Regulatory effect of transcription factors RORγt on the Th17/Treg balances in pregnant asthma mice

Objective 　To observe the expression changes in Th17-related cytokines and retinoid acid receptor-related orphan receptor gamma t (RORγt) in mouse model of asthma during pregnancy, and to explore the effects of dexamethasone (DXM) on Th17 cell secretory function and airway inflammation in mice. Methods 　Thirty female BALB/c mice were divided into three groups at random (n=10 per group): healthy pregnancy (HP) group, asthma in pregnancy (AP) group and dexamethasone (DXM) treated group (DXM group). The mice were treated as follows: those in AP and DXM groups were sensitized with an intraperitoneal injection of chicken ovalbumin-aluminum hydroxide [OVA/A1(OH)3] suspension on days 0, 7 and 14. The HP mice were sensitized with normal saline (NS) following the same protocol. All the mice were then bred with male BALB/c male mice. Pregnant mice in AP and DXM groups were exposed to 40-minute of 50g/L OVA aerosol for seven consecutive days starting from day 21. Dexamethasone in a dose of 1mg/kg was injected intraperitoneally in DXM mice 30 minutes before inhaling 50g/L OVA aerosol, while the HP group received NS only. The airway responsiveness to corresponding concentrations of inhaled acetylcholine was recorded 24h after the final OVA challenge. The index of airflow obstruction was expressed as enhanced pause (Penh) and its mean value after each nebulization. The airway tone is further expressed as the Penh(%) after being normalized by the baseline value. Total and differential cell counts in bronchoalveolar lavage fluid (BALF) were carried out in each sample. The histological changes in the lungs were assessed using light microscopy. The propotions of Th17 and Treg cell in mice peripheral blood cells were calculated and the Th17/Treg ratio was determined by flow cytometry. IL-17, IL-23, IFN-γand RORγt levels in the serum of mice were quantified by using ELISA technique. RT-PCR and Western blotting were performed to determine the levels of IL-17, RORγt, IL -23 and IFN-γin serum, and RORγt protein in lung tissues respectively. Results 　After being sensitized by OVA, the increased total and differential cell counts (Eos, Lym and Neu) in BALF, the increased airway hyper-responsiveness and the histological changes of lung tissues were in line with the features of asthma. In AP group, Th17 cells in peripheral blood significantly increased, while the Treg T cells significantly decreased (P < 0.05). The increased Th17/Treg ratio suggested that there was an imbalance of Th17/Treg ratio in this group. The change trend of cells in DXM group was the same as that in AP group (P < 0.01), while the Th17/Treg ratio in DXM group was higher than that in HP group, but lower than that in AP group (P < 0.05). The protein and mRNA expressions of IL-17, IL-23 and RORγt in peripheral blood and lung tissues were higher, and IFN-γlevel was lower in AP group than in HP and DXM group (P < 0.05). RORγt protein expression in the lung tissues was significantly higher in AP group (61.27%±3.11%) than in DXM (37.51%±1.93%) and HP (22.39%±0.86%) groups (P < 0.01), and a statistically significant difference was found between the latter two groups (P < 0.01). Correlation analysis showed that the expression of RORγt protein was positively correlated with the absolute numbers of eosinophils, lymphocytes and neutrophils (r=0.879, 0.759, 0.802, P＜0.01), and also positively correlated with IL -17 content in peripheral blood and mRNA expression of IL -17 (r=0.696, 0.863, P＜0.01) in lung tissues of AP group. Conclusions 　Up-regulation of Th17 cell secretory function and RORγt expression may be involved in the development and progression of asthma during pregnancy. DXM intervention can lead to improvement of asthma symptoms and inhibition of RORγt production, thus blocking the differentiation and proliferation of Th17 cells.