Mice lacking both TNF and IL-1 receptors exhibit reduced lung inflammation and delay in onset of death following infection with a highly virulent H5N1 virus

Abstract

Background: Highly pathogenic avian influenza viruses of the H5N1 subtype continue to cross the species barrier to infect humans and cause severe disease. It has been suggested that an exaggerated immune response contributes to the pathogenesis of H5N1 virus infection in mammals. In particular, H5N1 virus infections are associated with a high expression of the proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α).

Methods: We investigated the compounding affects of both cytokines on the outcome of H5N1 virus disease by using triple mutant mice deficient in 3 signaling receptors, TNF-R1, TNF-R2, and IL-1-RI.

Results: Triple mutant mice exhibited reduced morbidity and a significant delay in mortality following lethal challenge with a lethal H5N1 virus, whereas no such differences were observed with the less virulent A/PR/8/34 (H1N1) virus. H5N1-infected triple mutant mice displayed diminished cytokine production in lung tissue and a quantifiable decrease of macrophages and neutrophils in the lungs postinfection. Moreover, morphometric analysis of airway sections revealed less extensive inflammation in H5N1-infected triple mutant mice, compared with infected wild-type mice.

Conclusions: The combined signaling from the TNF or IL-1 receptors promotes maximal lung inflammation that may contribute to the severity of disease caused by H5N1 virus infection.

Figure 1. Morbidity and mortality in triple-mutant (TM) mice

Wild type C57B6 × 129 (WT) (n = 11) and TM mice (n = 7) were inoculated intranasally (i.n.) with 1,000 MID₅₀ of A/Hong Kong/483/97 (A and B) or A/PR/8/34 (C and D) virus and monitored daily for survival (A and C) and weight loss (B and D). Kaplan-Meier survival analysis was performed and significant differences in average time to death was observed between infected WT and TM mice (* p < 0.05). Lines represent average percent weight loss over time for each group and error bars show standard errors of the means.

Figure 2. Infectious virus in lung and brain tissues

WT and TM mice were inoculated i.n. with 1,000 MID₅₀ of HK/483 virus and whole lungs were harvested (n= 4 mice per time point) for virus titration on days 3 (A) and 5 p.i. (B). The limit of detection was 10^1.5 EID₅₀/ml. A significant increase in titers was observed from day 3 to 5 in the brains of both WT and TM infected mice (*p < 0.05, Students t test).

Figure 3. Lung cytokine levels

Lung cytokine levels from HK/483 infected mice (days 3 and 5 p.i.) were measured individually (n = 4 mice per group) by Bioplex Protein Array, in duplicate. Bars represent means from each infection group ± standard deviation (SD). The mean lung cytokine values among mock-infected WT mice are as follows; TNF-α (10.8 pg/ml) and RANTES (15.6 pg/ml) and the mean cytokine values among mock-infected TM mice are as follows; TNF-α (11.8 pg/ml) and RANTES (18.2 pg/ml). The following cytokines were not detectable in the mock-infected lungs of either mouse strain; G-CSF, IL-1-α, IL-3, IL-6, IL-12 (p70), IL-12 (p40), MIP-1-α, MCP-1, IL-17, KC, MIP-1-β, and IFN-γ. The minimum cytokine detection was <10 pg/ml. * p < 0.05 between TM and WT mouse groups for each day post-infection (Students t test).

Figure 4. Airway inflammation is reduced in mice infected with H5N1 virus

TM and WT mice were infected with HK/483 virus and 3 days later lungs were filled with fixative and sections were prepared for histopathological analysis. A) Inflamed airways in TM and WT mice. B) Inflammation throughout the conducting airways was quantified and percent surface area calculated as a measure of airway inflammation (n = 7-10 mice per group). C) Pulmonary inflammation was most prominent in larger and more central airways, but less in TM mice throughout the range of airway sizes. Airway diameter was measured throughout the lung sections and airway sizes binned within each genotype. Epithelial necrosis is shown in D). Inflamed airway sections were screened for areas of cellular necrosis and exposed basement membranes, and the fraction of inflamed airways that was denuded was calculated.

Figure 5. Effect of cytokine deficiency on lung immune cell populations following H5N1 virus infection

Mice were infected i.n. with 1,000 MID₅₀ of HK/483 virus (WT n= 8, TM n = 7, IL-1-R KO n = 5, TNF-R KO n = 4) or given PBS (WT n= 5, TM n = 8, IL-1-R KO n = 3, TNF-R KO n = 4). Lungs were removed on days 3 and 5 p.i. and single cell suspensions prepared from individual lungs. Total viable cells from individual lungs were counted on a hemocytometer by trypan blue exclusion prior to antibody labeling and cytometric analysis (A). Bars represent the average total number of live lung cells (× 10⁷ cells) per treatment group ± SD. Statistical differences between treatment groups were measured using the analysis of variance test (ANOVA). * P <0.05 between PBS inoculated mice and H5N1 infected mice in the same genetic group. (x002C6) P <0.05 between WT-H5N1 infected mice and cytokine deficient-H5N1 infected mice. † P <0.05 between WT-PBS inoculated mice and cytokine deficient-PBS inoculated mice. ‡ P <0.05 between TM-H5N1 infected mice and single cytokine deficient-H5N1 infected mice. Lung suspensions from individual mice were stained with fluorescent antibodies and analyzed by flow cytometry to determine immune cell composition following infection B). Numbers of macrophages (CD11b⁺, CD11c^-, Ly6G/C^-, CD3^-, CD4^-, CD8^-)(a), neutrophils (CD11b⁺, CD11c^-, Ly6G/C⁺, CD3^-, CD4^-, CD8^-) (b), dendritic cells (CD11b^-, CD11c⁺, Ly6G/C^-, CD3^-, CD4^-, CD8^-) (c) and T cell numbers (d and e) (CD11b^-, CD11c^-, Ly6G/C^-, CD3⁺ and CD4⁺ or CD8⁺) were determined by appropriate gating within the total lung cell population. Bars represent the average number of immune cells (× 10⁶ cells) per treatment group ± SD.