The TLR4 antagonist Eritoran protects mice from lethal influenza infection

Abstract

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 inflammation. Subsequently, we reported that Tlr4(-/-) mice are highly refractory to influenza-induced lethality, 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 antagonist-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.

Figure 1

Eritoran treatment protects mice from lethal influenza challenge. (A) Basic experimental protocol used to test Eritoran in mice infected with influenza. C57BL/6J mice were infected with mouse-adapted influenza, strain PR8 (~7500 TCID₅₀, i.n.; ~LD₉₀). Two days later, mice received placebo (vehicle only) or E5564 (Eritoran; 200 µg/mouse i.v.) once daily for 5 successive days (Days 2 to 6). (B) Mice were treated as shown in (A). In the left graph, survival was monitored daily (p <0.002). In the right graph, clinical scores (see Methods) were also measured daily. Each graph represents the combined results of 2 separate experiments, each with 5 mice/treatment/experiment. (C) Mice were infected as described in (A), but treated with Eritoran starting on Days 2, 4, or 6 post-infection. Left panel, survival (Day 2 and Day 4, p < 0.01; Day 6, p ≤ 0.05); right panel, clinical scores. Results are combined results from 2 to 3 separate experiments, with 5 mice/treatment group/experiment.

Figure 2

Eritoran treatment inhibits influenza-induced lung pathology and lung function. (A) Eritoran treatment improves lung pathology induced by PR8 infection. Representative H&E-stained sections were derived from mice treated as described in the text (4–5 mice/treatment group) sacrificed on Day 7 post-infection. Images are at 400× magnification. (B) Histological scoring for PR8-infected mice without or with Eritoran treatment ± s.e.m. (C) Pulse oximetry data collected on Day 6 post-infection ± s.e.m. (p < 0.001, comparing PR8 only with Mock-infected or PR8 + E5564; 4–6 mice/treatment group). (D) Eritoran treatment improves lung pathology of H3N2 infection of cotton rats. (E) Histological scoring for H3N2-infected cotton rats with or without eritoran treatment (n = 5 rats/treatment; data shown is means ± s.e.m.).

Figure 3

Treatment with Eritoran reduces lung viral titers. Mice were infected with PR8 and then either left untreated (NT) or eritoran (E5564)-treated as described in Fig. 1A and were sacrificed on Days 2, 4, 6 (A) and 7 (B) post-infection. (A, left panel). Results represent the combined results of 2 separate experiments (5 mice/treatment group/experiment) *, p < 0.05. (A, right panel). Day 7 post-infection (7 mice/treatment group); *, p < 0.001.

Figure 4

Eritoran treatment suppresses influenza-induced cytokine gene expression. Mice were treated as described in Fig. 1A and euthanized on Days 2 (3 h post-treatment), 4, and 6 post-infection (4 mice/treatment group/experiment; data presented are means ± s.e.m. from 2 separate experiments; p< 0.01 at each time point). Lungs were processed for total RNA and subjected to qRT-PCR for detection of specific gene expression.

Figure 5

Molecular requirements of Eritoran-induced protection. (A) WT, TLR4^−/−, CD14^−/−, TLR2^−/− mice were either untreated (closed circles), or treated with E5564 (open circles) or 2 days post-infection, for 5 successive days. WT data were combined from 5 separate experiments (5–6 mice/treatment/experiment), TLR4^−/− data from 3 separate experiments (5–6 mice/treatment/experiment), CD14^−/− mice (2 separate experiments; 5 mice/treatment group/experiment, and TLR2^−/− mice (2 separate experiments; 4–5 mice/treatment group/experiment). WT: untreated vs. E5564 treatment (p < 0.0001); TLR4^−/−: untreated vs. E5564 treatment (p = ns); CD14^−/−: untreated vs. E5564 treatment (p = ns); TLR2^−/−: untreated vs. E5564 treatment (p = ns). (B) Dose-dependent Eritoran inhibition of the transfer and binding of monomeric [³H]LOS from aggregated [³H]LOS to sCD14 and from [³H]LOS.sCD14 to MD2. Samples containing [³H]LOS aggregates (0.2 nM), His₆-sCD14 (~0.5 nM), and increasing concentrations as indicated of Eritoran or unlabeled LOS (left panel) or 2 nM [³H]LOS.sCD14, ca. 2 nM His₆-MD2, and increasing concentrations of Eritoran (or placebo) ± LBP (50 pM) and sCD14 (2 nM) (right panel) were incubated for 30 min at 37° C, followed by addition and incubation with NiFF Sepharose beads to capture His-tagged proteins. Formation of complexes of [³H]LOS with His₆-sCD14 (left) or MD2 (right) was assayed by measuring co-capture of [³H]LOS by NiFF Sepharose as previously described. Data are expressed as percent of co-capture of [³H]LOS observed in the absence of added Eritoran. Results shown represent the mean ± s.e.m. of 3 separate experiments with duplicate samples for each dose.