mRNA expression analysis
Strain specific analysis of IL4 and IL4RA gene expression in WT and B-hIL4/hIL4RA mice by RT-PCR. Mouse Il4 and Il4ra mRNA was detectable in splenocytes of wild-type (+/+) but not in homozygous B-hIL4/hIL4R mice (H/H) mice. Human IL4 and IL4RA mRNA was detectable only in homozygous B-hIL4/hIL4R mice but not in wild-type mice.
Strain specific IL4 expression analysis in homozygous B-hIL4/hIL4RA mice by ELISA.
Serum were collected from WT and homozygous B-hIL4/hIL4RA (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by ELISA with species-specific IL4 ELISA kit. Mouse Il4 was detectable in WT mice. Human IL4 was exclusively detectable in homozygous B-hIL4/hIL4RA mice but not WT mice.
Analysis of spleen leukocyte subpopulations by FACS
Analysis of subpopulation of T cells in lymph node by FACS
(A) Splenic B cells from C57BL/6 and B-hIL4/hIL4RA mice were cultured with LPS alone or together with 50ng/mL mIL-4/hIL-4. Culture supplements were harvested on day 6 for quantification of IgE by ELISA.(B) Splenic B cells from B-hIL4/hIL4RA mice were incubated with increasing doses of dupilumab (in house) for half of an hour before adding LPS and hIL-4 (50 ng/mL). Culture supernatant was harvested on day 6 for quantification of IgE by ELISA. The result show that B cells from B-hIL4/hIL4RA mice responded well to LPS and IL-4 with IgE production, as similar to C57BL/6 mice. Dupilumab (in house) could effectively block the expression of IgE.
IL-4 induced IgE secretion in B-hIL4/hIL4RA mice
Analysis of activity of B cells in spleen by FACS
Splenocytes were isolated from B-hIL4/hIL4RA mice and incubated with varying concentrations of IgG4 or dupilumab for half an hour. Adding human IL4 (50 pM) for 72 hours and analyzing the activity of B cells. The dupilumab group showed an effect to block B cell activation in B-hIL4/IL4RA mice and showed a dose-dependent effect; the control hIgG4 did not show an effect on block B cell activation.
The number of BALF immune cells in mouse asthma model
Analysis of immune cells in BALF by FACS. BALF immune cells were isolated from B-hIL4/hIL4RA mice (n=4 or n=5). The number and proportion of eosinophils were analyzed by flow cytometry under the treatment of PBS/dupilumab (in house). After treatment of dupilumab (in house), the number of CD45+ cells and eosinophils were much lower than the positive control in homozygous B-hIL4/hIL4RA mice.
OVA specific and total IgE production in serum and BALF of mouse asthma model
H&E staining in asthma-like model in B-hIL4/hIL4RA mice
H&E staining of asthma-like model in B-hIL4/hIL4RA mice. Lung tissues were collected at the study endpoint. H&E staining results showed that the lung tissues from B-hIL4/hIL4RA mice exposed to PBS aerosols did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammation in B-hIL4/hIL4RA mice. A significant reduction in eosinophils infiltration was observed in mice treated with dupilumab (in house).
Asthma model introduction
Establishment of asthma mouse model- HDM-induced acute asthma model
Modeling reagent：50 μg HDM in 50 μl PBS (intranasally, i.n.)
Modeling paradigm：
Efficacy assessment of anti-human IL4RA antibody in mouse asthma model induced with HDM
Detection of IgE levels in serum of asthmatic mouse model. Serum was taken at the end of the experiment and total IgE levels were measured using ELISA in serum. The results showed that the levels of total IgE in G2 model group were significantly increased compared with G1 control group, suggesting successful modeling. Total IgE levels were significantly lower after administration of dupilumab (in house) drug compared with the G2 modeling group.
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