Early-life viral infection and allergen exposure interact to induce an asthmatic phenotype in mice

Abstract

Background: Early-life respiratory viral infections, notably with respiratory syncytial virus (RSV), increase the risk of subsequent development of childhood asthma. The purpose of this study was to assess whether early-life infection with a species-specific model of RSV and subsequent allergen exposure predisposed to the development of features of asthma.

Methods: We employed a unique combination of animal models in which BALB/c mice were neonatally infected with pneumonia virus of mice (PVM, which replicates severe RSV disease in human infants) and following recovery, were intranasally sensitised with ovalbumin. Animals received low-level challenge with aerosolised antigen for 4 weeks to elicit changes of chronic asthma, followed by a single moderate-level challenge to induce an exacerbation of inflammation. We then assessed airway inflammation, epithelial changes characteristic of remodelling, airway hyperresponsiveness (AHR) and host immunological responses.

Results: Allergic airway inflammation, including recruitment of eosinophils, was prominent only in animals that had recovered from neonatal infection with PVM and then been sensitised and chronically challenged with antigen. Furthermore, only these mice exhibited an augmented Th2-biased immune response, including elevated serum levels of anti-ovalbumin IgE and IgG1 as well as increased relative expression of Th2-associated cytokines IL-4, IL-5 and IL-13. By comparison, development of AHR and mucous cell change were associated with recovery from PVM infection, regardless of subsequent allergen challenge. Increased expression of IL-25, which could contribute to induction of a Th2 response, was demonstrable in the lung following PVM infection. Signalling via the IL-4 receptor alpha chain was crucial to the development of allergic inflammation, mucous cell change and AHR, because all of these were absent in receptor-deficient mice. In contrast, changes of remodelling were evident in mice that received chronic allergen challenge, regardless of neonatal PVM infection, and were not dependent on signalling via the IL-4 receptor.

Conclusion: In this mouse model, interaction between early-life viral infection and allergen sensitisation/challenge is essential for development of the characteristic features of childhood asthma, including allergic inflammation and a Th2-biased immune response.

Figure 1

Experimental design. Diagrammatic representation of protocol for infection, sensitisation and inhalational challenge.

Figure 2

PVM infection. (A) PVM load assessed by PCR of lung tissue at 2-28 days post-infection. Levels of virus in samples from infected animals at days 2, 4 and 28 were at or below the limits of detection, as were levels in all samples from sham-infected control animals. (B) Mean weights of PVM-infected and naïve mice during the first 3 weeks of life. Significant differences shown as * = P < 0.05, ** = P < 0.01.

Figure 3

Histopathological changes. (A) A lymphoid aggregate adjacent to a bronchiole, in lung tissue from an animal from the virus-infected control group at 11 weeks of age. (B) An eosinophil within the tracheal epithelium (arrow) from a mouse from the virus/OVA group. (C) Reticulin-stained trachea from a naïve animal demonstrating normal thickness of subepithelial collagenous zone (black arrow) and epithelial layer (red arrow). (D) Trachea from a mouse infected with PVM at birth, then sensitised to OVA and challenged (virus/OVA group) demonstrating significant thickening of both the subepithelial collagenous zone (black arrows) and the epithelium (red arrow). (E) Left main bronchus from a naïve animal stained with PAS, showing that no mucin-producing cells are present (grade 0). (F) Left main bronchus from a mouse from the virus/OVA group, demonstrating numerous PAS-positive goblet cells in the epithelium (arrows) (grade 4). Scale bar = 150 μm in A, 25 μm in B and 50 μm in C-F.

Figure 4

Expression of Gob5. mRNA expression for Gob5 assessed by PCR of lung tissue. Significant difference compared to naïve animals shown as * = P < 0.05.

Figure 5

Development of AHR. (A) Airway responsiveness to increasing concentrations of β-methacholine, assessed by transpulmonary resistance (R_L). (B) Transpulmonary resistance assessed as area under the curve. Significant differences compared to naïve animals shown as * = P < 0.05, ** = P < 0.01, *** = P < 0.001; compared to virus-infected, OVA-challenged animals (Virus/OVA) shown as ## = P < 0.01.

Figure 6

Th2-biased immunological response. (A) Numbers of CD4⁺ T cells recovered from disaggregated lung tissue. (B) Numbers of eosinophils in tracheal epithelium. (C) Levels of OVA-specific IgE and IgG₁ in serum. (D-F) Levels of expression of mRNA for IL-4, IL-5 and IL-13 by CD4⁺ T-cells, relative to HPRT. Significant differences compared to virus-infected control animals shown as * = P < 0.05, ** = P < 0.01, *** = P < 0.001; compared to sham-infected, OVA-challenged animals (Sham/OVA) shown as # = P < 0.05, ## = P < 0.01.