Effect Of RNAi-mediated GATA-3 Gene Silencing On Differentiation And Inflammation Of Th17 Cells In Acute Mouse Model Of Asthma

PrefaceBronchial asthma is one of global diseases, and there is little difference both in area and ethnicity, or in age and sex. The Etiopathogenisis of bronchial asthma is complex. Asthma is chronic airway inflammation with the characteristics of intermittent and reversible airway obstruction, bronchial hyperresponsiveness, lymphocytes, eosinophil infiltration of airway submucosal. Regarding the induction of airway inflammation, it was widely recognized that it is a result of immune response in the bronchial airway. However, the underlying mechanism is not clear and requires further investigation. Many believe that imbalance of two types of T-helper cells, Th1 and Th2, is the underlying cause for asthma. Specifically, the increased activity of Th2 cells is a key event in asthma induction. Thl and Th2 are differentiated from a common precursor cells, ThO. Transcription factor GATA-3 regulates ThO differentiation in a specific manner. Such regulation is critical in controlling Thl and Th2. IFN-γand IL-4 are typical cytokines from Thl and Th2.In recent years, the roles of Th17 cells in airway inflammation are receiving increasing attention. Th17 cells,which can produce IL-17 cytokines, are a new lineage of T-cells that are controlled by the transcription factor RORyt. IL-17 is a cytokine that discovered recently, belongs to a unique type of multi-functional cytokines, which coordinates the local inflammation of tissues by inducing the release of proinflammatory factors and mobilizing neutrophils, so it plays an important role in inflammatory diseases and autoimmune diseases, especially in allergic airway inflammation in asthma.RNA interference is a kind of highly conserved gene silencing technology. RNAi technology is the same as gene knockout and gene silencing in functions, and more convenient than Gene knockout technology. In this study, we used acute mouse model of asthma to study the function of GATA-3 and RORyt in differentiation and inflammation of Th17 cells. One goal of this study is to elucidate the effect of RORyt on differentiation and inflammation of Th17 cells which is a subset of spleen CD4+T cells from acute mouse model of asthma. Furthermore, the other goal of our study is to detect the effect of downregulating GATA-3 expression by transfecting GATA-3-siRNA on expression of RORyt, and on differentiation and inflammation of Th17 cells from acute mouse model of asthma. Through this study, we hope to open new avenues and provide further experimental evidence for therapeutic treatment of asthma. Objective:To establish and improve acute mouse model of asthma and confirm reliability of the model with modified evaluation criterion.Methods:35 SPF female BALB/c mice were randomly divided into normal control group and acute asthma group, with 20 mice in acute asthma group and 15 mice in normal control group. The acute asthma model was established by sensitization with intraperitoneal injection of OVA and aerosol challenge with repeated inhalation of OVA in the acute asthma group. The control group received PBS as the substitution of OVA. After 24 hours of the last inhalation, asthmatic symptoms were observed; The changes in airway response were determined by lung resistance(RL) stimulated by acetylcholine(Ach); The white cell count, neutrophils(%), eosinophils(%), lymphocytes(%) of bronchoalveolar lavage fluid (BALF) were measured; Lung tissue sections were stained for general pathology; The total cellular count of spleen CD4+T cells and ratio of positive Th17 cells were detected; The levels of IL-4 and IL-17 concentration of BALF and splenocyte culture supernatant were examined.Results:(1) Asthma symptoms were more severe in acute asthma group, compared with normal control group.(2) Total RL was obviously increased in acute asthma group, compared with normal control group(p<0.01).(3) Total white cell count, Neu(%), Eos(%) and Lym(%) in the BALF of acute asthma group were more than those of normal control group (respectively p<0.01).(4) There were more extensive inflammatory cells infiltration around the bronchi in acute asthma group, compared with normal control group(p<0.01).(5) The total cellular count of spleen CD4+T cells and the ratio of positive Th17 cells in acute asthma group were obviously higher than those of normal control group(respectively p<0.01).(6) In either splenocyte culture supernatant or BALF, the levels of IL-4 and IL-17 concentration were significantly elevated in acute asthma group, compared with normal control group(respectively p<0.01).Conclusions:(1) The reliability of the improved and established acute mouse model of asthma was successfully confirmed.