MBD2 Serves As A Viable Target Against Idiopathic Pulmonary Fibrosis By Inhibiting Macrophage M2 Program

Background: Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive and fibrotic interstitial pneumonia with unknown causes,and its pathogenesis is still unclear and effective therapeutic strategies are limited.Macrophages play an important role in the pathogenesis of IPF,and the formation of alternatively activated macrophages(M2)is an important factor leading to the progression of IPF.It was reported that DNA methylation as an important epigenetic regulation mechanism plays an important role in the pathogenesis of IPF.MethylCp G-binding domain 2(MBD2)is responsible for the recognition and reading of methylated DNA,and there are evidences that DNA methylation gets involved in the macrophage activation,thus it is speculated that MBD2 may be involved in the pathogenesis of IPF by affecting macrophage activation.Objective: To explore the differences of DNA methylation level and MBD2 expression in lung tissue from the normal and the IPF patients and to reveal the potential relationship between MBD2 and M2 in human lung tissue;To dissect the role of MBD2 in bleomycin(BLM)-induced pulmonary fibrosis mouse model and its effect on macrophage alternative activation in vivo;To investigate the role of MBD2 in the alternative activation of macrophages in vitro and the mechanism;To investigate whether the liposome encapsulated si RNA targeting MBD2 has therapeutic effect on the lung fibrosis mouse model,which may provide a theoretical basis for the transformation from scientific research to the clinic.Methods: The DNA methylation level and the expression of MBD2 were measured in the lung tissues from control and the IPF patients,and the differences of MBD2 expression between two groups were tested by Western Blot.Immunofluorescence were performed by using the lung tissue sections and the BALF to explore the correlation between MBD2 and the alternatively activated macrophages.The macrophage-specific Mbd2 knockout mice and their littermates were used to establish the BLM-induced pulmonary fibrosis mouse model to research the effect of MBD2 on the phenotype of pulmonary fibrosis and the macrophage activation in vivo.Bone marrow derived-macrophages(BMDM)were extracted from macrophage-specific Mbd2 knockout mice and their littermates and then were differentiated into M2 by IL-4 to analyze the effect of MBD2 on the alternative activation of macrophages and the underlying mechanisms.The wild-type mice were administrated with BLM to induce the pulmonary fibrosis and the liposome-loaded Mbd2 si RNA was airway injected to observe whether the application of liposome-loaded si RNA targeting MBD2 had therapeutic effect on the mouse pulmonary fibrosis model.Results: We demonstrated that lungs originated from IPF patients and mice after bleomycin(BLM)-induced pulmonary fibrosis were characterized by the elevation of M2 macrophage activation and the altered DNA methylation along with overexpression of methyl-Cp Gbinding domain 2(MBD2),a reader responsible for the interpretation of DNA methylomeencoded information.Particularly,macrophage specific(Lyz M-Cre)conditional deletion of Mbd2 protected mice from BLM-induced lung injury and pulmonary fibrosis.Loss of Mbd2 significantly attenuated transforming growth factor ?(TGF-?)production and reduced M2 macrophage infiltration in the lung following BLM induction.Mechanistically,Mbd2 selectively bound to the Ship promoter in macrophages,by which it repressed Ship expression and enhanced PI3K/Akt signaling to promote macrophage M2 program.More importantly,the silencing of Mbd2 in lung tissues by si RNA-loaded liposomes through pulmonary administration are proposed to safely and efficiently target pulmonary macrophages and attenuate the BLM-induced lung fibrosis,which may serve as a novel modality for the disease treatment.Conclusion: Collectively,these data indicated that Mbd2 played an essential role for the initiation and progression of pulmonary fibrosis by affecting the macrophage alternative activation,and therefore,strategies aimed at silencing MBD2 could be a viable therapeutic approach for treatment of pulmonary fibrosis in clinical settings.