Inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal H1N1 influenza infection in mice

Abstract

Introduction: Factors implicated in influenza-mediated morbidity and mortality include robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction. Tumor necrosis factor-alpha (TNF-α) is an important pro-inflammatory cytokine present during influenza infection, but it is unclear whether direct inhibition of TNF-α can elicit protection against influenza infection.

Methods: In this study, the commercially available TNF-α inhibitor etanercept was used to inhibit TNF-α induced by lethal A/FM/1/47 (H1N1) influenza virus infection of mice. The effects of TNF-α inhibition on mouse survival, pathologic changes, immune cell infiltration, inflammatory cytokine secretion, Toll-like receptor expression, and activation of the NF-κB (nuclear factor kappa B) signaling pathway were evaluated.

Results: The intranasal delivery of etanercept provided significant protection against mortality (30% of mice survived up to 14 days after infection) in mice treated with etanercept. In contrast, no survivors were found beyond 6 days in mice treated with saline after lethal challenge with H1N1 influenza virus. It was observed that etanercept significantly reduced inflammatory cell infiltration (for example, macrophages and neutrophils), inflammatory cytokine secretion (for example, interleukin-6, TNF-α, and interferon gamma), and expression of Toll-like receptors (TLR-3, TLR-4, and TLR-7). Etanercept also downregulated and inhibited the cascade proteins of the NF-κB signaling pathway (for example, MyD88, TRIF, NF-κB, and p65), as well as enhanced host control of virus replication.

Conclusions: These findings indicate that etanercept, by blocking TNF-α, can significantly downregulate excessive inflammatory immune responses and provide protection against lethal influenza infection, making its use a novel strategy for controlling severe influenza-induced viral pneumonia.

Figure 1

Administration of etanercept protects against lethal H1N1 influenza virus infection and inhibits the immunopathologic effects associated with infection in vivo. Saline or 2.5 mg/kg of etanercept was administered i.n. to mice 2 hours after i.n. infection with 10 TCID₅₀ of influenza virus A/FM/1/47 (H1N1). (A, B) Mice were monitored for survival and body-weight loss. Data were derived from three separate experiments, with a total of 30 mice per group (10 mice per group each time). Survival curves show data until day 14 after infection, because further mortality was not observed at later time points. (C, D) Lung/body weight and inflammation score were evaluated on day 4 after infection. Mice were weighed (grams) and euthanized. Whole lungs were harvested, weighed (grams), and the corresponding lung/body index was calculated. The scores were calculated as follows: none, 0; ≤25%, 1; 26% to 50%, 2; 51% to 75%, 3; ≥76%, 4 lung involvement. #P ≤ 0.05 compared with saline recipients. (E through H) Histopathologic analysis on day 4 after infection was carried out by three separate pathologists who were blinded to the various experimental treatments.

Figure 2

Etanercept reduced inflammatory cells infiltration and dampened the production of pro-inflammatory cytokines. On days 2 and 4 after infection, mice were euthanized, and the lungs were harvested to be homogenized individually and assayed for the following. (A) Inflammatory cytokines (TNF-α, IFN-γ, IL-6) were analyzed with ELISA. (B through G) Major immune cells (macrophage/monocytes, neutrophils, NK cells, B cells, CD4⁺ Th cells, and CD8⁺ Tc cells) were analyzed with flow cell cytometry. Four samples were picked up from mice per group each time for assaying, and three replicated experiments were conducted. Data are presented as mean ± SD. #P ≤ 0.05 compared with saline recipients.

Figure 3

Etanercept inhibited the TLRs-NF-κB signaling pathway and enhanced host control of influenza replication. On days 2 and 4 after infection, mice were euthanized, and the lungs were harvested and assayed for the following: (A through C) Relative mRNA levels of TLR3, TLR4, and TLR7. (D through F) The cascade genes (MyD88, TRIF, NF-κB, p65) in the NF-κB pathway. (G) Influenza M gene, indicating the replication of influenza virus. (H through I) Immunohistochemical assay for phosphorylation of NF-κB p65 (dark yellow staining). The level of dark yellow staining indicates the activation of the NF-κB signaling pathway. Data are presented as mean ± SD. #P ≤ 0.05 compared with saline recipients.