Distinct Roles Of Short And Long TSLP Isoforms In House Dust Mite-Induced Asthmatic Airway Epithelial Barrier Disruption

Background:Allergic asthma is a common chronic nonspecific airway inflammatory diseases.Because the environment changes in last decade,the morbidity and mortality of asthma were significantly increased on a global scale and there are about 300 million asthma patients in the world.Airway epithelium has typically been thought to function mainly as the first defensive barrier by impeding the access of allergens.And the physical barrier function of airway epithelial cells is dependent on the integrity of the cell and the interaction of cell connection protein.Our previous study has proved the house dust mites(HDM)can destroy the airway epithelial barrier function.And how to restore the airway epithelial injury caused by allergens has become a focus on the prevention and treatment of asthma.Recent studies have shown that thymic stromal lymphocytes(TSLP)plays an important role in the prevention and treatment of asthma.TSLP is present in 2 distinct isoforms,short-and long-form TSLP(hereafter called sfTSLP and IfTSLP,respectively).In recent years the different role about sfTSLP and 1fTSLP has caused more and more attention.However,whether sfTSLP and 1fTSLP also play the different role in asthma haven’t yet reported.Aiming at this problem,in vivo and in vitro experiments we explore:1.Whether 1fTSLP contributes to HDM-induced airway epithelial barrier dysfunction.2.Whether synthetic sfTSLP can protect HDM-induced airway epithelial barrier dysfunction.3.Further identify the possible mechanism about the different functions of sfTSLP and 1fTSLP.Methods:1.In vitro experiment1.1 The human bronchial epithelial cell line 16HBE cultured.1.2 Using western blot、Real-time PCR、Trans Epithellal Electric Resistance、fluorescein isothiocyanate-dextran(FITC-dextran)and Immunofluorescence microscopy to study the effect of the different time and concentration HDM stimulation on 16HBE.1.3 Using Real-time PCR、Trans Epithellal Electric Resistance、fluorescein isothiocyanate-dextran(FITC-dextran)and Immunofluorescence microscopy to identify the effect of 1α,25-Dihydroxyvitamin D3(1,25D3)on the expression of sfTSLP and HDM-induced airway epithelial barrier dysfunction.1.4 Using western blot to identify whether mediation of HDM-induced 1fTSLP upregulation by the mitogen-activated protein kinase(MAPK)signaling cascade.1.5 Using western blot.Trans Epithellal Electric Resistance、fluorescein isothiocyanate-dextran(FITC-dextran)and Immunofluorescence microscopy to identify the effect of 1fTSLP on the airway epithelial barrier and the effect of STAT5 signaling pathways in the process.1.6 Using western blot、Trans Epithellal Electric Resistance、fluorescein isothiocyanate-dextran(FITC-dextran)and Immunofluorescence microscopy to identify the effect of sfTSLP on HDM-and 1fTSLP-induced airway epithelial barrier disruption.2.In vivo experiment2.1 BALB/c mice were randomly divided into 6 groups,(1)control group;(2)sfTSLP group;(3)1,25D3 group;(4)HDM group;(5)sfTSLP+HDM group,in which mice were pretreated with sfTSLP,followed by HDM;and(6)1,25D3+HDM group,in which the mice were pretreated with 1,25D3,followed by HDM.These treatment procedures were carried out daily for 5 consecutive days,for 8 consecutive weeks.2.2 Airway parameters were measured 24h after the last challenge.The blood was collected from eye artery,the supernatant was collected after centrifuging for IgE detecting.The bronchial alveolar lavage fluid(BALF)was collected,IFN-γ、IL-4、IL-5、IL-13、IL-33、TSLP(1fTSLP)in BALF were detected by ELISA.2.3 The left lung was grinding for Hematoxylin and eosin(H&E)and IHC of 1fTSLP,E-cadherin and β-catenin was used evaluate the comprehensive airway changes.The right lung was grinding for western blot analysis.Western blot was used to analyze the expression of target proteins.3.Statistical analysis was computed using SPSS(version 19.0).Results:1.HDM induced a significant upregulation of 1fTSLP and barrier disruption in 16HBE cells.2.1,25D3 upregulates stTSLP expression and inhibits HDM-induced barrier disruption in 16HBE cells.3.Mediation of HDM-induced 1fTSLP upregulation by the mitogen-activated protein kinase(MAPK)signaling cascade.4.Involvement of STAT5 phosphorylation in 1fTSLP-induced barrier dysfunction.5.Protective effect of sfTSLP on HDM-and 1fTSLP-induced airway epithelial barrier disruption.6.In the mouse model of HDM-induced asthma,sfTSLP and 1,25D3 inhibit the HDM-induced airway inflammation and airway epithelial barrier disruption by inhibiting phosphorylation of ERK1/2 and p38.Conclusions:1.HDM induced a significant upregulation of 1fTSLP via ERK and p38 signaling pathway.While the overexpression of 1fTSLP cause airway epithelial barrier disruption by STAT5 phosphorylation in 16HBE.2.In 16HBE,1,25D3 upregulates sfTSLP expression and sfTSLP can protect HDM-and IfTSLP-induced airway epithelial barrier disruption.3.In the mouse model of HDM-induced asthma,sfTSLP and 1,25D3 inhibit the HDM-induced airway inflammation and airway epithelial barrier disruption by inhibiting phosphorylation of ERK 1/2 and p38.