ITGB4 Deficiency In Airway Epithelia Induces Airway Remodeling Through Enhanced Activation Of EMTU

Asthma is a heterogeneous clinical syndrome of respiratory system which is characterized by chronic airway inflammation,airway remodeling,and airway hyperresponsiveness.Although the use of inhaled corticosteroids or combined long-acting beta-receptor agonists（LABA）has greatly improved clinical symptoms of asthma patients,long-term use of anti-inflammatory drugs has been shown to have limited effects on improving lung function and preventing asthma exacerbations of asthma patients.Previous studies have shown that airway remodeling persists after the subsides of airway inflammation.Airway remodeling can directly induce irreversible respiratory obstruction and airway hyperresponsiveness which is independent of airway inflammation.These observations suggest that airway remodeling is closely related to the clinicopathological process and therapeutic effect of asthma patients.However,the underlying mechanisms of airway remodeling and the relationship between airway inflammation and airway remodeling remain unclear.Airway epithelial cells（AECs）are the first barrier of lung tissue against external stimuli.AECs with impaired barrier function can directly induce airway remodeling by activating epithelial-mesenchymal trophic unit（EMTU）.Our previous studies have found that the expression of integrinβ4（ITGB4）in AECs is down-regulated in asthma patients.After ITGB4 silencing,the repair of AECs is slowed down and the apoptosis is increased.However,the effect of airway epithelial ITGB4 deficiency on airway inflammation and airway remodeling in asthma patients is poorly understood.Objective:The study aimed to identify the effect of epithelial ITGB4 deficiency on the barrier functions of AECs,airway inflammation and airway remodeling after allergy stimulation.Then,the molecular mechanism of EMTU activation and airway remodeling in the airway epithelial ITGB4deficiency mice after allergy stimulation was elucidated.Based on this,effective intervention strategies were explored to inhibit airway remodeling in asthma.Methods:（1）ITGB4-silenced human bronchial epithelial cell line（16HBE14o-）was constructed by siRNA.Silencing efficiency was detected by qPCR and WB.To evaluate the proliferation ability,the cell growth rate was detected by CCK8,the cell cycle was detected by flow cytometry and the expression of p21 was detected by qPCR and WB.The antioxidant capacity was assessed by the detection of reactive oxygen species and mitochondrial membrane potential.The barrier function was assessed by WB for the detection of tight junction protein 1（ZO-1）and adhesion junction protein E-cadherin（E-cad）and by the permeability assay of monolayer 16HBE14o-cells.AECs-specific conditional ITGB4 knockout mice(ITGB4^-/-)were constructed with doxycycline（Dox）from the 21st day of birth for 1 month,2 months and 3 months.The knockout efficiency of ITGB4 in AECs was detected by immunofluorescence staining for ITGB4 and Clara cell-secreted protein CCSP in lung tissue.The barrier function of AECs was assessed by immunohistochemical staining for proliferation marker Ki67,ZO-1 and E-cad.Airway inflammation was assessed by HE staining.Airway remodeling was assessed by PAS staining and Masson staining.（2）Juvenile ITGB4^-/-mice were constructed and asthma mice models induced by House dust mite（HDM）with different concentrations were established in adulthood.The airway hyperresponsiveness（AHR）was evaluated by the Buxco System.The level of airway inflammation was assessed by HE staining and qPCR for the detection of inflammatory factors in lung tissue.The level of airway remodeling was assessed by PAS staining,Masson staining and qPCR for the detection of remodeling-related molecules in lung tissue.（3）Juvenile ITGB4^-/-mice were constructed.HDM-induced asthma mouse models and the dexamethasone（DEX）-treated asthma models were established in adulthood.The inhibitory effect of DEX on airway inflammation was evaluated by HE staining,flow cytometry to classify inflammatory cells in lung tissue and ELISA to detect inflammatory factors in Broncho alveolar lavage fluid（BALF）.The AHR was evaluated by the Buxco System.The level of airway remodeling was assessed by PAS staining,Masson staining and qPCR for the detection of remodeling-related molecules in lung tissue.