| BackgroundAsthma is a very common disease in the world.It is now estimated that as many as 300 million people suffer from asthma and asthma attacks have increased by 1% per year.The burden of this disease to governments,health care systems,families,and patients is increasing worldwide,so there is a far-reaching society significance in the asthma research.Asthna is a chronic inflammatory disease of the airways in which many inflammatory cells play a role,in particular,mast cells,eosinophils,T lymphocytes.The recruitment of inflammatory cells and the release of cytokines and mediators of inflammation are the major reason which cause and keep the airway inflamemation of asthma,and futher cause the tissue damage and airway disfunction.Among these inflammatory cells,T lymphocytes play a pivotal role in the pathogenesis of asthma.According to the function in immune response,we divided T lymphocytes into three subsets,help T cell(Th),cytotoxic T cell(Tc) and regulatory T cell(Treg).Th ceils divided into Th1 cell and Th2 cell.Long time ago,asthma has believed as a chronic inflammatory disease of the airways orchestrated by activated Th2 cells.Th1 has believed as a protected factor of asthma.The immunotherapy aim to allergy could change the Th2 cell phenotype into Thl cell phenotype,and the clinical symptoms has improved.The individual which polarize to Th1 had a few possibility to suffer from asthma.So the theory of Th1/Th2 disequilibrium has got common received in asthma.In recent years,the concept of a disturbed Th1/Th2 balance has failed to adequately explain many(pre)clinical observations.Some research found Th1 cells could aggratate allergy and asthma.Cytokine of Th1 cell INF-γis related with airway responsiveness and eosinophils.Treg cells not only repress the Th1 cells,but also repress the Th2 cells.So Treg cells can explain the pathogenesis better than the Th1/Th2,and become a focus in asthma reseach.Th17 cells have recently emerged as a new independent T cell subset that different from Th1,Th2 cells.There has a complicated relationship between Th17 and Treg cells.Th17 cells related with asthma.Since the disequilibrium of Th1/Th2 is an important immunologic abnormality in asthma,and also an important link in astham pathogenesis,so is there also exist a disequilibrium of Treg/Th17? This issue need us to have a further reseach.ObjectiveTo identify the unbalanced T cell-specific subpopulation Treg and Th17 in immune dysregulation of asthma,and the change of the gene and protein expression ratio of T cell-specific inflammation transcription factors Foxp3,RORγt in lung tissues,and to investigate the association between the unbalanced Treg/Th17 and the airway inflammation in asthmatic mice.Methods1.To establish and assess a modified mouse asthma model.Thirty female BALB/c mice were randomly divided into a control group,an asthmatic group and a dexamethasone group.We immunized mice with OVA/alum (containing 20μg of OVA bound to 20mg of aluminum hydroxide),made up in 0.2ml sterile saline,injected intraperitoneally and subcutaneously on days 0,7 and 14. On days 21-27,mice were exposed to aerosolized OVA(5%) for 45 min.The control group were received an intraperitoneal and subcutaneous injection of 0.2 ml saline and inhaled saline.Airway responsiveness was measured by non-invasive whole-body plethysmography(WBP) method and the change of airway histology was observed by hematoxylin/eosin(HE) stain.Concentrations of IL-5 and IFN-γin bronchoalveolar lavage fluid(BALF) were measured by enzyme-linked immunosorbent assay(ELISA).2.To identify the unbalanced T cell-specific subpopulation Treg and Th17 in immune dysregulation of asthma.Concentrations of IL-10 and IL-17 in serum and in BALF were measured by ELISA.The mRNA expressions of IL-10 and IL-17 in the lungs were detected by RT-PCR.The frequencies of CD4~+CD25~+Foxp3~+ and CD4~+IL-17~+ cells were assessed by intracellular cytokine staining and flow cytometric analysis.3.To identify the unbalanced T cell-specific transcription factors Foxp3,RORγt in immune dysregulation of asthma.The mRNA expressions of IL-10,IL-17,Foxp3 and RORγt in the lungs were detected by RT-PCR.The expressions of Foxp3 and RORγt in tissues were determined immunohistochemical staining photographic analysis.The protein expressions of Foxp3 and RORγt in the lungs were detected by Western blot.Results1.The mice of asthmatic group demonstrated symptoms of acute asthma when exposed to aerosolized OVA,such as breathing deeply and fast,standing still,bowing the back,urinary and fecal incontinence,and so on;the mice of control group didn't show these symptoms of acute asthma.Airway responsiveness was determined before and after aerosolizing of methacholine(3.125,6.25,12.5,25,50mg/ml),and airway responsiveness of the asthmatic group were significantly elevated compared with those of the control group(P＜0.01).Numerous inflammatory cells,including eosinophils,lymphocyte and neutrophils et al,infiltrated around the bronchioles and blood vessels,the airway epithelium was thickened,and mucus had accumulated in the lumen of bronchioles,interalveolar septum collapse,construction disorder,alveolar space and interstitial congestion.In bronchioalveolar Lavage Fluid,numbers of total cells,eosinophils,lymphocyte and neutrophils were were much higher in the asthma group than those in the control group(P＜0.01).The levels of IL-5 in BALF were much higher in the asthma groups than those in the control group(P＜0.01);In contrast,the levels