Experimental protocol for development of adjuvant-free murine chronic model of allergic asthma

Abstract

Background: Mouse models of allergic asthma play a crucial role in exploring of asthma pathogenesis and testing of novel anti-inflammatory drugs. Widely used acute asthma models usually developed with adjuvant (aluminum hydroxide (alum)) do not reproduce one of the main asthma feature - airway remodeling while chronic asthma model mimic the pathophysiology of human disease. Moreover, the use of alum causes distress in experimental animals and impedes the test of adjuvant-containing drugs. In this study, we aimed to develop a chronic adjuvant-free asthma model with pronounced asthmatic phenotype.

Methods: Female BALB/c mice were divided into 3 groups. The first group was sensitized with intraperitoneal injections of ovalbumin (OVA) emulsified in aluminum hydroxide on days 0, 14, 28 followed by two stages of intranasally challenge with OVA on days 41-43 and 62-64. The second group was subcutaneously sensitized with the same dose of OVA without adjuvant and challenged on the same days. The third group (negative control) included mice which did not received any kind of treatment (i.e. sensitization and challenge). Serum levels of OVA-specific IgE, IgG2a and IgG1 antibodies were detected by ELISA. Airway hyper-responsiveness was measured by non-invasive plethysmography on days 44 and 65. Bronchoalveolar lavage fluids (BALF) sampled in all groups on days 45 and 66 were analyzed by light microscopy. The left lung was removed for histological analysis. The IL-4 and IFNγ mRNA expression in BALF cells was evaluated by RT-PCR.

Results: The OVA-specific IgE antibody response was two-fold increased in mice from adjuvant-free group compared to the adjuvant group that reflects reorientation of immune response towards Th2 phenotype. At the same time, the level of OVA-specific IgG1 and IgG2a antibodies was increased in the adjuvant group. Airway hyperresponsiveness to methacholine in mice of both experimental groups was two-fold higher than in control. Analysis of cell composition in BAL has shown a significant increase in eosinophil count in both experimental groups that indicate the development of allergic inflammation. Lung histology revealed airway remodeling in both experimental groups including goblet cell hyperplasia/metaplasia, thickening of airway walls, collagen deposition in the wall of distal airways. Additionally, the tendency to develop hypertrophy of bronchial smooth muscle layer was observed. Study of gene expression in BAL cells revealed the increase of IL-4 level in both adjuvant and adjuvant-free groups while IFNγ expression in both experimental groups was similar to control group.

Conclusion: We have developed a chronic adjuvant-free mouse asthma model which possesses all necessary features of the disease including airway remodeling and is more suitable for pre-clinical evaluation of novel therapeutic approaches including adjuvant-containing drugs.

Keywords: Adjuvant; Chronic allergic asthma; Cytokines; Inflammation; Mouse model.