration of EP protects against lethal sepsis.
This work was supported in part by National Institutes of Health Grants ROI GM57226 and ROI GM62508 and the Defense Advanced Research Planning Agency (N65236-00-1-5434).
TNF, tumor necrosis factor
HMGB1, high mobility group B-1
MAPK, mitogen-activated protein kinase
EP, ethyl pyruvate
This paper was submitted directly (Track II) to the PNAS office.
- 1. Angus D. C., Linde-Zwirble, W. T., Lidicker, J., Clermont, G., Carcillo, J. & Pinsky, M. R. (2001) Crit. Care Med.29, 1303-1310. [
- 2. Marshall J. C. (2001) Crit. Care Med.29, S99-S106. [
- 3. Tracey K. J., Beutler, B., Lowry, S. F., Merryweather, J., Wolpe, S., Milsark, I. W., Hariri, R. J., Fahey, T. J., III, Zentella, A., Albert, J. D., et al. (1986) Science234, 470-474. [
- 4. Wang H., Bloom, O., Zhang, M., Vishnubhakat, JM., Ombrellino, M., Che, J., Frazier, A., Yang, H., Ivanova, S., Borovikova, L., et al. (1999) Science285, 248-251. [
- 5. Abraham E., Anzueto, A., Gutierrez, G., Tessler, S., San Pedro, G., Wunderink, R., Dal Nogare, A., Nasraway, S., Berman, S., Cooney, R., et al. (1998) Lancet28, 929-933. [
- 6. Fisher C. J., Dhainaut, J. F., Opal, S. M., Pribble, J. P., Balk, R. A., Slotman, G. J., Iberti, T. J., Rackow, E. C., Shapiro, M. J., Greenman, R. L., et al. (1994) J. Am. Med. Assoc.271, 1836-1843. [
- 7. Abraham E., Arcaroli, J., Carmody, A., Wang, H. & Tracey, K. J. (2000) J. Immunol.165, 2950-2954. [
- 8. Andersson U., Wang, H., Palmblad, K., Aveberger, AC., Bloom, O., Erlandsson-Harris, H., Janson, A., Kokkola, R., Zhang, M., Yang, H., et al. (2000) J. Exp. Med.192, 565-570.
- 9. Wang H., Vishnubhakat, J. M., Bloom, O., Zhang, M., Ombrellino, M., Sama, A. & Tracey, K. J. (1999) Surgery (St. Louis)126, 389-392. [
- 10. Fink M. P. (2002) Curr. Opin. Clin. Nutr. Metab. Care5, 167-174. [
- 11. Sims C. A., Wattanasirichaigoon, S., Menconi, M. J., Ajami, A. M. & Fink, M. P. (2001) Crit. Care Med.29, 1513-1518. [
- 12. Tawadrous Z., Delude, R. L. & Fink, M. P. (2002) Shock17, 473-477. [
- 13. Yang R., Gallo, D. J., Baust, J. J., Uchiyama, T., Watkins, S. K., Delude, R. L. & Fink, M. P. (2002) Am. J. Physiol.283, G212-G221. [
- 14. Wichmann M. W., Haisken, J. M., Ayala, A. & Chaudry, I. H. (1996) J. Surg. Res.65, 109-114. [
- 15. Hesse D. G., Tracey, K. J., Fong, Y., Manogue, K. R., Palladino, M. A., Jr., Cerami, A., Shires, G. T. & Lowry, S. F. (1988) Surg. Gynecol. Obstet.166, 147-153. [
- 16. Lee J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., et al. (1994) Nature (London)372, 739-746. [
- 17. Derijard B., Raingeaud, J., Barrett, T., Wu, I. H., Han, J., Ulevitch, R. J. & Davis, R. J. (1995) Science267, 682-685. [
- 18. Zhang C., Baumgartner, R. A., Yamada, K. & Beaven, M. A. (1997) J. Biol. Chem.272, 13397-13402. [
- 19. Dong Z., Qi, X. & Fidler, I. J. (1993) J. Exp. Med.177, 1071-1077.
- 20. Hambleton J., McMahon, M. & DeFranco, A. L. (1995) J. Exp. Med.182, 147-154.
- 21. Cohen P. S., Nakshatri, H., Dennis, J., Caragine, T., Bianchi, M., Cerami, A. & Tracey, K. J. (1996) Proc. Natl. Acad. Sci. USA93, 3967-3971.
- 22. Ishikawa Y., Mukaida, N., Kuno, K., Rice, N., Okamoto, S. & Matsushima, K. (1995) J. Biol. Chem.270, 4158-4164. [
- 23. Vincenti M. P., Burrell, T. A. & Taffet, S. M. (1992) J. Cell. Physiol.150, 204-213. [
- 24. Brown M. C., Tomaras, G. D., Vincenti, M. P. & Taffet, S. M. (1997) J. Interferon Cytokine Res.17, 295-306. [
- 25. Narravula S. & Colgan, S. P. (2001) J. Immunol.166, 7543-7548. [
- 26. Czura C. J., Wang, H. & Tracey, K. J. (2001) J. Endotoxin Res.7, 315-321. [
- 27. Wang H., Yang, H., Czura, C. J., Sama, A. E. & Tracey, K. J. (2001) Am. J. Respir. Crit. Care Med.164, 1768-1773. [