（4）The level of EMTU activation in lung tissue was evaluated by immunohistochemical staining for Ki67,E-cadherin,fibroblast marker Vimentin and myofibroblast markerα-SMA.The EMTU co-culture models of ITGB4-silenced 16HBE14o-cells and lung fibroblasts（HLF-1）were constructed in vitro.The activation level and collagen synthesis of HLF-1 cells in the co-culture model were evaluated by WB.The differentially expressed growth factors during EMTU activation in AECs were screened by qPCR in vitro which was further verified by qPCR,WB and immunofluorescence staining in vivo.The key signaling pathway of ITGB4 deficiency-regulated EMTU activation in AECs was further clarified by WB.（5）The inhibitors were used to block the key molecules that regulate EMTU activation in HDM-stressed ITGB4^-/-mice.The level of EMTU activation in lung tissue was assessed by immunohistochemical staining for Ki67,E-cadherin,Vimentin andα-SMA.The AHR was evaluated by the Buxco System.The level of airway inflammation was assessed by HE staining.The level of airway remodeling was assessed by PAS staining and Masson staining.Results:（1）ITGB4-specific siRNA significantly inhibited the expression of ITGB4 in 16HBE14o-cells.ITGB4 silencing slowed down the proliferation of 16HBE14o-cells,decreased antioxidant capacity and damaged epithelial barrier function.Juvenile ITGB4^-/-mice were constructed successfully.Epithelial ITGB4 deficiency in juvenile mice did not directly induce spontaneous airway remodeling but led to damage of the airway epithelial barrier.（2）Juvenile ITGB4^-/-mice were more prone to AHR,airway inflammation and airway remodeling in response to HDM stimulation with low concentration.After HDM stimulation with the same concentration,ITGB4^-/-mice had increased airway reactivity,enhanced airway inflammation and aggravated airway remodeling compared with control mice(ITGB4^+/+mice).（3）DEX intervention only partially suppressed the increased airway reactivity and aggravated airway remodeling in HDM-stressed ITGB4^-/-mice although the enhanced airway inflammation was almost completely suppressed.After DEX blocked airway inflammation,the HDM-stressed ITGB4^-/-mice still exhibited increased airway reactivity and aggravated airway remodeling compared to HDM-stressed ITGB4^+/+mice.（4）Under HDM stimulation,ITGB4^-/-mice exhibited enhanced EMTU activation compared with ITGB4^+/+mice,which was not completely suppressed by DEX.In vitro EMTU co-culture models,ITGB4 silencing in 16HBE14o-cells induced increased activation of HLF-1 cell and collagen synthesis after HDM stress and up-regulated the expression of fibroblast growth factor（FGF2）,which was not completely inhibited by DEX.Similar results of FGF2 expression were observed in AECs from ITGB4^-/-mice.Besides,ITGB4 silenced 16HBE14o-cells hyperactivated SHP2/JNK/c-Jun signaling pathway which further increased the expression of FGF2 after HDM stimulation.Both SHP2inhibitor and JNK inhibitor could effectively reduce the expression of FGF2 by inhibiting c-Jun activation and its nuclear translocation.（5）JNK inhibitor could effectively inhibit the enhanced EMTU activation,increased airway reactivity,aggravated airway remodeling and enhanced airway inflammation in HDM-stressed ITGB4^-/-mice.JNK inhibitor could effectively inhibit the enhanced EMTU activation,increased airway reactivity and aggravated airway remodeling in HDM-stressed ITGB4^-/-mice.Conclusions:（1）ITGB4 deficiency in airway epithelia during childhood disrupts the barrier function of AECs.（2）ITGB4 deficiency in airway epithelia during childhood enhances the HDM-stressed airway inflammation and airway remodeling in adulthood.（3）ITGB4 deficiency in airway epithelia directly aggravates airway remodeling independent of airway inflammation under HDM stimulation.（4）ITGB4 deficiency in airway epithelia upregulates FGF2expression by enhancing the activation of SHP2/JNK/c-Jun signaling pathway,which further potentiates EMTU activation after HDM stimulation.（5）Both JNK inhibitor and FGF2 inhibitor can alleviate the aggravated airway remodeling in HDM-exposed ITGB4^-/-mice through blocking the enhanced EMTU activation.