The Study Of The Role Of IDO Regulation The Differentiation Of Th17/Treg In The Immune Tolerance Of Asthma

Background: Asthma is a serious global health problem affecting all age groups.The prevalence of asthma is increasing in many countries,especially among children.Research has suggested that the prevalence of asthma increased dramatically approximately 50% per 10 years in childhood asthma in the last twenty years.The global cost has increased by 30% for the control of abrupt deterioration and daily symptoms in the asthma patients,and it has brought significant impact on people's work,studies and lives.Therefore,it is of great practical significance to carry out the research of asthma control and prevention.The mainstay treatment for asthma is inhaled corticosteroids,but inhaled corticosteroids can only control asthma not cure asthma.The World Health Organization(WHO)and the European Academy of Allergy and Clinical Immunology(EAACI)recommended that the allergen specific immunotherapy(SIT)was the only effective treatment for asthma.In recent years,it was confirmed that the imbalance of Th1 / Th2 cells,especially Th2 cell activation played a critical role in the immunologic mechanism in the pathogenesis of asthma;Th17 cytokines played an important role in the immune mechanism of asthma,especially in neutrophilic asthma;Regulatory T cells(Treg)had the function of inhibiting the activity of Th2 cells and played an important role in the asthma control.Immune tolerance is an important part of SIT,in which Treg play a key role,but the mechanisms are incompletely understood.Studies also confirmed indoleamine2,3-dioxygenase(IDO),the rate-limiting enzyme of tryptophan catabolism,played an important role in the peripheral tolerance,can induced T cell inhibition and Treg activation,was the molecular switch of Th17 cell to Treg under certain conditions,and it was helpful for the formation of tracheal immune tolerance in allergen-specific immunotherapy.Therefore,we hypothesized that IDO mediate tryptophan catabolism may regulate the differentiation of Th17 and Treg cells,and play a role in the immune tolerance of asthma.Objective: 1.To establish a mouse model of asthmatic immune tolerance and evaluate.2.To study the the role of IDO mediated the differentiation of Th17 and Treg cells induced asthmatic immune tolerance with allergen specific immunotherapy.Methods: 1.Establishment a mouse model of asthmatic immune tolerance and evaluation.Eighteen female BALB/c mice were randomly divided into asthmatic immune tolerance model group,asthma control group and normal control group,each group of 6 mice.Those mice in asthmatic immune tolerance model group were sensitized with subcutaneous injection in abdomen of 10?g ovalbumin(OVA)on day 0 and 7,were induced immune tolerance with subcutaneous injection in abdomen of 1mg ovalbumin(OVA)daily from day 21 to day 25,were challenged with 1%OVA on day 35,36 and 37.On day 42,mice were sacrificed and specimens were obtained.Asthma control group were treated with the same procedure except induction of immune tolerance with subcutaneous injection in abdomen of 1mg ovalbumin(OVA)daily from day 21 to day 25.And normal control group were sensitized and challenged with equal amount of saline as a substitute.The airway responsiveness was measured by noninvasive lung functional instrument 24 hours after the last challenge.On day 42,mice were sacrificed and specimens were obtained.The lung tissue were obtained and histopathologic evaluation were done through hematoxylin & eosin(H&E).The number of totle cells and eosinophilia cells in bronchoalveolar lavage fluid(BALF)were counted by using hemacytometer.Frequencies of OVA specific Ig E in peripheral blood serum,IL-4 and IL-10 in BALF of each group were measured by enzyme-linked immunosorbent assay.2.The role of IDO regulate the differentiation of Th17 / Treg in asthmatic immune tolerance(1)Experimental groups: twenty-four famle BALB/c mice were randomly divided into asthmatic immune tolerance group,asthma group and normal group,asthmatic immune tolerance combined with 1-methyltryptophan group,each group of 6 mice.Those mice in the group of asthmatic immune tolerance combined with 1-methyltryptophan were sensitized with subcutaneous injection in abdomen of 10?g ovalbumin(OVA)on day 0 and 7,were subcutaneous injected in abdomen of 1mg ovalbumin(OVA)combined with 2mg 1-methyltryptophan daily from day 21 to day 25,were challenged with 1%OVA on day 35,36 and 37.On day 42,mice were sacrificed and specimens were obtained.The method of modeling and evaluation of other groups was performed as previously described.(2)Frequencies of OVA specific Ig E in peripheral blood serum,IL-4,IL-10 and IL-17 in BALF of each group were measured by enzyme-linked immunosorbent assay.The propotions of Th17 and Treg cells in CD4 T lymphocytes in the peripheral blood were calculated by flow cytometry.