(2) The modified evaluation criterion of acute asthmatic mouse model was successful. Objective:To investigate the change of RORyt expression in acute mouse model of asthma, and to elucidate the effect of RORyt on differentiation and inflammation of Th17 cells which is a subset of spleen CD4+T cells from acute mouse model of asthma.Methods:Spleen mononuclear cells from acute mouse model of asthma were removed red blood cells, and, followed by isolation of CD4+T cells with immuno-magnetic beads, they were cultivated in RPMI-1640 with 10% Fetal Bovine Serum, stimulated by ConA and PMA. After 24 hours, all cells were detected the expression of RORyt mRNA by real-time PCR, the level of RORyt protein by Western blotting, the ratio of positive Th17 cells by flow cytometer (FCM), and the production of IL-17 by ELISA. The correlation between RORyt expression and the ratio of positive Th17 cells, and IL-17 production were analyzed.Results:(1) The expression of RORyt mRNA and protein in spleen CD4+T cells from acute asthma group were significantly elevated, compared with normal control group(respectively p<0.01). (2) The ratio of positive Th17 cells from acute asthma group was obviously higher than that of normal control group(p<0.01).(3) The more extensive production of IL-17 in splenocyte culture supernatant was found in acute asthma group, compared with normal control group(p<0.01)(4) The RORyt mRNA expression of spleen CD4+T cells from acute asthma group was positively correlated with the ratio of positive Th17 cells and the production of IL-17(respectively r=0.854,0.872;p<0.01).(5) The RORyt protein expression of spleen CD4+T cells from acute asthma group was positively correlated with the ratio of positive Th17 cells and the production of IL-17(respectively r=0.902,0.891;p<0.01).Conclusions:(1) RORyt expression is significantly increased in spleen CD4+T cells from acute mouse model of asthma.(2) The differentiation and inflammation of Th17 cells are significantly elevated in acute mouse model of asthma.(3)RORyt expression is associated with differentiation and inflammation of Th17 cells in acute mouse model of asthma. Objective:To detect the effect of downregulating GATA-3 expression by transfecting GATA-3-siRNA on expression of RORγt, and on differentiation and inflammation of Th17 cells from acute mouse model of asthma.Methods:Spleen CD4+T cells from acute mouse model of asthma were transfected with three chemosynthesis siRNA sequences by transient electroporation. After cultivating and stimulation with cytokine, the best efficient siRNA-GATA-3 was choosed by detection GATA-3 expression. This siRNA-GATA-3 was trasnsfected into Spleen CD4+T cells from asthma group. Cells, divided into three groups:control group, untransfected asthmatic group and transfected asthmatic group, were measured the expression of RORγt and GATA-3 mRNA by real-time PCR, the level of RORγt and GATA-3 protein by Western blotting, the ratio of positive Th17 cells by FCM and the production of IL-17 by ELISA. The correlation between GATA-3 expression and RORγt expression, the ratio of positive Th17 cells, and IL-17 production were analyzed.Results:(1) siRNA-GATA-3-201 had better silencing effect than other siRNA sequences(p<0.01).(2) The expression of GATA-3 mRNA and protein in spleen CD4+T cells from transfected asthmatic group were lower than those of controls (respectively p<0.01).(3) The expression of RORyt mRNA and protein in spleen CD4+T cells from transfected asthmatic group were higher than those of controls (respectively p<0.01).(4) The ratio of positive Th17 cells from transfected asthmatic group was significantly increased, compared with controls (respectively p<0.01).(5) The IL-17 production of splenocyte culture supernatant from transfected asthmatic group was significantly elevated, compared with controls (respectively p<0.01).(6) The GATA-3 mRNA expression on spleen CD4+T cells from transfected asthmatic group was negatively correlated with RORyt mRNA expression, the ratio of positive Th17 cells and the production of IL-17 (respectively r=-0.622,-0.692,-0.877;p<0.01).(7) The GATA-3 protein expression on spleen CD4+T cells from transfected asthmatic group was negatively correlated with RORyt protein expression, the ratio of positive Th17 cells and the production of IL-17 (respectively r=-0.894,-0.728,-0.886; p<0.01).Conclusions: (1) Application of RNA interference technique in asthmatic mouse model may implement target gene silencing.(2) RNAi-mediated GATA-3 gene silencing could increase RORyt expression in spleen CD4+T cells from acute mouse model of asthma.(3) RNAi-mediated GATA-3 gene silencing could promote the differentiation and inflammation of Th17 cells in acute mouse model of asthma.(4) RNAi-mediated GATA-3 gene silencing could increase RORyt expression, which could promote the differentiation and inflammation of Th17 cells in spleen CD4+T cells from acute mouse model of asthma.