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.