Inactivated influenza virus vaccine is efficient and reduces IL-4 and IL-6 in allergic asthma mice

Abstract

Background: Allergic asthma is a globally respiratory inflammatory disease. Influenza virus is a respiratory pathogen that causes yearly epidemics and results in high rates of morbidity and mortality. Patients with allergic asthma had a more severe symptom and a higher mortality when they were infected with influenza virus. Hence, influenza vaccination is recommended for patients with asthma.

Objectives: We evaluated the efficacy and effects of influenza vaccination on allergic asthma in a mouse model.

Methods: Ovalbumin-immunized mice were inoculated with inactivated influenza virus A/Puerto Rico/8/34 (PR8) as vaccines and morbidity or mortality and allergic asthma features of these mice were analyzed.

Results: Mice inoculated with inactivated PR8 induced high levels of anti-PR8 IgG2a and upregulation of Toll-like receptor (TLR) 7. Vaccinated allergic mice were healthy when they were challenged with live influenza virus while none of non-vaccinated allergic mice survived. Furthermore, inactivated influenza virus vaccine induced neither extra airway inflammation nor asthma features such as IgE, airway hyper-reactivity, and eosinophilia in allergic mice. Particularly, decreased frequency of immune cell infiltrated airways and Th2 cytokines IL-4 and IL-6 production in the bronchoalveolar lavage fluid were noted in vaccinated allergic mice. These results suggested that inactivated influenza virus vaccine is efficient to protect allergic mice from further influenza infection, and it does not exacerbate but reduces IL-4 and IL-6 of allergic asthma.

Conclusion: Influenza vaccination is essential and efficient for allergic subjects to protect influenza virus infection.

Keywords: IL-4; IL-6; PR8; asthma; vaccine.

Figure 1

Intramuscular inoculation of inactivated PR8 influenza virus (10 HAU/mouse) induced the highest production of anti‐PR8 IgG2a and upregulated TLR7 expression. (A, B) Mice were inoculated with inactivated PR8 influenza virus on days 0 and 7 by different routes. (A) Serum anti‐PR8 IgG2a was measured on days 0, 7 and 14 by ELISA. (B) BAL total cells were counted. i.m., intramuscular; i.n., intranasal; s.c. subcutaneous. N = 4–5 mice per group. (C) Mice were received intramuscular injection of PBS or inactivated PR8 influenza virus. After 18 hours, the muscles near the site of injection were isolated and expression of TLR‐7 was detected by quantitative RT‐PCR. Relative mRNA was calculated by normalizing the values of the TLR7 to that of β‐actin. *P < 0·05; ***P < 0·005.

Figure 2

Inactivated influenza virus vaccine protected allergic mice from influenza infection. (A) Experimental protocol. Mice were immunized with OVA/Alum or PBS on days 0, 7, 14, and 21, inoculated with inactivated PR8 influenza virus or PBS on days 28 and 35, and challenged with live influenza virus on day 42. (B) Sera were collected on day 27 and OVA‐specific IgE were measured by ELISA. (C) Sera were collected on day 41 and PR8‐specific IgG2a were measured by ELISA. (D) Sera were collected on day 41 and PR8‐specific IgG1 were measured by ELISA. (E) Survival of mice challenged with live influenza virus was measured. Mice were observed for 20 days after influenza infection. N = 10 mice per group. *P < 0·05; **P < 0·01; ***P < 0·005.

Figure 3

Inactivated influenza vaccination did not change serum levels of anti‐OVA IgE, airway hyper‐reactivity, and inflammatory cell infiltration of allergic asthma mice. (A) Experimental protocol. Mice were immunized with OVA/Alum on days 0, 7, 14, and 21, inoculated with inactivated PR8 influenza virus or PBS on days 28 and 35, and then challenged with intranasally OVA on days 42 and 43. Mice were euthanized on day 44. (B) Serum levels of anti‐OVA IgE were measured by ELISA. (C) Airway function was measured by lung resistance (RL). (D) BAL total cell numbers and (E) the absolute numbers of macrophages (MΦ), neutrophils (Neu), eosinophils (Eos), and lymphocytes (Lym) were obtained by counting cells on a cytocentrifuged preparation. The absolute numbers were calculated by multiplying the respective frequencies by the absolute number of BAL cells. N = 9 mice per group.

Figure 4

Inactivated influenza vaccination reduced the frequency of infiltrative airways of allergic asthma mice. Mice were immunized with OVA/Alum, inoculated with inactivated PR8 influenza virus or PBS, and challenged with intranasally OVA. Mice were euthanized on day 44 as shown in Figure 3A. (A) Representative H&E stain of lung tissue. (B) The percentages of infiltrative airways in mice. N = 9 mice per group. *P < 0·05.

Figure 5

Inactivated influenza vaccination reduced IL‐4 and IL‐6 in the BALF of allergic asthma mice. Mice were immunized with OVA/Alum, inoculated with inactivated PR8 influenza virus or PBS, and challenged with intranasally OVA. Mice were euthanized on day 44 as shown in Figure 3A. BALF cytokines (A) IL‐4, (B) IL‐5, (C) IL‐6, (D) eotaxin, and (E) IFN‐γwere determined by ELISA. N = 9 mice per group. **P < 0·01; ***P < 0·005.