The Toll-Like Receptor 4 Antagonist Eritoran Protects Mice from Lethal Filovirus Challenge

Abstract

The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa, which has seen intermittent reemergence since it was officially declared over in February of 2016, has demonstrated the need for the rapid development of therapeutic intervention strategies. Indirect evidence has suggested that the EBOV infection shares several commonalities associated with the onset of bacterial sepsis, including the development of a "cytokine storm." Eritoran, a Toll-like receptor 4 (TLR4) antagonist, was previously shown to result in protection of mice against lethal influenza virus infection. Here, we report that eritoran protects against the lethality caused by EBOV and the closely related Marburg virus (MARV) in mice. Daily administration of eritoran reduced clinical signs of the disease and, unexpectedly, resulted in reduced viral titers. Analysis of peripheral blood indicated that eritoran reduced granulocytosis despite an apparent increase in the percentage of activated neutrophils. Surprisingly, the increased survival rate and reduced viremia were not accompanied by increased CD3⁺ T lymphocytes, as lymphopenia was more pronounced in eritoran-treated mice. Overall, a global reduction in the levels of multiple cytokines, chemokines, and free radicals was detected in serum, suggesting that eritoran treatment may alleviate the severity of the "cytokine storm." Last, we provide compelling preliminary evidence suggesting that eritoran treatment may alter the kinetics of cytokine responses. Hence, these studies are the first to demonstrate the role of TLR4 in the pathogenesis of EBOV disease and indicate that eritoran is a prime candidate for further evaluation as a clinically viable therapeutic intervention strategy for EBOV and MARV infections.IMPORTANCE A hallmark of bacterial sepsis is the uncontrolled activation of the TLR4 pathway, which is the primary cause of the pathological features associated with this disease. Considering the importance of TLR4 signaling in bacterial sepsis and the remarkable pathological similarities associated with infections caused by filoviruses Ebola virus (EBOV) and Marburg virus (MARV), we assessed the ability of eritoran, a TLR4 antagonist, to protect mice against these viruses. Here, we show that eritoran effectively promotes survival of mice of filovirus infection, as 70% and 90% of mice receiving daily eritoran treatment survived lethal EBOV and MARV infections, respectively. Eritoran treatment resulted in a remarkable global reduction of inflammatory mediators, which is suggestive of the mechanism of action of this therapeutic treatment. These studies are the first to show the critical importance of the TLR4 pathway in the pathogenesis of filovirus infection and may provide a new avenue for therapeutic interventions.

Keywords: Ebola virus; Marburg virus; Toll-like receptor 4; cytokine storm; viral hemorrhagic fever.

FIG 1

Eritoran protects mice from lethal EBOV challenge. (A) Overview of studies investigating the use of eritoran as a therapeutic for EBOV infection. C57BL/6J mice were challenged via i.p. route with 1,000 PFU of mouse-adapted EBOV. Mice received 10 daily injections of eritoran or placebo (vehicle) via the i.p. route. (B) Survival curves from two independent experiments consisting of groups of 5 mice per group. (C) Illness scores assigned as described in Materials and Methods. (D) Weight change following EBOV challenge. (E) Viremia on day 6 postinfection. (B to D) Mean values of two independent experiments of 5 mice per group ± standard errors. (E) Mean values ± standard errors based on 4 mice in placebo group and 5 mice in eritoran-treated group. See also Fig. S1 and S2 in the supplemental material.

FIG 2

Eritoran treatment increases neutrophil activation. Analysis of peripheral blood in mock-infected, placebo-treated, and eritoran-treated infected mice by flow cytometry. (A) Absolute neutrophil counts. (B) Percentages of CD11b⁺ neutrophils positive for CD64. (C) Percentages of Ly6G/Lyc6C neutrophils positive for CD69. See also Fig. S4.

FIG 3

Eritoran reduces expression of Th1- and Th17-associated cytokines by T lymphocytes. (A) Absolute counts of CD3⁺ T-cell subsets in white blood cells. (B to E) Percentages of the indicated T-cell populations positive for the indicated markers of activation. (B) CD3⁺ CD8⁺ T cells positive for IFN-γ. (C) CD3⁺ CD4⁺ T cells positive for IFN-γ. (D) CD3⁺ CD4⁺ T cells positive for IL-4. (E) CD3⁺ CD4⁺ T cells positive for IL-17. Mean values ± standard errors based on 5 mice per group on day 6 postinfection. Histograms are representative of one mouse from each group. See also Fig. S5.

FIG 4

Eritoran treatment reduces cytokine storm and the levels of free radicals. (A) Heat map of serum cytokine levels in placebo- and eritoran-treated mice following normalization to mock levels, day 6 postinfection. (B to F) Serum levels of inflammatory mediators, including Th1-associated cytokines (B), Th2-associated cytokines (C), chemokines (D), cytokines associated with stem cell and progenitor differentiation and survival of T lymphocytes and NK cells (E), and reactive oxygen species/reactive nitrogen species (F). Average fold difference versus uninfected mice, mean values ± standard errors based on 5 mice per group (mock and eritoran) or 4 mice per group (placebo). *, P < 0.05 for eritoran compared to placebo. See also Table S1.

FIG 5

Eritoran protects mice from lethal MARV challenge. C57BL/6J mice were challenged via the i.p. route with 1,000 PFU of mouse-adapted MARV. Mice received 10 daily injections of eritoran or placebo (vehicle) via the i.p. route. (A) Survival curves generated from MARV-infected mice treated with placebo or eritoran. (B) Illness scores assigned as described in Materials and Methods. (C) Weight change following MARV challenge. Mean values from two independent experiments of 5 mice per group ± standard errors (A to C).