Influenza B Virus (IBV) Immune-Mediated Disease in C57BL/6 Mice

Abstract

Influenza B viruses (IBV) primarily infect humans, causing seasonal epidemics. The absence of an animal reservoir limits pandemic concern, but IBV infections may cause severe respiratory disease, predominantly in young children and the elderly. The IBV disease burden is largely controlled by seasonal influenza vaccination; however, immunity due to vaccination is sometimes incomplete, a feature linked to antigenic mismatches. Thus, understanding the features that contribute to disease pathogenesis is important, particularly immune-mediated versus virus-mediated outcomes. Unexpectedly, C57BL/6 (B6) mice intranasally infected with a low multiplicity of infection of B/Florida/04/2006 developed substantial morbidity and mortality. To address the cause, B6 mice were treated daily with dexamethasone to dampen the immune and pro-inflammatory response to IBV infection, allowing the determination of whether the responses were immune- and/or virus-associated. As expected, dexamethasone (DEX)-treated mice had a lower pro-inflammatory response and reduced lung pathology despite the presence of high viral lung titers, but mortality was comparable to PBS-treated mice, indicating that mortality may be linked to lung virus replication. The results showed that the immune response to IBV is the major cause of morbidity, mortality, lung pathology, and viral clearance. Importantly, the results suggest that a robust lung CTL response and associated leukocyte influx contribute to disease.

Keywords: IBV; dexamethasone; host–pathogen interaction; immune-mediated disease; influenza B virus; mouse model.

Figure 1

Weight loss (A) and survival (B) of B/Florida/04/2006-challenged C57BL/6 (B6) mice. B6 mice were i.n. infected with 10⁶ or half-log dilutions down to 10¹ PFU of B/Florida/04/2006. Weight loss and survival were recorded daily for ten days post-infection. Points represent the mean +/− SEM of the proportion of original weight (n = 5 mice/group).

Figure 2

Viral lung titers (A), weight loss (B), and survival (C) of B/Florida/04/2006-challenged B6 mice. B6 mice were i.n. challenged with 10³ PFU B/Florida/04/2006. Viral lung titers were determined on days 1, 2, 4, 6, or 8 pi by virus plaque assay. Dots represent the mean PFU/mL of lung homogenate +/− SEM. The limit of detection for viral plaque assay was 50 PFU/mL. Weight loss and survival were recorded daily for 8 days post-infection. Points represent mean +/− SEM of the proportion of original weight. * p < 0.05 between the PBS and DEX-treated mice by Multiple Mann-Whitney U tests (n = 5 mice/group).

Figure 3

Total BAL cells during IBV infection. Mice were treated with PBS or 10 mg/kg DEX daily (starting on day −1 before infection), and BAL cells were collected at indicated days pi with IBV B/Florida/04/2006. Counts were performed using a hemocytometer and Trypan blue exclusion. Data represent the mean + SEM of live BAL cells/mL (n = 3–5 mice/group/time point). No significance between PBS and DEX-treated mice on any day post-infection was detected by multiple Mann–Whitney U Tests.

Figure 4

BAL cell phenotypes during IBV infection. BAL cell phenotypes from mice treated with PBS (black bars) or 10 mg/kg DEX (gray bars) and uninfected, untreated mice (white bars) were stained for (A) CD3⁺ T cells, (B) CD3⁻/CD11b⁺/Ly6G⁻ macrophages, and (C) CD3⁻/CD11b⁻/Ly6G⁺ neutrophils, as determined by flow cytometry. Bars represent the mean +/− SEM of total BAL leukocytes on days 4, 6, and 8 pi (DPI). * p < 0.05 by two-way analysis of variance (ANOVA) with Bonferroni’s correction compared with uninfected, untreated mice. (n = 3–5 mice/group/time point).

Figure 5

BAL T cell response during IBV infection. BAL T cells from mice treated with PBS or 10 mg/kg DEX and infected with 10³ PFU IBV were collected on day 8 pi and analyzed by flow cytometry. (A) Extracellular surface staining to determine percentages of CD8⁺ and CD4⁺ cells of CD3⁺ gated lymphocytes. A portion of BAL cells was stimulated with PMA/Ionomycin as described in materials and methods, and functional analyses of cytokine production were determined for (B) granzyme B and (C) IFNγ. Bars represent the mean +/− SEM, * p < 0.05, ** p < 0.01 by multiple Mann–Whitney U test.

Figure 6

PCR lung transcript expression. Mouse lung transcript expression was performed from lungs of mice treated with PBS or 10 mg/kg DEX and infected with 10³ PFU IBV were collected (A) 2 and (B) 8 days post-infection (dpi). Bars represent the mean +/− SEM fold change of transcripts determined by ΔΔCt, normalized to ACTB, and uninfected, untreated controls, where * p < 0.05 by multiple Mann–Whitney U Tests.

Figure 7

H&E staining of lungs: 40× (left) and 200× (right) magnification photomicrograph of a lung of a mouse that was treated with PBS (top) or DEX (bottom) and infected with IBV on day 8 pi. Bronchiolitis (asterisks), perivascular inflammation (arrows), vasculitis, (arrowheads).