The Receptor For Advanced Glycation End Products Contributes To TDI-Induced Airway Inflammation Through Stabilization Of β-catenin

BackgroundToluene diisocyanate-induced asthma is characterized by airway neutrophil and eosinophil inflammation as well as airway remodeling.Yet its mechanisms are very poorly defined.Studies have indicated that both the receptor for advanced glycation end products and β-catenin take important parts in the development of asthma,but their roles in TDI-induced asthma are currently not addressed.MethodsPartⅠ:To establish a TDI-induced murine asthma modelBriefly,the mice were dermally sensitized on the dorsum of both ears with 0.3%TDI on days 1 and 8.And then on days 15,18 and 21,they were challenged via the airways by means of compressed air nebulization.According to the concentration of TDI used for challenge and the times of challenge,mice were randomly divided into the following groups:①1%TDI1 group,which refers to mice challenged with 1%TDI on day 15;② 1%TDI2 group,which refers to mice challenged with 1%TDI on days 15 and 18;③1%TDI3 group,which refers to mice challenged with 1%TDI on days 15,18 and 21;④3%TDI1 group,which refers to mice challenged with 3%TDI on day 15;03%TDI2 group,which refers to mice challenged with 3%TDI on days 15 and 18;⑥3%TDI3 group,which refers to mice challenged with 3%TDI on days 15,18 and 21.Control mice were sensitized and challenged with the vehicle of TDI by the same times.Soon after the last airway challenge,airway reactivity to methacholine,airway inflammation,serum IgE and Thl/Th2-related cytokines were analyzed to see if an asthma model is established.Part Ⅱ:Blocking RAGE signaling in TDI-induced asthma modelWith the method justified in PartⅠ,we sensitized and challenged the experimental mice with TDI to first generate an asthma model.Inhibitors of RAGE,FPS-ZM1 and the RAGE antagonist peptide(RAP),were injected i.p.after each challenge.Sham mice received the same volume of vehicle by comparison.Airway resistance was measured in vivo and bronchoalveolar lavage fluid was analysed.Lungs were examined by histology and immunohistochemistry.Western blotting and quantitative PCR were also used.Part Ⅲ:Blocking β-catenin signaling with selective antagonists in TDI-induced asthmaMale BALB/c mice were sensitized and challenged with TDI to establish a chemical induced asthma model.Inhibitors of p-catenin,XAV-939,and ICG-001 were respectively given to the mice through intraperitoneally injection.Sham mice received the same volume of vehicle by comparison.Airway resistance was measured in vivo and bronchoalveolar lavage fluid was analysed.Lungs were examined by histology and immunohistochemistry.Western blotting and quantitative PCR were also used.ResultsPart Ⅰ:Asthma responses could be induced by challenging mice three times with 3%TDI through compressed air nebulization following dermal sensitizationResults showed that in 1%TDI1 group,1%TDI2 group and 3%TDI1 group,no significant increased airway reactivity,little airway inflammation,no higher titers of IL-4,IFN-γ,IgE were found.Though the mice challenged with 1%TDI for three times had slight inflammatory infiltration around the airway,larger numbers of neutrophils in the bronchoalveolar lavage fluid and raised IgE level in the serum,we detected no increased airway reactivity and no higher IL-4 nor IFN-y in this group when compared with control.Similarly,mice challenged twice with 3%TDI exhibited mild peribronchial inflammation and more neutrophils in,yet neither airway reactivity nor levels of IL-4,IFN-y and serum IgE were increased in those mice compared with control.Surprisingly,those mice challenged with 3%TDI for three times developed typical asthma responses:airway hyperresponsiveness,pronounced inflammatory accumulation around the airways,a large number of neutrophils and a slight number of eosinophils in the bronchoalveolar lavage fluid,higher levels of total serum IgE and IL-4,IFN-γ in supernatants of primary cultured lymphocytes.Part Ⅱ:Blockade of the RAGE axis with specific antagonists not only attenuated TDI-induced airway inflammation but also blunted the activation of p-catenin signalingAfter mice were challenged with 3%TDI for three times,we found that the expression of RAGE and of its ligands HMGB1,S100A12,S100B,HSP70 was increased compared withe vehicle-exposed lungs.Immunohistochemistry showed that changes of RAGE and its ligands were most preeminent in the airway epithelium.All these increases were inhibited by FPS-ZM1 or RAP.Either antagonist blunted airway reactivity,airway inflammation and goblet cell metaplasia,and decreased release of Th2 cytokines.TDI exposure decreased level of membraneβ-catenin,phosphorylated Akt(Ser473),inactivated GSK3β(Ser9),dephosphorylated β-catenin at Ser33/37/Thr41,which controls its cytoplasmic degradation,increased phosphorylated p-catenin at Ser552,raised cytoplasmic and nuclear levels of β-catenin and up-regulated its targeted gene expression(MMP2,MMP7,MMP9,VEGF,cyclin Dl,fibronectin),all of which were partly reversed by RAGE inhibition.Part III:Blocking β-catenin signaling could attenuated TDI-induced airway inflammation and airway hyperresponsivenessTDI exposure led to a significantly increased activity of β-catenin.Treatment with either XAV-939 or ICG-001 effectively inhibited activation of β-catenin and downregulated mRNA expression of β-catenin targeted genes in TDI-asthmatic mice,paralleled by dramatically attenuatedTDI-induced hyperresponsiveness and inflammation of the airway,alleviated airway goblet cell metaplasia and collagen deposition,decreased Th2 inflammation,as well as lower levels of TGFβ1,VEGF,HMGB1,and IL-1β.ConclusionsⅠ.Airway challenge through compressed air nebulization following dermal sensitization was proved to be effective in generating a TDI-induced murine asthma model.Ⅱ.The receptor for advanced glycation end products is a central mediator of TDI-induced asthma and is required for TDI-induced β-catenin stabilization.Ⅲ.β-catenin signaling is responsible for TDI-induced allergic airway inflammation and airway hyperresponsiveness.