IL-4/IL-13 Upregulates SHH Expression In Allergic Airway Epithelia Contributing To Goblet Cell Metaplasia

Bronchial asthma is a common and frequently-occurring disease of the respiratory system,and its pathogenesis is unknown.Because of its refractory nature,WHO has classified asthma as a lifelong disease.The pathophysiology of asthma is mainly manifested by airway inflammation,airway remodeling,and airway hyperresponsiveness.Mucous cell metaplasia,defined as the reversible transformation of airway epithelia to mucous cells,occurs in many chronic airway diseases,such as asthma,cystic fibrosis,and chronic obstructive pulmonary disease(COPD).It is characterized by mucous hypersecretion and associated with the morbidity and mortality of these pathological conditions.SHH is one of the major secretory ligand molecules of the HH signaling pathway.It exhibits high expression during lung development,and its expression gradually decreases as the lung matures.At present,abnormally high expression of SHH is found in many lung diseases,such as lung injury,pulmonary fibrosis,and asthma.In asthma,inducible factors and mechanisms of abnormally high expression of SHH,and the effect of SHH on the progression of asthma have not been reported.Objective: 1.The expression pattern of SHH in asthma 2.The inducing factors and mechanisms of SHH high expression 3.The role of SHH on the progression of asthma and its mechanismMethods: 1.Establishing asthma models induced by OVA and HDM in mice,respectively,and detect the expression of SHH and PTCH1 by immunofluorescence and immunohistochemistry.2.Explore the SHH expression and HH signaling activity inducing by Th2 type inflammatory factors(IL-4,IL-13,and IL-5)by q PCR,western blotting,and luciferase activity in human 16 HBE cell line.3.Th2 type inflammatory factors were intratracheally injected to C57 mice,and then,the expression of SHH and the activity of HH signaling were detected by immunohistochemistry,western blotting,and Elisa in lungs.4.Inhibitor of downstream signaling of inflammatory factors was administrated to OVA-induced mouse asthma models,then,we detected the SHH expression by western blotting and immunofluorescence in the lungs.5.In the OVA-induced mouse asthma model,cyclopamine at 1 and 6 mg/m L were inhaled,and then we tested the pulmonary inflammation cell infiltration by cell counting and HE staining,goblet cell metaplasia and mucin protein Muc5 ac expression by immunofluorescence and luciferase activity.6.To knockdown of SMO in airway epithelial cell,an adenovirus expressing Cre recombinase was instilled in the trachea of the OVA-induced SMOflox/flox mouse asthma model,then pulmonary inflammation cell infiltration,goblet cell metaplasia,and the expression of mucin protein Muc5 ac were detected by cell counting,HE and PAS staining,immunofluorescence,and immunohistochemistry.7.The adenovirus expressing Cre recombinase was instilled in the trachea of the OVA-induced SMO-M2 mouse asthma model to overexpress the activated form of SMO in airway epithelial cells.Then,we examined the pulmonary inflammation cell infiltration,goblet cell metaplasia,and the expression of Muc5 ac.8.SMO agonist purmorphamine or N-SHH were administrated to 16 HBE cells to detect the expression of Muc5 ac and the key regulatory protein FOXA2 and SPDEF.The lentivirus express sh Gli1 and sh Gli2 were used to knock down Gli1 and Gli2,respectively,and then we tested the roles of Gli in the activation of Muc5 ac.9.Mice were received an intratracheal instillation of N-SHH-neutralizing antibody(SHH-Nab)at 0.5 or 2.0 g/mouse.2 hours after each OVA aerosolization,we detected the inflammatory cell infiltration,goblet cell metaplasia,and Muc5 ac expression in the lung.Results: 1.The expression of SHH was high in lung tissues of the OVA-induced mouse model and mainly aggregated in airway epithelial cells.High expression of SHH in airway epithelial cells was also found in HDM-induced mouse asthma models.At the same time,the SHH content in BALF of asthmatic patients was significantly more than that of the foreign body inhalation group.Immunofluorescence staining showed that SHH was mainly expressed in carat cells with CC10 positive.2.IL-4 and IL-13 but not IL-5,significantly induced SHH transcription and protein expression in 16 HBE cells in a dose-dependent manner.IL-4 and IL-13 also induced the expression and the activity of Gli in 16 HBE cells.SHH neutralizing antibody significantly inhibited the expression and the activity of Gli luciferase induced by IL-4.The inhibitors of JAK1/2 and STAT6,downstream signaling molecules of IL-4/IL-13,significantly inhibited the expression of SHH induced by IL-4.Overexpression of JAK1/2 and its activated forms and STAT6 induced SHH and Gli1 expression and Gli luciferase activity significantly.CHIP experiments demonstrated that STAT6 binds to the Shh promoter region directly to mediate Shh transcription.These results indicated that IL-4/IL-13 induced SHH expression and secretion mainly through the JAK1/2-STAT6 signaling,in turn,these SHHs activated the HH signaling in 16 HBE.3.IL-4 and IL-13 at 400 ng/m L induced the expression of SHH and the phosphorylation of STAT6 significantly in mouse airway epithelial cells.4.AS,the STAT6 inhibitor,significantly inhibited the overexpression of SHH in airway epithelial cells induced by OVA in vivo.5.Aerosolized inhalation of 1 and 6 mg/m L of cyclopamine dose-dependently inhibited OVA-induced lung inflammation,goblet cell metaplasia,and Muc5 ac expression in a mouse asthma model.Cyclopamine significantly inhibited goblet cell metaplasia and Muc5 ac expression but not lung inflammation at a dose of 1 mg/m L.6.Knockout of SMO in airway epithelial cells showed a significant inhibitory effect on goblet cell metaplasia and Muc5 ac expression,without effecting pulmonary inflammation induced by OVA.7.Activation SMO in airway epithelial cells induced goblet metaplasia and Muc5 ac expression without effecting pulmonary inflammation in a low-inflammatory asthma model induced by OVA.8.Purmorphamaine,N-SHH,and overexpression Gli2 and △ N-Gli2 significantly induced the expression of Muc5 ac or its promoter-luciferase activity while knockdown Gli1 or Gli2 inhibited this induction.CHIP experiments confirmed that Gli2 directly binds to the promoter region of Muc5 ac.Both Purmorpharmaine and N-SHH reduced the expression of FOXA2 and increased the expression of SPDEF.Knockdown of Gli1 or Gli2 or cyclopamine blocked this effect.9.SHH neutralizing antibodies significantly inhibited lung inflammation,goblet cell metaplasia and Muc5 ac expression in mouse asthma models induced by OVA.Conclusions:1.Abnormally high expression of SHH was shown in airway epithelial cells in both mouse asthma models and asthmatic children.2.Th2 inflammatory factors including IL-4 and IL-13 are the main factors that induce the expression of SHH.IL-4 and IL-13 induce SHH expression through the JAK1/2-STAT6 signaling in airway epithelial cells.STAT6 directly binds to the promoter region of Shh to mediate the transcription of Shh.3.High expression of SHH in airway epithelial cells activates its own HH signaling,thereby promotes goblet cell metaplasia and mucus protein secretion in the asthma mouse model.4.Activated HH signaling regulates mucin protein expression through the Gli transcription factor,which directly binds to the promoter region of Muc5 ac.HH signaling also regulates the expression of Muc5 ac by up-regulating SPDEF and down-regulating FOXA2 respectively.