Mice with asthma are more resistant to influenza virus infection and NK cells activated by the induction of asthma have potentially protective effects

Abstract

Purpose: This study was conducted in order to investigate whether the virulence of the influenza virus infection is affected by asthma in mice.

Methods: Mice with asthma or control mice were infected with influenza virus. The survival rate, body weight, virus titer, cytokine profile, and cell infiltration in bronchoalveolar lavage fluid (BALF) were measured. The NK cell cytotoxicity was determined by a co-culture system with YAC-1 cells, and the effects of NK cells were observed by depletion of NK cells using anti-asialoGM1 serum. The virus-specific CD8(+) T cell killing assay was also performed.

Results: When asthmatic or control mice were infected with non- and sub-lethal doses of influenza virus, the asthmatic mice were more resistant to the virus than control mice with regard to the survival rate, the remission of body weight loss, and the virus burden. Anti-viral cytokines and the NK cell number were increased in the BALF of asthmatic mice before the infection. The NK cell cytotoxicity in the asthmatic mice was significantly enhanced compared to that in control mice, and the depletion of NK cells in asthmatic mice was abrogated both the improved survival rate and the recovery of the body weight loss. The antigen-specific CD8(+) T cell killing activity in asthmatic mice was also significantly increased following the infection compared to that in control mice.

Conclusion: NK cell activated by the induction of asthma and the subsequently activated antigen-specific CD8(+) T cells could promptly eliminate the viral-infected cells, thus leading to improvements in the morbidity and mortality of influenza virus infection.

Fig. 1

Asthmatic model mice exhibit increased OVA-specific IgE in their serum and histopathological changes characterized by allergic asthma. C57BL/6 mice were intraperitoneally sensitized with OVA or PBS as a control every other day for 2 weeks, then allowed to rest for 3 weeks. The OVA-sensitized mice or control mice were then intranasally challenged with OVA or PBS on days −3, −2 and −1, respectively. Bloods, trachea tissues, and lungs were collected from the mice on day 0. a The OVA-specific IgE in the serum was measured by an ELISA assay. b The trachea tissues (upper panels) and lungs (bottom panels) were stained with HE. Six mice were used in each group and similar observations were obtained from each mouse

Fig. 2

The survival rate and recovery of body weight loss were improved in asthmatic mice during influenza virus infection. C57BL/6 mice were sensitized and challenged with OVA or PBS. Subsequently, the mice were infected with 10 (a, b), 100 (c, d), or 1,000 pfu (e, f) of influenza virus. The survival rate (a, c, e) and body weight (b, d, f) were monitored daily until day 20. The body weight data of the dead mouse were excluded from the analysis from the day of death onwards. Twelve mice were evaluated for each group. Similar results were obtained from three independent experiments. *p < 0.05 compared to the body weight of control mice on the same day. These results are representative of three independent experiments

Fig. 3

The virus titer in asthmatic mice is lower than that in control mice after influenza virus infection. C57BL/6 mice were sensitized and challenged with OVA or PBS. The mice were then infected with 100 pfu of the influenza virus. The virus titers in the BALF were calculated by the TDID₅₀ assay on days 2, 4, and 6 after influenza virus infection. Six mice were used in each group. *p < 0.05 compared to the virus titer of the control mice. Similar results were obtained from two independent experiments

Fig. 4

Differences were observed in the cytokine production between asthmatic mice and control mice during influenza virus infection. C57BL/6 mice were sensitized and challenged with OVA or PBS. Subsequently, the mice were infected with 100 pfu of the influenza virus. BALF samples from the mice were collected on day 0 before viral infection and on days 2, 4, and 6 after influenza virus infection, and the cytokine concentrations in the BALF supernatants were measured by ELISA assays. Six mice were used in each group. These results are representative of two independent experiments. *p < 0.05 compared to the cytokine production of control mice. These results are representative of two independent experiments

Fig. 5

There are changes in the frequency and number of NK cells in the lungs and spleen following the induction of asthma. C57BL/6 mice were sensitized and challenged with OVA or PBS. The BALF samples or spleens from the mice were collected on day 0 before influenza virus infection. The total numbers of cells in the BALF (a) or spleen (d) were counted under a microscope with 0.25% trypan blue staining. The frequencies of NK cells in the BALF (b) or spleen (e) were analyzed by FCM. The NK1.1-positive and CD3-negative lymphocytes were identified to be NK cells. The numbers of NK cells in the BALF (c) or spleen (f) were calculated by total cell counting. Twelve mice were evaluated in each group. Similar results were obtained from two independent experiments

Fig. 6

NK cell cytotoxicity and the effects of NK cell depletion during influenza virus infection. C57BL/6 mice were sensitized and challenged with OVA or PBS. The cells in the spleen were considered to be effector cells and were prepared from asthmatic mice or control mice on day 0 before the infection. The titrated effector cells and 1 × 10⁵ cells ml⁻¹ of YAC-1 cells were co-cultured at various effector/target ratios, as indicated in the figure. The cytotoxicity percentages were calculated as described in the “Materials and Methods” (a). Asthmatic mice were injected i.v. with normal rabbit serum or anti-asialoGM1 serum on days −4 and 0 before the infection. The survival rate (b) and body weight (c) were monitored daily until day 20. The body weight data of the mice that died were excluded from the day of death onwards. Twelve mice were used in each group. Similar results were observed from two independent experiments. *p < 0.05 compared to the body weight of control mice on the same day. These results are representative of two or three independent experiments

Fig. 7

The antigen-specific cytotoxicity of CD8⁺ T cells following influenza virus infection. C57BL/6 mice were sensitized and challenged with OVA or PBS. Subsequently, the mice were infected with 100 pfu of influenza virus. An FCM analysis and an in vivo killing activity assay were performed on day 6 after the infection. Cells in the spleen were incubated with H-2D^b Influenza NP tetramer followed by staining with both an anti-CD8 Ab and an anti-CD3 Ab and analyzed by FCM. Cells that were CD8-positive, CD3-positive, and influenza NP tetramer-positive were identified as antigen-specific CD8⁺ T cells (a). The number of antigen-specific CD8⁺ T cells was calculated by total cell counting (b). Representative FACS data of the in vivo killing assay from non-infected control or asthmatic mice and infected control or asthmatic mice are shown (c). The percentages of in vivo killing activity for asthmatic mice or control mice were calculated as indicated in the “Materials and Methods” (d). Five mice were used in each group. Similar results were obtained from two independent experiments