Abstract: Objective To explore the specific immunotherapeutic effect of Der p1 T cell epitope fusion protein on asthma in a mouse model. Methods Total 30 female BALB/c mice were randomly divided into three groups: control group, asthma group and specific immunotherapy group(SIT) treated with Der p1 T epitope fusion protein. The mice in asthma and SIT groups were sensitized with intraperitoneal injection of 200 μl(10 μg) crude extracts of house dust mite on day 0, 7 and 14, while the mice in control group were given with intraperitoneal injection of the same volume of PBS solution. Then the mice in asthma and SIT groups were challenged by spray inhalation of house dust mite extracts (0.5 μg/ml) for 30 minutes every day started from 21st day. The asthmatic symptoms of the challenged mice were observed and recorded for 7 consecutive days. Control group was given with the same volume of PBS. The mice in SIT group were treated with intraperitoneal injection of 20 μg Der p1 T cell epitope fusion protein in total volume of 200 μl 30 min before each challenge for 7 consecutive days. Mice in control group and asthma group were injected intraperitoneally with the same volume of PBS. The blood was collected from each mouse and sera were collected 24 hours after the last challenge, mice were euthanized and bronchoalveolar lavage fluid was collected by trachael intubation. ELISA kit was used to detect the serum levels of antigen-specific IgE and IgG2a and cytokines, including IL-10, IL-4, IFN-γ and IL-17A in the bronchoalveolar lavage fluid. Flow cytometry analysis was used to determine the change of Th1/Th2 and Th17/Treg cells. Lung tissues were taken for histological observation of the pathological changes. Results After being sensitized and challenged, the mice in the control group only had a short period of mild restlessness. The mice in asthma group showed obvious symptoms of restlessness, wheezing and out of breath. After immunotherapy, the mice in the SIT group had reduced asthmatic symptoms, significant improvement than mice in asthma group without treatment. The results of ELISA showed that the amount of specific IgE antibody in asthma group was (31.49 ± 4.32) IU/ml, which was significantly higher than that in control group [(8.53 ± 1.92) IU/ml] (P < 0.01) and in SIT group [(16.68 ± 2.45) IU/ml] (P < 0.01). The amount of antibody IgG2a in asthma mice was (19.56 ± 3.89) μg/ml, which was lower than that in control group [(42.43 ± 2.07) μg/ml] (P < 0.01) and in SIT group [(36.96 ± 5.04) μg/ml] (P < 0.01). The levels of IFN-γ and IL-10 in asthma group were (134.23 ± 22.49) pg/ml, (22.43 ± 8.27) pg/ml, respectively, which are significantly lower than those in control group [(212.36 ± 33.21) and (72.84 ± 21.42) pg/ml, respectively] (P < 0.05, 0.01). However, after being treated with SIT, the levels of IFN-γ and IL-10 had significantly recovered compared to the asthma group without SIT treatment, as (183.76 ± 24.66) pg/ml, (61.05 ± 7.97) pg/ml, respectively (P < 0.05, 0.01). The levels of IL-4 and IL-17A in asthma group were (165.45 ± 34.59) pg/ml, (464.21 ± 41.36) pg/ml, respectively, which are significantly higher than those in control group [(21.21 ± 5.26) and (115.74 ± 30.82) pg/ml, respectively] (P < 0.05, 0.01). Treatment with SIT reduced IL-4 and IL-17A levels to (64.15 ± 17.33) pg/ml, (271.61 ± 27.07) pg/ml, respectively, with significant difference compared to that in asthmat group without treatment (P < 0.05, 0.01). Consistently, the results of flow cytometry showed that the percentages of Th1 and Treg cells in CD4⁺ T lymphocytes of spleen in asthma group were 2.8% and 4.9% respectively, which were significantly lower than those in control group (3.7% and 10.3%) (P < 0.05, 0.01). The treatment with SIT significantly stimulated Th1 and Treg cells to 3.1% and 8.8%, respectively, with statistical significance compared to asthma group without treatment (P < 0.05, 0.01). The percentages of Th2 and Th17 cells in CD4⁺ T lymphocytes of spleen in asthma group were 2.8% and 2.2% respectively, which was significantly higher than those in control group (1.0% and 0.3%) (P < 0.01). The treatment of SIT reduced Th2 cells and Th17 cells to 1.7% and 0.6% respectively, with significant difference compared to asthma group without treatment (P < 0.01). Pathological results showed that there was significant inflammatory cell infiltration around the bronchioles, broken muscle fiber and the collapsed epithelial cells were observed in asthma group. These pathological changes were much improved in mice with SIT treatment with reduced bronchus wall thickness and inflammatory cells infiltration. Conclusion Der p1 T epitope fusion protein has significant immunotherapeutic effect on asthma in a mouse model.

Key words: Dermatophagoides pteronyssinus, Der p1 T cell epitome fusion protein, Specific immunotherapy, Asthma