Peroxisome Proliferator-Activated Receptor γ(PPARγ) Agonist Down Regulates IL-17 Expression From CD4~+T-lymphocytes In Acute Asthma

Peroxisome Proliferator-Activated Receptorγ(PPARγ) agonist down regulates IL-17 expression from CD4+ T-lymphocytes in acute asthmaBackground:Asthma is one of the most common inflammatory diseases and its incidence is dramatically increasing from the last twenty years.Eosinophils,Macrophages,T-lymphocytes, mast cells and epithelial cells are involved in the process of inflammation. Recently it has been suggested that Th 17 and its cytokines(IL-17)are involved in the pathogenesis of asthma in mouse model.PPARγhas been shown to play an important role in the control of inflammatory responses of airways in acute asthma.Objectives:1)To confirm the Ovalbumin sensitized and airway challenged acute asthma model.2) To evaluate the down regulating effect of PPARγagonist on IL 1.7 expression isolated from CD4+ T-lymphocytes from the spleen of acute asthmatic mice.Methods:Mice were divided into control (normal)group and acute asthmatic group. We used mouse model of acute asthma induced by sensitization and airway challenge with ovalbumin and only by PBS in control (normal)group. The acute asthma model was confirmed by evaluating the behavioral changes and asthmatic symptoms occurred in mice and through the pulmonary function test with methacholine provocation, level of inflammatory cells and IL-17 in BALF and lung tissue histology. On day 21st,all the mice were sacrificed and spleen was taken out.CD4+ T-lymphocytes from the spleen of control and asthmatic groups were isolated,purified by immuno-magnetic beads method.The CD4+ T-lymphocytes viability was assessed by trypan blue staining and the level of Th-17 cells in CD4+ T-lymphocytes assessed by flow cytometry. The level of IL-17 in CD4+ T-lymphocytes culture supernatant was measured by ELISA. The correlation between BALF IL-17 concentration and BALF neutrophil(%)was assessed. The isolated CD4+ T-lymphocyte were divided into Nontrol group (Con),Asthmatic control group (Acon),Asthmatic rosiglitazone group (Aros)and Asthmatic Dexamethasone group (Adex).After the addition of rosiglitazone and dexamethasone, and then these cells were cultured for 24 hours,IL-17 protein and IL-17 mRNA of CD4+ T-lymphocytes from all the groups were detected by Western Blot(WB)and real-time Polymerase Chain Reaction(rt-PCR)respectively.Results:I Confirmation of acute asthma model1 Changes in mice after sensitization and nebulization with OVAOVA sensitized group mice showed excitation, ear grasping, sneezing at the beginning and drowsiness, lack of activities and reduced eating habit later after subsequent airway challenge with OVA. Control (Normal)group mice sensitized and nebulized only with PBS showed normal sensitivity, activity, behaviour and eating habit.2 BALF examinationTotal cellular counts,neutrophils,eosinophils,macrophages and lymphocytes level increased in acute asthmatic group as compared to normal group, all P<0.01 respectively.BALF IL-17 level increased in acute asthmatic group as compared to normal group, p<0.01.3 Pulmonary Function Test (PFT) interpretationWith the subsequent increase in methacholine doses, the progressive increase in lung and airway resistance recorded in acute asthmatic group as compared to normal group, all p <0.01 respectively.4 Lung histology Acute asthmatic group showed significant increase in inflammatory cells like eosinophils,neutrophils macrophages as compared to normal group, p<0.01.ⅡThe level of CD4+ T-lymphocytes in normal and asthmatic group After 24 hours of culture, the acute asthmatic group showed higher concentration of CD4+ T-lymphocytes than the normal group,(4.164±0.260 X 1010/L) vs(1.138±0.130 X 10/L,p <0.01.III Level of Th-17 cells in CD4+ T-lymphocytes in normal and asthmatic group after 24 hours of culture The acute asthmatic group showed higher level of Th 17 cells than the normal group, p<0.01.IV Level of IL-17 in CD4+ T lymphocytes culture supernatant after 24 hours CD4+ T-lymphocytes derived from the acute asthmatic mice showed obvious increase in IL-17 concentration than the normal group, p<0.01.V Impact of Rosiglitazone and Dexamethasone in IL-17 protein and mRNA expression on cultured CD4+T-lymphocytes1)Expression of IL-17 protein from CD4+ T-lymphocytes in all groups detected by Western BlotAsthmatic group(Acon)showed increased level of IL-17 protein expression than that of other groups, p<0.01 respectively. The Aros and Adex group showed decreased IL-17 protein expression from CD4+ T-lymphocytes than the Acon group, all p<0.01.But the Aros and Adex group showed no difference in IL-17 mRNA expression from CD4+ T-lymphocytes,p>0.05. 2)Expression of IL-17 mRNA from CD4+ T-lymphocytes in all groups detected by real-time PCR Asthmatic group(Acon)showed increased level of IL-17 mRNA expression than that of other groups,p<0.01 respectively. The Aros and Adex group showed decreased IL-17 mRNA expression from CD4+ T-lymphocytes than the Acon group, all p<0.01.But the Aros and Adex group showed no any differences in IL-17 mRNA expression from CD4+ T-lymphocytes, p>0.05.Conclusion:1.Acute asthma model was successfully established.2.IL-17 was involved in neutrophilic airway inflammation in acute asthma.3.PPAR y agonist down regulates the IL-17 expression from CD4+ T-lymphocytes in acute asthma.4. Dexamethasone also found to down regulate the IL-17 expression from CD4'T-lymphocytes in acute asthma.