A pharmacologically immunosuppressed mouse model for assessing influenza B virus pathogenicity and oseltamivir treatment

Abstract

Immunocompromised patients are highly susceptible to influenza virus infections. Although neuraminidase inhibitor (NAI) therapy has proved effective in these patients, the treatment regimens require optimization, which can be partly addressed via animal models. Here, we describe a pharmacologically immunosuppressed mouse model for studying the pathogenesis of influenza B viruses and evaluating the efficacy of antiviral treatment. We modeled clinical regimens for dexamethasone and cyclophosphamide to immunosuppress BALB/c mice that were then inoculated with B/Phuket/3073/2013 (Yamagata lineage) or B/Brisbane/60/2008 (BR/08, Victoria lineage) virus. Although both viruses caused morbidity and mortality in immunosuppressed mice, BR/08 was more virulent, consistently inducing greater morbidity and 100% lethality in mice inoculated with at least 10³ TCID₅₀/mouse. The replication of both viruses was prolonged in the lungs of immunosuppressed mice, but the extent of pulmonary inflammation in these mice was markedly less than that in immunocompetent animals. Most of the examined cytokines, including IFN-γ, IL-1β, and RANTES, were significantly decreased in the lungs of immunosuppressed mice, as compared to immunocompetent animals, until at least 10 days post-infection. Treatment with the NAI oseltamivir for 8 or 16 days increased the mean survival time and reduced virus spread in the lungs of immunosuppressed mice challenged with a lethal dose of BR/08 but did not completely provide protection or decrease the virus titers. Our data suggests that the synergy of the viral load and aberrant immune responses is a key contributor to the severity of infection, as well as the limited efficacy of oseltamivir, which in immunosuppressed mice curtails virus release without clearing infected cells.

Keywords: Antiviral treatment; Immune responses; Immunosuppressed mice; Influenza B virus; Oseltamivir; Pathogenesis.

Figure 1.. Morbidity and mortality of immunocompetent and immunosuppressed mice inoculated with influenza B viruses.

Female 6- to 8-week-old immunocompetent and immunosuppressed (DEX+CP-treated) BALB/c mice (n = 5/group) were anesthetized with isoflurane and inoculated intranasally with influenza B viruses at a range of doses (10²–10⁵ TCID₅₀/mouse in 30 μL). The graphs show the weight loss (A, C, E, and G) and survival (B, D, F, and H) of immunocompetent mice inoculated with PH/13 (A, B) or BR/08 (C, D) viruses and of immunosuppressed mice inoculated with PH/13 (E, F) or BR/08 (G, H) viruses. Control uninfected animals (black line) received sterile PBS (A-D) or DEX+CP (E-H). The horizontal dotted line in panels A, C, E, and G indicates the endpoint for mortality (25% loss of initial weight). The probabilities of survival were determined by Kaplan-Meier and log-rank tests. Abbreviations: DEX+CP, dexamethasone and cyclophosphamide; PH/13, B/Phuket/3073/2013; BR/08, B/Brisbane/60/2008.

Figure 2.. Viral load in respiratory tract of immunocompetent and immunosuppressed mice inoculated with influenza B viruses.

Immunocompetent and immunosuppressed (DEX+CP-treated) BALB/c mice were anesthetized with isoflurane and inoculated intranasally with 10² TCID₅₀/mouse (A, B) or 10⁵ 50 TCID₅₀/mouse (C) of influenza PH/13 or BR/08 virus. Virus titers were determined in the lungs and nasal turbinates of mice (n = 3/group) at 3, 6, 10, and 16 dpi by TCID₅₀ assays in MDCK cells. The bars represent the mean virus titers ± SDs in mouse nasal turbinates (A) and lungs (B, C). *P < 0.05, **P < 0.01, and ***P< 0.001 for comparisons of immunocompetent and immunosuppressed mice by one-way ANOVA with Bonferroni’s multiple comparison post-test.

Figure 3.. Lymphocyte and neutrophil counts in immunocompetent and immunosuppressed mice inoculated with influenza B viruses.

Immunocompetent and immunosuppressed (DEX+CP-treated) BALB/c mice were anesthetized with isoflurane and inoculated intranasally with 10² TCID₅₀/mouse of influenza PH/13 or BR/08 virus. Blood was collected from uninfected control (PBS-treated) mice and from DEX+CP-treated uninfected and virus-inoculated mice (n = 5/group) by the retro-orbital route at −1, 3, 6, 10, 13, 17, and 24 dpi. Total lymphocyte (A), neutrophil (B), CD4⁺ cell (C), CD8⁺ cell (D), CD19⁺ cell (E), and NK cell (F) counts were determined with an automatic cell counter, and lymphocyte populations were differentiated by flow cytometry. Bars represent the mean values ± SDs. *P < 0.05, **P < 0.01, and ***P < 0.001 for comparisons of immunocompetent and immunosuppressed mice one-way ANOVA with Bonferroni’s multiple comparison post-test.

Figure 4.. Induction of pulmonary cytokine/chemokine responses in immunocompetent and immunosuppressed mice inoculated with influenza B viruses.

