Asthma is a chronic inflammatory disorder of the airway characterized by variable airflow obstruction, airway hyperresponsiveness (AHR) and airway inflammation. The main clinical symptoms of asthma are shortness of breath, wheezing, coughing, and increased mucus secretions upon exposure to allergens. The pathogenesis of asthma is caused by complex interactions between genetic, epigenetic, and environmental factors. Different asthma clinical endotypes with different pathological changes are mediated by several types of airway cells and cells involved in the immune responses, including airway epithelial cells, eosinophils, T cell subsets, and so on. In particular, Th2 cells have been thought to predominate in high eosinophilic asthma, with raised levels of IL-4, IL-5, and IL-13.
The classical murine model of asthma is ovalbumin-induced airway inflammation model. Mice are sensitized by multiple intraperitoneal injections of ovalbumin and then challenged by inhalation of aerosolized ovalbumin. IgE and eosinophils level are found higher in mice in this model and histological staining shows increased airway mucus and inflammatory leukocyte infiltration. Biocytogen provides a robust and validated ovalbumin-induced murine model of asthma. As a key differentiating factor, Biocytogen also generated and validated this model in B-hIL4/IL4RA mice where human IL-4 and IL-4 receptor (IL-4RA) replaces their mouse counterpart via genomic knock-in. B-hIL4/IL4RA mice thus allow direct testing of asthma therapeutics targeting human IL-4 pathway in mice.
Generation of B-IL4/IL4R mice and its phenotypic analysis
Asthma model generation and validation
Acute murine asthma model scheme
B-hIL4/hIL4RA mice between 5 and 8 weeks of age were acclimatized for at least 1 week before initiation of sensitization and challenge with ovalbumin (OVA) in the creation of the acute murine model of asthma.
Bronchoalveolar lavage fluid (BALF) eosinophilia in OVA-induced mouse asthma model in B-hIL4/IL4RA mice
Upon ovalbumin sensitization and challenge, B-hIL4/hIL4RA mice showed a considerable increase of infiltration of leukocytes including eosinophils and neutrophils. This represented a clear allergic phenotype at day 26 in BALF when compared with mice not sensitized or challenged.
Figure 1. BALF cell count
Mice (n=5/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, BALF cells were collected and eosnophils were counted with flow cytometry.
BALF eosinophilia in mouse asthma model
Ovalbumin sensitization and challenge induced a clear allergic phenotype at day 26 characterized by considerable increase of eosinophilia in BALF when compared with mice that were not sensitized or challenged.
IgE production in mouse asthma model
Level of OVA-specific IgE antibody was approximately five-fold higher in OVA-challenged B-hIL4/IL4RA mice compared with sham-challenged B-hIL4/IL4RA mice.
H&E stain in mouse asthma model
Histology of lung of B-hIL4/hIL4RA mice sensitized and challenged with OVA or PBS. Examination of H&E stained lung tissue of B-hIL4/hIL4RA mice revealed patchy peribronchial inflammatory infiltrates, composed primarily of eosinophils and lymphocytes. In contrast, sham-challenged mice exposed to PBS showed normal lung histology.
Examples for Asthma drug efficacy evaluation using B-hIL4/hIL4RA mice model
Anti-human IL-4 (dupilumab) Efficacy Evaluation in B-hIL4/hIL4Ra mice
Group Alum/ovalbumin sensitization Challenge Drug Treatment G1 PBS PBS Blank Control G2 OVA OVA Blank Control G3* OVA OVA Dupilumab G4 OVA OVA Dupilumab
All drugs were administered i.p.
The experimental design for OVA challenge asthma model in B-hIL4/hIL4RA mice and treatment with or without dupilumab.G1: Mice were challenged with PBS and treated without any drug. G2: Mice were challenged with OVA and treated without any drug. G3: Mice were challenged with OVA and treated with dupilumab before challenged as a prophylactic treatment. G4: Mice were challenged with OVA and treated with dupilumab after challenged as a therapeutic treatment.
Dupilumab abolishes inflammatory cell infiltrates in BALF in acute OVA-induced asthma model of B-hIL4/hIL4RA mice
BALF immune cells were isolated from homozygous B-hIL4/hIL4RA mice (n=7). The proportions of eosinophils subpopulations were determined by flow cytometry in B-hIL4/IL4RA mice treated with or without dupilumab. Prophylactic treatment before OVA challenge (G3) with dupilumab almost completely abolished the infiltration of eosinophils as opposed to untreated group (G2). Therapeutic treatment after OVA challenge (G4) with dupilumab could also obviously reduce the infiltration of eosinophils.
Flow cytometry analysis shows near elimination of inflammatory cells in BALF by dupilumab treatment
BALF immune cells were isolated from homozygous B-hIL4/hIL4RA mice (n=7). The number of CD45+ cells and eosinophils were determined by flow cytometry in B-hIL4/IL4RA mice treated with or without dupilumab. Prophylactic treatment before OVA challenge (G3) with dupilumab almost completely abolished the infiltration of eosinophils as opposed to untreated group (G2). Therapeutic treatment after OVA challenge (G4) with dupilumab could also obviously reduce the infiltration of eosinophils.
Dupilumab eliminates IgE production against OVA in mouse asthma model
Serum were collected at the study endpoint. OVA-specific IgE antibody levels were analyzed. The results showed that IgE levels were dramatically decreased in the dupilumab treatment group compared with the untreated group.
H&E staining indicates reduced inflammation in dupilumab-treated B-hIL4/IL4RA mice
Airways from B-hIL4/hIL4Ra mice exposed to PBS aerosols (G1)  did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammation in B-hIL4/hIL4RA mice, as well as an increase in the level of mucus secretion (G2). A reduction in inflammatory infiltrates and mucus secretion was observed in mice treated with dupilumab (G3, G4).