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- Materials and methods
Allergic asthma is a respiratory inflammatory disease characterized by reversible airway obstruction, airway hyper-responsiveness, airway inflammation, and mucus hypersecretion in atopic patients. The common hallmarks of allergic asthma are the production of allergen-specific IgE and the increased presence of eosinophils and T helper 2 (Th2) cells in the airway. Th2 cells play a major role in the development of asthma by producing cytokines such as interleukin (IL)-4, IL-5, and IL-13 to enhance IgE class switching, mast cell production, goblet cell hyperplasia, mucus hypersecretion and the maturation, activation, and accumulation of eosinophils.[1, 2] In addition, increased activity of Th17 or Th9 as well as decreased regulatory T cells represents additional mechanisms to contribute to asthma which may skew the system toward an increased Th2 response.
Due to the great improvement of detection techniques including the development of highly sensitive and specific molecular diagnosis, there is considerable evidence that respiratory viral infection is linked to the initial development as well as exacerbations of asthma.[4, 5] Many different viruses are associated with these episodes, particularly respiratory syncytial virus, influenza viruses, parainfluenza viruses, and rhinoviruses.[3, 6] Despite extensive association of common types of respiratory viruses with asthma, there is no evidence yet to prove viral infection is a cause of asthma, but suggests that there may be common susceptibilities to both viral infection and asthma.[3, 7-10] Influenza viruses, an orthomyxovirus, are single-stranded negative sense RNA viruses with three major types A, B, and C, and multiple subtypes. Upon infection of host cells by influenza virus, viral RNAs are sensed by pattern recognition receptors (PRRs), such as Toll-like receptor 7 (TLR7).[11, 12] TLR7 is important not only for the activation of the innate antiviral response but also for the induction of adaptive immunity. The recognition of influenza viruses by plasmacytoid dendritic cells through TLR7 results in their activation of costimulatory molecules and production of IFN-α,[11, 12] which is a critical cytokine for establishing an antiviral state and bridging the innate and adaptive immune systems. In addition, whole inactivated influenza virus also activates dendritic cells through the engagement of TLR7.[15, 16] Of note, influenza A virus infection represents a significant public health threat, particularly in the case of children, the elderly, and those with underlying diseases, all of whom are at a significantly increased risk of disease complications and death following influenza virus infection.[17-19] In fact, atopy itself may have more severe respiratory viral infection and associated wheezing, particularly in rhinovirus infection,[20, 21] suggesting that virus–allergy interaction is at work in at least some asthmatics.[20, 22-24]
While yearly influenza vaccination is widely recommended for patients with asthma, the efficacy of protection of influenza infection and the benefits of influenza vaccination in preventing asthma exacerbations are unclear. In this study, we examined the efficacy and effects of influenza vaccination on an ovalbumin (OVA)-immunized mouse model of allergic asthma by using inactivated influenza virus vaccine. To rule out the possible effects induced by commercial vaccine formulation such as excipients, chicken egg components, and formaldehyde, we chose UV-inactivated virus as the vaccine. Our results demonstrated that influenza vaccinated OVA-immunized mice got protection from influenza virus infection while non-vaccinated mice did not. Moreover, influenza vaccination did not induce extra airway inflammation but reduced cell infiltration to airways and Th2 cytokines IL-4 and IL-6 production in the bronchoalveolar lavage fluid (BALF) of OVA-immunized and challenged mice.
- Top of page
- Materials and methods
In this study, we determined the efficacy and effects of inactivated influenza virus vaccine on an OVA-immunized mouse model of allergic asthma. Our results demonstrated that mice inoculated with inactivated influenza virus vaccine induced high levels of anti-PR8 IgG2a and upregulation of TLR7. Vaccinated allergic mice were healthy while non-vaccinated allergic mice were all dead after subsequent influenza virus challenge. Inactivated influenza virus vaccine neither induced airway inflammation nor changed asthma features such as IgE, airway hyper-responsiveness, and eosinophilia in allergic mice. Of note, decreased frequency of airways infiltrated with immune cells was observed in vaccinated allergic mice. Th2 cytokines IL-4 and IL-6 production in the BALF of allergic mice inoculated with inactivated influenza virus was decreased. These results suggested that UV-inactivated influenza virus vaccine is efficient in protecting allergic mice from influenza infection, and it does not exacerbate but reduce some features of allergic asthma.
