Effect Of Short Thymic Stromal Lymphopoietin On Toluene Diisocyanate(TDI)-induced Airway Inflammation And The Mechanism Involved

Background and objective:Diisocyanatesare important sensitizers that are used in the production of rigid or flexible polyurethane foam;they are also used as hardeners in urethane spray paints and adhesives.Diisocyanates have been the most common cause of occupational asthma in many industrialized areas.toluene diisocyanate(TDI)is one of the primary types ofdiisocyanates.TDI-induced asthma is characterizedby hyperresponsiveness and inflammation of the airways.This inflammation is associated with infiltrationof lymphocytes,eosinophils,and neutrophils into thebronchial lumen.However,the mechanisms of TDI-induced asthma are still poorly understood.Thymic stromal lymphopoietin(TSLP)is an IL-7-like cytokine.TSLP is expressed mainly by epithelial cells(ECs)and epidermal keratinocytes(KCs)and is constitutively expressed in the thymus and intestinal ECs.Recent studies revealed that other types of cells such as mast cells,smooth muscle cells,fibroblasts,dendritic cells(DCs),trophoblasts,and cancer or cancer-associated cells also express TSLP.TSLP produced in response to environmental and endogenous factors contributes to disorders and homeostasis.TSLP can drive the development of strong allergic Th2 responses with release of IL-4,IL-5,IL,13 and TNF-a through upregulation of OX40 ligand expression on TSLP-treated DCs.Promotion of TSLP expression by IgE and Th2-related cytokines(IL-4,IL-13,IL-25,and IL-33)indicates amplification cycles.Whereas TSLPR-/-(knockout)mice are protected from airway inflammation after ovalbumin challenge,lung-specific transgenic expression of TSLP,coupled with antigenic stimulation,leads to airway eosinophilic inflammation,goblet cell metaplasia and remodeling in the murine lung.TSLP is overexpressed in the airways of asthmatic patients and is associated with Th2 inflammation and degree of airflow obstruction.Moreover,treatment with AMG 157,a human anti-TSLP monoclonal immunoglobulin G21ambda,reduced allergen-induced bronchoconstriction and indexes of airway inflammation before and after allergen challenge.Therefore,TSLP is closely related to the pathogenesis of asthma.However,recent studies indicate that human TSLP has two variants:long TSLP(1TSLP)and short TSLP(shTSLP).They share the common structures,but exert different functions.LTSLP is also called full-length TSLP(fl-TSLP).The molecule weight is 25 KDa.In most of previous studies,they did not distinguish between these TSLP variants.Harada et alfirst demonstrated the functional analysis of TSLP variants in human bronchial epithelial cells,indicating the long TSLP variant highly expression after poly(I:C)/dsRNA stimulation,which contributed to Th2-polarized immunity.The short TSLP ameliorated experimental colitis in vivo,indicating its anti-inflammatory properties.Meanwhile,in vitro analysis,shTSLP also exerted antimicrobial activities.More importantly,one of our latest study also indicated that shTSLP prevented epithelial barrier disruption in house dust mite induced asthma model.These studies helped us furtherly understand the TSLP variants,especially the shTSLP.However,the roles shTSLP on asthma have been still poorly addressed.Here we wanted to examine whether shTSLP exert anti-inflammatory activities in TDI-induced asthma murine model.Airway epithelial cells form a barrier to the outside world andare at the front line of mucosal immunity.Many cytokines could be secreted when epithelial cells sensitized by various allergens,such as house dust mite(HDM).Cytokines including IL-25,IL-33and TSLP can initiate and maintain the airway inflammation.Our previous studies demonstrated that HMGB1-RAGE axis played a critical role in TDI-induced airway inflammation.Here we also wanted to investigate the effects of TDI-HSA on TSLP isoforms and HMGB1-RAGE signaling and the possible involvement of shTSLP.Methods1:In vivo experiments:1.1 Male BALB/c mice were sensitized and challenged with TDI to generate a chemical-induced asthma model.Synthetic short TSLPpeptides were given intranasally(i.n.)or intraperitoneally(i.p.)before each challenge.1.2Assessment of airway hyperresponsiveness(AHR),and quantification of total serum IgE,and measurement of Thl-related IFN-y and Th2-related IL-4,IL-5,IL-13 in supernatants of cultured lymphocytes and the analysis of BAL fluids and pulmonary histopathological examination,andthe immunohistochemistry and western blot analysis of HMGB1,RAGE,TSLP and p-STAT3,p-STATA5 expression in the lung.2 In vitro experiments2.1Normal human bronchial epithelial cells line 16HBE14o-(16HBE)cells were cultured in RPMI-1640 medium.Cells treated by TDI-HSA or rhHMGB1 with or without the pretreatment of short TSLP.2.2 Western Blot and Real-Time PCR to detect the expressions of HMGB1,RAGE,1TSLP and signaling pathway proteinsand 1TSLP and shTSLP mRNA in 16HBE cells2.3Immunofluorescence microscopy to detect the expression and localization of HMGB1-RAGE in 16HBE cells3 Statistical analysesSPSS 20.0 software was used for the statistical analyses.Statistical analysis was performed by one-way analysis of variance(ANOVA)and post hoc tests were analyzed by Bonferonni(equal variances assumed)or Dunnett's T3(equal variances not assumed)post hoc tests for multiple comparisons.Results were presented as means ± Standard Deviation(SD).P<0.05 was considered statistically significant.Results1 In vivo experiments1.1 TDI-induced asthmatic mice exhibited a significant increase in AHR,which could be partly recovered by short TSLP.The level of total serum IgE increased by TDI was markedly inhibited by short TSLP.And short TSLP peptides also decreased the secretion of Thl-related IFN-y and Th2-related IL-4,IL-5 and IL-13 in supernatants of cultured lymphocytes in TDI asthmatic mice.Higher amounts of neutrophils and eosinophils were found after sensitization and challenge with TDI,which were significantly decreased by short TSLP peptides.Similarly,histologic analysis revealed typical pathologic features of TDI-induced airway disease.Numerous inflammatory cells infiltrated around the bronchus,and the lining airway epithelium was thickened and proliferated in TDI group.Mice treated with short TSLP showed marked reductions in extravasation of inflammatory cells in peribronchial and perivascular regions and the proliferation of bronchial epithelial cells.1.2HMGB1,RAGE and mouse TSLP expression were increased in lungs of TDI-immunized mice and decreased after short TSLP treatment.It was the p-STAT5(Y694)that significantly increased in TDI mice and decreased after short TSLP treatment.2 In vitro experiments2.1 Both TDI-HSA and rhHMGB1 promoted RAGE and long TSLP expression andactivated STAT5(Y694),Akt(S473)and p38 MAPK(T180/Y182)signals in 16HBE cells.2.2Short TSLP inhibited long TSLP and RAGE expression and STAT5(Y694),Akt(S473)and p38 MAPK(T180/Y182)activation in 16HBE cells.2.3 Short TSLP decreased the nucleocytoplasmic translocation of HMGB1 and the colocalization of HMGB-RAGE in 16HBE cells.ConclusionsThe shTSLP attenuates TDI-induced airway inflammation,and the mechanisms involved could be related to the inhibitions of HMGB1,RAGE and TSLP expression and STAT5(Y694)phosphorylation.