of IFN-γwas markedly lower in the asthma group as compared with the control group(P＜0.01).2.Airway responsiveness was determined before and after aerosolizing of methacholine(3.125,6.25,12.5,25,50mg/ml),and airway responsiveness of the asthmatic group were significantly elevated compared with those of the control group and dexamethasone group(P＜0.01).Airway responsiveness of the dexamethasone group were significantly reduced compared with those of the asthmatic group(P＜0.01);there was no difference of airway responsiveness among the dexamethasone group and the control group before and after aerosolizing of methacholine(3.125,6.25,12.5mg/ml)(P＞0.05),but after aerosolizing of methacholine(25,50mg/ml),airway responsiveness of the dexamethasone group were significantly elevated compared with those of the control group(P＜0.01).In bronchioalveolar Lavage Fluid,numbers of total cells,eosinophils,lymphocyte and neutrophils were were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).The levels of IL-17 in BALF and serum were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the levels of IL-10 was markedly lower in the asthma group as compared with the Dexamethasone and control groups(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).The levels of IL-17 mRNA expression were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the expression of IL-10 mRNA was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).The frequencies of CD4~+CD25~+Foxp3~+ cells in asthmatic group were significantly lower than those of control group(P＜0.01),The frequencies of CD4~+CD25~+Foxp3~+ cells in Dexamethasone group were higher than those of asthma group(P＜0.01),but still lower than those of control group(P＜0.01);The frequencies of CD4~+IL-17~+ cells in asthmatic group were significantly higher than those of control group(P＜0.01),The frequencies of CD4~+IL-17~+ cells in Dexamethasone group were lower than those of asthma group(P＜0.01),but still higher than those of control group(P＜0.01).The ratio of CD4~+CD25~+Foxp3~+ ceils/CD4~+IL-17~+ cells in asthmatic group were significantly lower than those of control group(P＜0.01),The ratio of CD4~+CD25~+Foxp3~+ cells/CD4~+IL-17~+ cells in Dexamethasone group were higher than those of asthma group(P＜0.01),but still lower than those of control group(P＜0.01);The ratio of CD4~+CD25~+Foxp3~+ cells/CD4~+IL-17~+ cells was negatively correlated with airway responsiveness,the number of eosinophils, lymphocytes and neutrophils,respectively.The ratio of CD4~+CD25~+Foxp3~+ cells/ CD4~+IL-17~+ cells was negatively correlated with the gene and protein expressions of IL-17 in lung tissues,but positively correlated with the gene and protein expressions of IL-10 in lung tissues,respectively.3.The levels of RORγt mRNA expression were much higher in the asthma groups than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the expression of Foxp3 mRNA was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).The RORγt protein level were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the Foxp3 protein level was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).In the control group,the ratios of gene and protein expressions of Foxp3 and RORγt were significantly increased compared with the asthmatic group(P＜0.01);In the dexamethasone group,the ratios of gene and protein expressions of Foxp3 and RORγt were significantly decreased compared with the control group(P＜0.01),and there was also a difference of the ratios of gene and protein expressions of Foxp3 and RORγt between the dexamethasone group and asthmatic group(P＜0.01).The ratio of protein expression of Foxp3 and RORγt was negatively correlated with airway responsiveness,the numbers of eosinophils,lymphocytes and neutrophils in lung tissues,respectively.The ratio of protein expression of Foxp3 and RORγt was negatively correlated with the gene and protein expressions of IL-17 in lung tissues respectively,but positively correlated with the gene and protein expressions of IL-10 in lung tissues.Conclusions1.We improved the sensitizing agent and the method of sensitization,and established a modified mouse asthma model successfully.Non-invasive whole-body plethysmography(WBP) method can detect airway responsiveness conveniently.This successful modified mouse asthma model provides useful tools in the subsequent research of asthma.2.In asthmatic group,the unbalanced T cell-specific subpopulation Treg/Th17 contributes to both high frequencies of CD4~+IL-17~+ and low frequencies of CD4~+CD25~+Foxp3~+ cell,and the ratios of CD4~+CD25~+Foxp3~+ cell/CD4~+IL-17~+ cell were low,the ratios may evaluate the immune disbalance of asthma objectively.The ratio of CD4~+CD25~+Foxp3~+ cell/CD4~+IL-17~+ cell were negatively correlated with airway inflammation.Imbalance of Treg and Th17 may play a key role in the formation of airway inflammation of asthma.3.In asthmatic group,the unbalanced T cell-specific transcription factors Foxp3 and RORγt contributes to both high expression of RORγt and low expression of Foxp3,and the ratios of gene and protein expressions of Foxp3/RORγt were low.The ratio of protein expressions of Foxp3/RORγt were negatively correlated with airway inflammation.Imbalance of transcription factors Foxp3 and RORγt may play a key role in the unbalance of Treg and Th17 in airway inflammation of asthma.4.Dexamethasone treatment inhibits the airway inflammation of asthma by regulate the balance between Treg and Th17. |
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