T-bet Affects The Proliferation And Inflammation Of Th17 Cells Through IL-23 Recptor In Acute Aathma

Part I Establishment of Acute Asthmatic Mouse Model and Confirmation of its ReliabilityObjective:To confirm the reliability of established acute asthmatic mouse model with modified evaluation criterion.Methods:35 SPF female BALB/c mice were randomly divided into control group and asthmatic group. The asthmatic model was reproduced by sensitization with intraperitoneal injection of OVA, followed by repeated inhalation of OVA. The control group received only PBS in the same manner. After 24 hours of the last inhalation, asthmatic symptoms were observed. The changes in airway responseveness were determined by lung resistance(RL) stimulated by acetylcholine(Ach); the white cell count, neutrophils(%), eosinophils(%), lymphocytes(%) were measured from bronchoalveolar lavage fluid (BALF); Lung tissue sections were stained for general pathology. Spleenic mononuclear cells from acute asthmatic mouse model were removed red blood cells, followed by being isolated CD4+T cells using immuno-magnetic beads. Spleenic CD4+T cells were cultivated in RPMI-1640 with 10% Fetal Bovine Serum, stimulated by ConA and PMA. After 24 hours, the total spleenic CD4+T cells and ratio of positive Thl7 cells were detected; the levels of IL-4 and IL-17 concentration obtained from BALF and spleenic CD4+T culture supernatant were examined.Results:(1) Asthmatic symptoms were more severe in asthmatic group as compared with controls.(2) Total Lung Resistance was obviously increased in asthmatic group as compared with controls (p<0.01).(3) More extensive inflammatory cells infiltrated around the bronchi and mucus deposition in airway lumen were found in asthmatic group as compared with controls (p<0.01).(4) White cell count, Neu(%), Eos (%) and Lym(%) in the BALF of asthmatic group were obviously increased in sthmatic group as compared with controls (respectively p<0.01).(5) Spleenic CD4+T cell count and the ratio of positive Th17 cells were obviously increased in asthmatic group as compared with controls (p<0.01).(6) IL-4 and IL-17 concentration were significantly elevated in BALF and spleenic CD4+T culture supernatant from asthmmtic group as compared with controls (respectively p<0.01).(7) IL-17 concentration in BALF form asthmatic group positively correlated with Neu(%) (r=0.903,P<0.01).Conclusions:Acute asthmatic mouse model reproduces successfully according to modified evaluation criteria. PartⅡEffect of IL-23 Receptor on Proliferation and Inflammation of Th17 Cells from Acute Asthmatic Mouse ModelObjective:To detect the effect of interleukin-23 receptor(IL-23 Receptor) on proliferation and inflammation of Th17 cells, a subset of spleenic CD4+T cells from acute asthma mouse model.Methods:Spleenic CD4+T cells isolated using immuno-magnetic beads and cultured in RPMI-1640 with 10% Fetal Bovine Serum, stimulated by ConA and PMA. After 24 hours, the expression of IL-23 receptor mRNA and IL-23 receptor protein were detected by real-time PCR and Western blotting respectively; the ratio of positive Th17 cells was detected by flow cytometer (FCM); IL-17 concentration was detected by ELISA. Finally, the correlation between IL-23 receptor expression with the ratio of positive Th17 cells and IL-17 concentration were analyzed.Results:(1) The expression of IL-23 receptor mRNA in spleenic CD4+T cells from asthmatic group was significantly elevated as compared with controls (p<0.01).(2) The expression of IL-23 receptor protein in spleenic CD4+T cells from asthmatic group was significantly elevated as compared with controls (p<0.01).(3) The ratio of positive Thl7 cells in asthmagc group was obviously higher than that of controls (p<0.01).(4) The more extensive IL-17 concentration was found in asthmtic group as compared with controls (p<0.01)(5) The IL-23 receptor mRNA expression in asthmatic group positively correlated with the ratio of positive Th17 cells(r=0.789, P<0.05) and the IL-17 concentration (r=0.788, P<0.05)。(6) The IL-23 receptor protein expression in asthmatic group positively correlated with the ratio of positive Th17 cells (r=0.828, p<0.01) and the IL-17 concentration (r=0.802, P<0.05)。Conclusions:IL-23 receptor expression is significantly increased in spleenic CD4+T cells from acute asthmatic mouse model and associates with proliferation and inflammation of Th17 cells.PartⅢDown-regulating of T-bet Affects the Promotion the Poliferation and Inflammation of Th17 Cells througth IL-23 Receptor in acute asthmaObjective:To investigate the effect of down-regulating T-bet on proliferation and inflammation of Th17 cells from acute asthmatic mouse model.Methods:Spleenic CD4+T cells from acute asthmatic mouse model were transient transfected with three chemosynthesis siRNA sequences by Lipofectamine2000. After culture and stimulation with cytokine(ConA and PMA), the best efficient siRNA-T-bet choosn by detection of T-bet expression, was trasnsfected into spleenic CD4+T cells from asthmatic group. Cells divided into three groups:control group, untransfected asthmatic group and transfected asthmatic group. The expression of IL-23 receptor and T-bet mRNA were analyzed by real-time PCR. The expression of IL-23 receptor and T-bet protein were analyzed by Western blotting. The ratio of positive Th17 cells was measured by FCM. The IL-17 concentration was detected by ELISA. Finally, the correlation between T-bet expression with IL-23 receptor expression, the ratio of positive Th17 cells and IL-17 concentration were analyzed.Results:(1) siRNA-T-bet-429 had better silencing effect than other siRNA sequences (p<0.01). (2) The expression of T-bet mRNA in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of T-bet mRNA in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(3) The expression of T-bet protein in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of T-bet protein in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(4) The expression of IL-23 receptor mRNA in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of IL-23 receptor mRNA in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(5) The expression of IL-23 receptor protein in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of IL-23 receptor protein in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(6) The ratio of positive Th17 cells in transfected asthmatic and transfected siRNA-T-bet-429 group significantly elevated as compared with controls (p<0.01). The ratio of positive Th17 cells in transfected siRNA-T-bet-429 group significantly elevated as compared with transfected asthmatic group (p<0.01).(7) The IL-17 concentration of spleenic CD4+T culture supernatant in transfected asthmatic and transfected siRNA-T-bet-429 group significantly elevated as compared with controls (p<0.01). The IL-17 concentration of spleenic CD4+T culture supernatant in transfected siRNA-T-bet-429 group significantly elevated as compared with transfected asthmatic group (p<0.01).(8) The T-bet mRNA expression in transfected siRNA-T-bet-429 group negatively correlated with IL-23 receptor mRNA expression (r=-0.786,p<0.05), and the ratio of positive Thl7 cells (r=-0.882, P