IFN-γ Attenuates Eosinophilic Inflammation but Is Not Essential for Protection against RSV-Enhanced Asthmatic Comorbidity in Adult Mice

Abstract

The susceptibility to respiratory syncytial virus (RSV) infection in early life has been associated with a deficient T-helper cell type 1 (Th1) response. Conversely, healthy adults generally do not exhibit severe illness from RSV infection. In the current study, we investigated whether Th1 cytokine IFN-γ is essential for protection against RSV and RSV-associated comorbidities in adult mice. We found that, distinct from influenza virus, prior RSV infection does not induce significant IFN-γ production and susceptibility to secondary Streptococcus pneumoniae infection in adult wild-type (WT) mice. In ovalbumin (OVA)-induced asthmatic mice, RSV super-infection increases airway neutrophil recruitment and inflammatory lung damage but has no significant effect on OVA-induced eosinophilia. Compared with WT controls, RSV infection of asthmatic Ifng^-/- mice results in increased airway eosinophil accumulation. However, a comparable increase in eosinophilia was detected in house dust mite (HDM)-induced asthmatic Ifng^-/- mice in the absence of RSV infection. Furthermore, neither WT nor Ifng^-/- mice exhibit apparent eosinophil infiltration during RSV infection alone. Together, these findings indicate that, despite its critical role in limiting eosinophilic inflammation during asthma, IFN-γ is not essential for protection against RSV-induced exacerbation of asthmatic inflammation in adult mice.

Keywords: RSV; asthma; comorbidity.

Figure 1

Prior RSV infection has no significant impact on acute bacterial clearance during secondary S. pneumoniae infection. C57BL/6 WT mice were infected with RSV or X31 (IAV) and 7 days later super-challenged with SPn. (A) Lung SPn burdens, (B) Representative dot plots (top panel) and numbers (bottom panel, mean ± SE, n ≥ 5) of airway alveolar macrophages (AM), monocytes (Mo), neutrophils (Neu), and eosinophils (Eos), and (C) levels of TNF-α, IL-6, and IFN-γ (mean ± SE, n ≥ 5) at 24 h after SPn infection. Each dot represents one mouse; * p < 0.05, *** p < 0.001, one-way ANOVA with Tukey’s multiple comparisons’ test. Data shown are representative of two independent experiments.

Figure 2

RSV infection of WT mice with OVA-induced asthma increases eosinophilic and neutrophilic inflammation. (A) Scheme of the experimental design. (B) Lung RSV titers, (C) airway protein and (D) LDH levels, (E) numbers of airway eosinophils (Eos) and neutrophils (Neu) in the BALF at day 7 after RSV infection of OVA-induced asthmatic C57BL/6 WT mice. Corresponding control mice were treated with PBS. AU, Arbitrary Unit; * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey’s multiple comparisons’ test. Data shown are representative of two independent experiments.

Figure 3

IFN-γ inhibits both eosinophilic and neutrophilic inflammation during RSV infection of OVA-induced asthmatic mice. (A) Numbers of eosinophils (Eos) and neutrophils (Neu) and (B) LDH levels in the BALF at day 7 after RSV infection of C57BL/6 WT and Ifng^−/− mice. (C) Numbers of airway eosinophils and neutrophils at day 7 after RSV infection of OVA-induced asthmatic C57BL/6 WT, Ifng^−/−, and Ifnar^−/− mice. ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey’s multiple comparisons’ test. Data shown are representative of two independent experiments.

Figure 4

RSV infection of WT mice with HDM-induced asthma induces moderate levels of eosinophilic and neutrophilic inflammation. (A) Scheme of the experimental design. (B) Lung RSV titers and (C) numbers of airway eosinophils and neutrophils at day 7 after 10⁵ PFU RSV infection of HDM-induced asthmatic C57BL/6 WT mice. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey’s multiple comparisons’ test. Data shown are representative of at least two independent experiments.

Figure 5

IFN-γ is necessary for limiting HDM-induced eosinophil infiltration regardless of RSV infection. Numbers of airway eosinophils and neutrophils in C57BL/6 WT and Ifng^−/− mice during (A) HDM-induced asthma alone and (B) RSV infection of HDM-induced asthmatic mice. * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s multiple comparisons’ test. Data shown are representative of at least two independent experiments.