Mice
IL-25-deficient mice(Il25-/-)were obtained from Tsinghua university, which have been described in the previous studies18, 19. The genotyping primers were as follows: forward, 5′-CTGCTCCAGTCAGCCTCTCT-3′; reverse1, 5′AGCAGCTGGGCAAGTGAC-3′; reverse2,5′ AGGTGGAGAAAGTGCCTGT-3′. All mice were bred and maintained under specific pathogen-free conditions in Shanghai Biomodel Organism Science and Technology Development Co., Ltd. Sex-matched littermate WT and Il25-/- mice 6 to 8 weeks of age were mainly used for experiments.
The HDM-induced mouse asthma model was established as described previously20. Briefly, mice were sensitized intranasally with 100 μg house dust mite extracts (HDM) (Greer Labs, low endotoxin) in 40 μL PBS on day 0 and challenged intranasally with 10 μg HDM on days 7 to 11, harvested on day 14. In some experiments for investigating the role of eosinophils in the sensitization period, mice were sensitized with 100 μg HDM before the first challenge and lungs were harvested for flow cytometry analysis at indicated times. To study the effect of IL-25, mice were treated intranasally with 1μg IL-25(R&D) in 40 μL PBS and lungs were digested in HBSS media containing type 1A collagenase (Sigma) to obtain the single cell suspension for flow cytometry analysis 24 hours later. Histopathologic analysis was done to identify asthma allergic inflammation in mouse models. BALF was centrifuged and supernatants were frozen at -80℃ for subsequent cytokine analysis. Cytokines IL-4, IL-5, IFN-γ and IL-13 levels in BALF and serum IgE levels were measured by ELISA according to the manufacturer’s instructions (BioLegend). All animal studies were approved by the Ruijin Hospital Animal Ethics Committee.