Immunocompetent and immunosuppressed (DEX+CP-treated) BALB/c mice were anesthetized with isoflurane and inoculated intranasally with 10² TCID₅₀/mouse of influenza PH/13 or BR/08 viruses. The expression levels of IFN-γ (A), IL-12(P40) (B), IL-1α (C), IL-12(P70) (D), IL-1β (E), IP-10 (F), IL-10 (G), and TNF-α (H) were assayed in lung homogenates at 3, 6, and 10 dpi. Bars represent mean values ± SDs (n = 3/group/time-point). *P < 0.05, and ***P < 0.001 for comparisons of PH/13 and BR/08 virus–infected immunocompetent mice; and ^#P < 0.05 ^##, P < 0.01, and ^###P < 0.001 for comparisons of PH/13 and BR/08 virus–infected immunocompetent and immunosuppressed mice by one-way ANOVA with Bonferroni’s multiple comparison post-test. The level of cytokine/chemokine expression in control uninfected mice is indicated by a black dotted line for immunocompetent mice and by a red dotted line for immunosuppressed mice.

Figure 5.. Histopathologic changes in the lungs of immunocompetent and immunosuppressed mice inoculated with influenza B viruses.

Immunocompetent and immunosuppressed (DEX+CP-treated) BALB/c mice were anesthetized with isoflurane and inoculated intranasally with 10² TCID₅₀/mouse of influenza PH/13 or BR/08 virus. Pulmonary lesions were evaluated at 6 or 16 dpi (n = 2/group). Mouse lungs were fixed in 10% neutral buffered formalin and stained with hematoxylin-eosin (HE) (A, E, I, and M), subjected to immunohistochemical (IHC) staining with anti–influenza B antiserum (B, F, J, and N), or analyzed by histomorphometry at 6 dpi (C, G, K, and O) or 16 dpi (D, H, L, and P). Representative images for each treatment group are shown (magnification: ×20 [HE and IHC] or ×2 [histomorphometry]). In the histomorphometry images, the total lung areas examined are outlined in green; areas of active infection with antigen-positive cells are shown in red; and areas of inactive infection with lesions but negligible antigen are shown in yellow. The percentage of the total lung area represented by the lesions is indicated for each image.

Figure 6.. Histopathologic changes in the lungs of immunocompetent and immunosuppressed mice inoculated with influenza BR/08 virus and treated with oseltamivir.

Immunocompetent and immunosuppressed (DEX+CP-treated) BALB/c mice were anesthetized with isoflurane and inoculated intranasally with a 5 MLD₅₀ dose of BR/08 virus. The NAI oseltamivir was administered by oral gavage at a dose of 20 mg/kg/day (0.1 mL/mouse) twice daily for 8 (immunocompetent mice) or 16 (immunosuppressed mice) days. Control animals received sterile water for 8 days. Pulmonary lesions were evaluated at 6 dpi (n = 2/group). Mouse lungs were fixed in 10% neutral-buffered formalin and stained with HE (A, D, G, and J), subjected to IHC staining with anti–influenza B antiserum (B, E, H, and K), or analyzed by histomorphometry (C, F, I, and L). Representative images for each treatment group are shown (magnification: ×20 [A, B, D, E, G, H, J, and K] or ×2 [C, F, I, and L]). The total lung areas examined are outlined in green; areas of active infection with antigen-positive cells are shown in red, and areas of inactive infection with lesions but negligible antigen are shown in yellow. The percentage of the total lung area represented by the lesions is indicated for each image.

Figure 7.. Pulmonary cytokine/chemokine responses in immunocompetent and immunosuppressed mice inoculated with influenza BR/08 virus and treated with oseltamivir.

Immunocompetent and immunosuppressed (DEX+CP-treated) BALB/c mice were anesthetized with isoflurane and inoculated intranasally with influenza BR/08 virus and treated with oseltamivir as described in the legend for Figure 6. The expression levels of IFN-γ (A), IL-6 (B), GM-CSF (C), IL-9 (D), IL-1α (E), IL-12(P70) (F), IL-1β (G), IL-15 (H), IL-5 (I), and TNF-α (J) were assayed in lung homogenates at 6 and 10 dpi. Bars represent mean values ± SDs (n = 3/group/time-point). *P < 0.05, **P < 0.01, and ***P < 0.001 for comparisons of untreated and oseltamivir-treated immunocompetent mice; ^{^^^}P < 0.001 for comparison of untreated and oseltamivir-treated immunosuppressed mice; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 for comparisons of untreated and DEX+CP–treated mice; and ^$$P < 0.01, and ^$$$P < 0.001 for comparison of oseltamivir-treated immunocompetent and immunosuppressed mice by one-way ANOVA with Bonferroni’s multiple comparison post-test. The level of cytokine/chemokine expression in control uninfected mice is indicated by a black dotted line for immunocompetent mice, by a red dotted line for immunosuppressed mice, and by a green dotted line for immunosuppressed, oseltamivir-treated mice.