| Background:Pulmonary fibrosis(PF)is a severe chronic interstitial disease caused by a variety of causes.The main features are early diffuse alveolitis and continuous alveolar damage.In the later stage,a large number of fibroblasts proliferate and extracellular matrix(ECM)abnormally aggregate,leading to the destruction of lung structure and eventually leading to PF.Inflammation has been considered to be the primary cause of pulmonary fibrosis,and epithelial-mesenchymal transition(EMT)is an important pathogenesis that promotes pulmonary fibrosis.Macrophages are a class of specialized immune cells that play an important role in maintaining immune homeostasis.In the process of pulmonary fibrosis,macrophages also play an important role,according to the microenvironment and external stimuli polarization into two different functional states,classic activated macrophages(M1)and alternative activated macrophages(M2)in order to exert their immunoregulatory effects.Wnt signaling is a key signaling pathway for the body to maintain tissue stability and damage repair,and it plays a very important role in immune regulation.Interleukin 17A(IL-17A)is an immunomodulator capable of inducing macrophage polarization and is involved in host defense and various immune related diseases.A compelling body of studies revealed that both Wnt signaling and IL-17 were abberrently expressed in pulmonary fibrosis.Objective:The aim of this study was first investigated the mechanism of Wnt/β-catenin signaling on IL-17A-induced macrophage polarization,and it’s effect on mesenchymal properties of lung epithelial cells.Methods:1.The mouse macrophage RAW264.7 was pretreated with conditioned medium,Wnt/β-catenin signaling inhibitor XAV939 and IL-17A.The surface morphology of macrophages was observed by scanning electron microscopy.Wnt/β-catenin signaling pathway-related proteins β-catenin,active-β-catenin(ABC),TCF-4,Cyclin D1 and c-Myc;Macrophage polarized marker proteins iNOS and Argl;macrophage regulatory factors p-STAT1 and p-STAT3 and their downstream molecules p21,BCL-XL and signaling pathway regulator SOCS3;inflammatory signaling pathway MAPK-related proteins p-JNK,p-P38 and p53 were detected by western blot.QRT-PCR was used to detect the expression levels of MCP-1,Fizzl and Ym-1.Enzyme-linked immunosorbent assay(ELISA)was used to detect inflammatory factors IL-6,IL-10 and TNF-α in cell culture supernatants.And the macrophage polarization-related membrane markers CD86 and CD206 were detected by flow cytometry.2.Isolation of primary mouse Tracheal epithelial cells,construct a air-liquid interface culture model,and then co-culture with macrophages,using Wnt3a conditioned medium,XAV939,IL-17A and IL-17A antibodies,using western blot to detect Epithelial-mesenchymal transition marker proteins a-SMA,Collagen I and Vimentin in whole protein extracts from epithelial cells.Results:1.After treatment with IL-17A,the expression of β-catenin,ABC,TCF-4,Cyclin D1 and c-Myc was up-regulated in Wnt/β-catenin cascade,indecate that IL-17A can effectively activate Wnt/β-catenin signaling,and IL-17A shows synergistic ability to enhance Wnt/β-catenin signaling by Wnt3a activation.2.Activation of Wnt/β-catenin signaling in macrophage RAW264.7 inhibits IL-17A-induced M1 polarization and reduce IL-17A-repressed M2 polarization.Molecular mechnism studies have show that an activation of Wnt/β-catenin signaling inhibited the IL-17A-induced macrophage M1 polarization,in part by reducing p-STAT3 expression,while reduced the IL-17A-supressed macrophage M2 polarization by augmenting p-STATl expression.At the same time,Wnt/β-catenin signaling can activate the p38 MAPK signaling pathway through synergy with IL-17A.3.During EMT process,Wnt3a can inhibit Vimentin and a-SMA expression,and can effectively inhibit IL-17A-induced Vimentin expression.IL-17A antibody has no significant effect on Vimentin expression,but can induce a-SMA expression.Both Wnt3a and IL-17A could induce Collagen I expression,but IL17A antibody had no significant effect on Collagen I expression.That’s indicate that activation of Wnt/β-catenin signal can slow down EMT process caused by IL-17A.Conclusion:IL-17A can effectively activate Wnt/β-catenin signaling and cooparate with Wnt3a;Further studies found that an activation of Wnt/p-catenin signaling inhibited the IL-17A-induced macrophage M1 polarization,in part by reducing p-STAT3 expression,while reduced the IL-17A-supressed macrophage M2 polarization by augmenting p-STAT1 expression,during this process,Wnt/(3-catenin signaling can interact with IL-17A to activate the p38 MAPK signaling pathway;In addition,Wnt3a is able to slow down EMT process caused by IL-17A.Taken together,Wnt/β-catenin signaling can inhibit IL-17A-induced macrophage M1 polarization and reduce IL-17A-inhibited macrophage M2 polarization slow down EMT progression in lung epithelial cells. |
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