A growing number of epidemiologic studies have shown that certain respiratory viral infections such as respiratory syncytial virus, rhinovirus, and influenza virus are associated with increased atopy to common allergens and an overall increased risk of asthma.[7-9, 17, 20, 21, 23] In the recent study on H1N1 influenza virus, Kloepfer et al. reported that increased susceptibility to H1N1 infection and greater hospitalization rates for those with asthma were observed and that H1N1 accounted for 23% of episodes of loss of asthma control during the peak viral season. Previous studies demonstrated that respiratory viral Th1 responses did not down regulate Th2 immune response but can positively regulate Th2 responses and exacerbate allergic Th2-type diseases.[7, 9, 17] Moreover, respiratory viral infection in atopic patients initiates an atopy-dependent cascade that amplifies and sustains airway inflammation and then drives cumulative airway tissue damage.[8, 23] Here, our study showed that allergic mice were more vulnerable to influenza infection like allergic patients, while vaccination can protect them from infection. A survey study in pediatric patients shows that influenza vaccination is associated with fewer asthma exacerbations. After controlling for several potential confounding variables, administration of influenza vaccine is associated with a protective effect against indicators of asthma exacerbations. Hence, these findings reinforce the influenza vaccination is essential and efficient for allergic subjects to prevent further infection.
Chirkova et al.'s study shows that intranasal administration with live attenuated influenza vaccine during the remission phase of bronchial asthma did not enhance allergic inflammatory changes in the lung, OVA-specific IgE, and IL-4 production by spleen lymphocytes. In addition, after additional OVA exposure, histological and immunological changes in these mice were the same as in the control group. Using intranasal instillation of inactivated influenza virus before allergen sensitization, Minne et al. show that vaccination significantly reduced the serum levels of total and OVA-specific IgE as well as allergen induced airway hyper-reactivity. Moreover, only slight and short-term local inflammatory responses are shown in intranasal instillation of inactivated influenza virus, whereas immunogenic efficacy of vaccine is still maintained. These results, coupled with our data herein, suggest that influenza vaccination is safe and does not exacerbate but reduce when encountering subsequent contact of asthma sufferers with allergen.
While Th2 cells promote airway inflammation in asthma, it has been proposed that Th1 cells, which secrete IFN-γ, protect against allergic disease by dampening the activity of Th2 effector cells, which secrete IL-4, IL-5 and IL-6. The immune balance controlled by Th1 and Th2 is crucial for immunoregulation, and its imbalance causes various immune diseases including allergic asthma.[2, 34] In this study, we found BALF Th2 cytokines IL-4 and IL-6 were significantly decreased in vaccinated allergic mice. However, no increased production of IFN-γ was observed. In addition, we found a strong OVA/alum immunized Th2-associated immune response did affect following immune responses against influenza virus to produce more IgG1. However, the increased anti-PR8 IgG1 neither enhance allergic immune response nor reduce the efficacy of vaccination. Hence, the decreased IL-4 in vaccinated allergic mice was not due to the counter balance by Th1 cytokine. IL-4, which induces differentiation of Th2 cell and class switching of B cell, plays an important role in allergic asthma. IL-6, a proinflammatory cytokine, prevents Th1 differentiation by upregulating suppressor of cytokine signaling 1 (SOCS1) expression in activated CD4+ T cells, thereby interfering with signal transducer and activator of transcription 1 (STAT1) phosphorylation induced by IFN-γ In addition, IL-6 induces the production of IL-4 in activated CD4+ T cells, which promotes the differentiation of these cells into effector Th2 cells; these observations suggest that decrease in both IL-4 and IL-6 in vaccinated mice in this study may be due to the decrease in IL-6 which then leads to the decrease of IL-4.
Many pathogens, including bacteria and viruses, have been shown to be recognized by Toll-like receptors (TLRs) of innate immune cells. Influenza virus is recognized by TLR7.[11, 12] In this study, we found that high levels of TLR7 expression in mice inoculated with inactivated PR8 virus. This is consistent with previous study that the immunogenicity of the whole inactivated vaccine against influenza is dependent on MyD88-dependent TLR7 signaling. In addition, administration of TLR7 agonists reduces serum IgE, IL-4, airway hyper-reactivity, and airway inflammation in murine models of allergic airway disease.[37-40] These protective effects of TLR7 agonists in allergic asthma are mediated through type I interferons, IL-12 and IL-10, or TGF-β. Taken together, inactivated influenza virus vaccination induces the expression of TLR7, which then reduces IL-4 in allergic asthma.
In summary, our findings clearly indicate that vaccination with inactivated influenza virus can protect allergic mice from influenza infection. In contrast to exacerbation, influenza vaccination seems to downregulate the allergic airway inflammatory responses maybe through TLR7 signaling.