(3)Activites of Indoleamine 2,3 dioxygenase(IDO)in asthmatic immune tolerance group,asthma group and normal control group were measured by high-performance liquid chromatography.Results: 1.Establishment of mouse model of asthmatic immune tolerance Animal behavior:Mice in asthmatic immune tolerance group had mild asthmatic symptoms.Mice in asthma control group presented typical asthma symptoms,such as irritability,tachypnea,cyanosis;Mice in normal control group had no specific symptoms.Airway responsiveness: Airway responsiveness of mice in asthmatic immune tolerance group were significantly lower than those in the asthma control group(p<0.05).Lung H&E staining:The Inflammation of lung tissues and the ratio of eosinophils in asthmatic immune tolerance group were significantly reduced than those in the control group(p<0.05).Counts of the number of totle cells and ratios of eosinophilia cells in bronchoalveolar lavage fluid(BALF)in asthmatic immune tolerance group were significantly lower than those in the asthma control group.Frequencies of OVA specific Ig E in peripheral blood serum and IL-4 in BALF in asthmatic immune tolerance group were significantly lower than those in the asthma control group(p<0.05).Frequencies of IL-10 in BALF in asthmatic immune tolerance group were significantly higher than those in theasthma control group(p<0.05).2.IDO regulates Th17/Treg differentiation were assumed to be involved in asthmatic immune tolerance by allergen specific immunotherapy. Airway responsiveness: Airway responsiveness in asthmatic immune tolerance group were significantly lower than those in asthma group(p<0.05),but higher than the control group.Airway responsiveness in asthmatic immune tolerance combined with 1-methyltryptophan group were significantly higher than those in asthmatic immune tolerance group(p<0.05),but lower than asthma group.Lung H&E staining:The Inflammation of lung tissues in asthmatic immune tolerance group were significantly decreased than those in asthma group(p<0.05),but were increased than the control group.The Inflammation of lung tissues in asthmatic immune tolerance combined with 1-methyltryptophan group were significantly increased than those in asthmatic immune tolerance group(p<0.05),but were decreased than asthma group.Counts of the number of totle cells and the ratios of eosinophils in bronchoalveolar lavage fluid(BALF):Those in asthmatic immune tolerance group were significantly reduced than those in asthma group(p<0.05),but were elevated than the control group.Those in asthmatic immune tolerance combined with 1-methyltryptophan group were significantly elevated than those in asthmatic immune tolerance group(p<0.05),but were reduced than asthma group.Frequencies of OVA specific Ig E in peripheral blood serum,IL-4,IL-10 and IL-17 in BALF were measured by enzyme-linked immunosorbent assay:Frequencies of OVA specific Ig E,IL-4,and IL-17 in asthmatic immune tolerance group were significantly lower than those in asthma group(p<0.05),but were higher than the control group.Frequencies of OVA specific Ig E,IL-4,and IL-17 in asthmatic immune tolerance combined with 1-methyltryptophan group were significantly higher than those in asthmatic immune tolerance group(p<0.05),but were lower than asthma group.Frequencies of IL-10 in asthmatic immune tolerance group were significantly higher than those in asthma group(p<0.05),were higher than the control group.Frequencies of IL-10 in asthmatic immune tolerance combined with 1-methyltryptophan group were significantly lower than those in asthmatic immune tolerance group(p<0.05),but were higher than asthma group.The propotions of Th17 and Treg cells in CD4 T lymphocytes in the peripheral blood were calculated by flow cytometry: The propotions of Th17 cells in CD4 T lymphocytes in asthmatic immune tolerance group were significantly lower than those in asthma group(p<0.05),but were higher than the control group.Those in asthmatic immune tolerance combined with 1-methyltryptophan group were significantly higher than those in asthmatic immune tolerance group(p<0.05),but were lower than asthma group.The propotions of Treg cells in CD4 T lymphocytes in asthmatic immune tolerance group were significantly higher than those in asthma group(p<0.05),were higher than the control group.Those in asthmatic immune tolerance combined with 1-methyltryptophan group were significantly lower than those in asthmatic immune tolerance group(p<0.05),but were higher than asthma group.Activites of Indoleamine 2,3 dioxygenase(IDO)were measured by high-performance liquid chromatography: Activites of Indoleamine 2,3 dioxygenase(IDO)in asthmatic immune tolerance group were significantly increased than those in asthma group(p<0.05),but were increased than the control group.Conclusions: 1.Continuous subcutaneous injected high dose of OVA in sensitized mice established a mouse model of asthma immune tolerance.2.Th17/Treg imbalance exists in asthma,Th17/Treg imbalance can be reversed by the treatment of SIT which can induced immune tolerance in asthma.3.IDO regulating the differentiation of Th17/Treg can induce asthma immune tolerance,and regulating of the expression of IDO shows promise for the prophylaxis and treatment of asthma.