Influenza A infection enhances antigen-induced airway inflammation and hyperresponsiveness in young but not aged mice

Abstract

Background: Although morbidity and mortality rates from asthma are highest in patients > 65 years of age, the effect of older age on airway inflammation in asthma is not well established.

Objective: To investigate age-related differences in the promotion of allergic inflammation after influenza A viral respiratory infection on antigen-specific IgE production, antigen-induced airway inflammation and airway hyperresponsiveness in mice.

Methods: To accomplish this objective, the following model system was used. Young (6 week) and aged (18 months) BALB/c mice were first infected with a non-lethal dose of influenza virus A (H/HKx31). Mice were then ovalbumin (OVA)-sensitized during the acute infection (3-days post inoculation) and then chronically underwent challenge to the airways with OVA. Forty-eight hours after the final OVA challenge, airway hyperresponsiveness (AHR), bronchoalveolar fluid (BALF) cellular and cytokine profile, antigen-specific IgE and IgG1, and lung tissue inflammation were measured.

Results: Age-specific differences were noted on the effect of a viral infection, allergic sensitization, airway inflammation and airway hyperresponsiveness. Serum OVA-specific IgE was significantly increased in only the aged mice infected with influenza virus. Despite greater morbidity (e.g. weight loss and sickness scores) during the acute infection in the 18-month old mice that were OVA-sensitized, there was little effect on the AHR and BALF cellular differential. In contrast, BALF neutrophils and AHR increased, but eosinophils decreased in 6-week mice that were OVA-sensitized during an acute influenza infection.

Conclusion: With increased age in a mouse model, viral infection prior to antigen sensitization affects the airway and systemic allergic response differently. These differences may reflect distinct phenotypic features of allergic inflammation in older patients with asthma.

Keywords: aging; asthma; influenza; murine model.

Figure 1

Experimental protocol. Mice (6-weeks, 18-months) were infected with influenza A 3 days prior to antigen sensitization. Mice were sensitized intraperitoneally (i.p) with 100μg OVA absorbed with 2mg alum in 0.4ml PBS on days 0 and 7. Ten days after the last sensitization dosing, mice were anesthetized i.p. with ketamine and xylazine and challenged intratracheally (i.t.) for a total of four times with 100μg OVA on days 17, 24, 31 and 32. Control mice consisted of: age-matched antigen sensitized and challenged alone, virally infected alone (day -3), and naïve mice. Forty-eight hours after the final OVA challenge the mice were sacrificed.

Figure 2

Mouse morbidity. Following infection with a sub-lethal dose of Influenza A, mice were evaluated for weight loss [(A) young and (B) aged] and sickness score (C). Data expressed as mean ±SEM. */+/# p<0.05, **/++/## p<0.01, ***/+++/### p<0.001. *OVA/OVA-infected acute compared to naïve, +infected-only compared to naïve, #OVA/OVA-compared to naïve.

Figure 3

Effect of viral infection on specific antigen-induced immunoglobulin production. Sera were collected forty-eight hours after the final OVA-challenge and antigen-specific (A) IgE (B) IgG₁ was determined by ELISA (Serum dilutions: IgG1-1:250,000, IgE-1:10). Data expressed in mean ±SEM from combined data from 2 independent experiments. * p<0.05, **p<0.01, ***p<0.001.

Figure 4

Effect of antigen sensitization during an acute Influenza A infection on antigen-induced BALF inflammation. Forty-eight hours after the final OVA-challenge, BALF was collected for total cell count and differential. Data expressed as mean ±SEM from combined data from 2 independent experiments for total (A) cell count, (B) neutrophil, (C) eosinophil, (D) macrophage, and (E) lymphocyte counts. * p<0.05, **p<0.01, ***p<0.001.

Figure 5

Effect of influenza A infection on antigen induced cytokine production. BALF supernatant was assayed for a panel of cytokines and chemokines [(A) IL-4 (B) IL-5 (C) IL-6 (D) KC (E) IL-13 (F) TNFα] and chemokines [(G) RANTES (H) Eotaxin 1 (I) Eotaxin 2] using a multiplex-assay. Data expressed in mean ±SEM from combined data from 2 independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Effect of influenza A infection on antigen induced cytokine production. BALF supernatant was assayed for a panel of cytokines and chemokines [(A) IL-4 (B) IL-5 (C) IL-6 (D) KC (E) IL-13 (F) TNFα] and chemokines [(G) RANTES (H) Eotaxin 1 (I) Eotaxin 2] using a multiplex-assay. Data expressed in mean ±SEM from combined data from 2 independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 6

Impact of aging, antigen sensitization and challenge and viral infection on lung histopathology (A) Lung histopathology was evaluated by H&E (to access the degree of total lung inflammation), periodic-acid-Schiff (PAS) (to measure mucus metaplasia; magenta), Picrosirius red visualized under polarizing light (to measure collagen deposition) and immunohistochemistry for α-smooth muscle actin (α-SMA) (for contractile elements in the airway wall; brown). All images were taken at ×200 magnification. (B) Perivascular inflammation (PV), (C) Peribronchial inflammation (PB), and (D) mucus metaplasia (MM) of lung tissues were graded as described in the “Methods” section.

Figure 6

Impact of aging, antigen sensitization and challenge and viral infection on lung histopathology (A) Lung histopathology was evaluated by H&E (to access the degree of total lung inflammation), periodic-acid-Schiff (PAS) (to measure mucus metaplasia; magenta), Picrosirius red visualized under polarizing light (to measure collagen deposition) and immunohistochemistry for α-smooth muscle actin (α-SMA) (for contractile elements in the airway wall; brown). All images were taken at ×200 magnification. (B) Perivascular inflammation (PV), (C) Peribronchial inflammation (PB), and (D) mucus metaplasia (MM) of lung tissues were graded as described in the “Methods” section.

Figure 7

Effect of antigen sensitization during an acute influenza A infection on antigen-induced airway hyperresponsiveness. Forty-eight hours after the final OVA-challenge, AHR was determined by APTI after acetylcholine injection. Data were means ±SEM from combined data from 3-4 independent experiments (n=5-7/group). * p<0.05, **p<0.01, ***p<0.001.