Epigenetic Regulation Of Interleukin 6 By Histone Acetylation In Macrophages And Its Role In Paraquat Induced Pulmonary Fibrosis

Background and objectivePulmonary fibrosis is a chronic,progressive inflammatory disease of pulmonary interstitial,the occurrence of which is about 1/10 000 and the survival rate is 30-50%at 5 years after diagnosis.The main pathological change associated with this disease is lung interstitial,which is characterized by cell proliferation and ECM excessive accumulation.The mechanisms involved remain undefined;however,growing evidences indicate that inflammatory,epithelial injury,and apoptosis,especially EMT may play an important role in the development of pulmonary fibrosis.EMT is a process that loss of epithelial cells characteristics(e.g.,intercellular junctions and apical-baso-lateral polarity)but gain mesenchymal functions(e.g.,produce ECM components,migration,and invasion).During the EMT process,the mesenchymal markers such as a-SMA and vimentin are overexpressed,while epithelial markers such as E-cadherin and cytokeratin are low expressed.PQ is a highly toxic herbicide,which is widely used in numerous developing countries around the world.Ingestion of PQ leads to multiple organ damage especially in the lung,including epithelial cell destruction,pulmonary edema,and inflammation,which are considered to be mediated by the overproduction of reactive oxygen species.PQ induced progressive pulmonary fibrosis,the most serious lung damage is often associated with high mortality,appears as early as several days to several weeks after PQ inges-tion.However,the underlying molecular mechanisms of which remain elusive.IL-6,as a multifunctional cytokine,is involved in the pathogenesis of various autoimmune and chronic inflam-matory diseases through different signaling pathways.It is produced by various cells,but the main sources are macrophages and monocytes.Macrophage,a key component of immune responses,is a predominant regulator of inflammation in diseases.IL-6 that is released from macrophages into the extracellular space can also influence fibrosis and inflammation via paracrine actions on other cell types.IL-6 exerts its biological activities through IL-6R and gp130.When IL-6 binds to mIL-6R(membrane-bound form of IL-6R),homodimerization of gp130 is induced and form a high-affinity functional receptor complex of IL-6,IL-6R and gp130.The homodimerization of receptor complex activates JAKs that then phosphoryl-ate tyrosine residues in the cytoplasmic domain of gp130.IL-6 can simultaneously generate functionally distinct or sometimes contradictory signals through its receptor complex,IL-6Ralpha and gpl30.The final physiological output is thought to be a consequence of the diverse signaling pathways generated by a given ligand.IL-6 induction promotes collagen depo-sition in multiorgans such as kidney,heart,and skin.The cytokine IL-6 is elevated in mice and humans with pulmonary fibrosis.However,its impact on fibrosis and regulatory mechanisms are not well understood.Epigenetics refer to alterations of gene expression without changes in the DNA sequence.Histone modification,as an epigenetic mechanism,including acetylation,methylation,phosphorylation,deamination,P-N-acetylglucosamine,ADP ribosylation,ubiquitination,and sumoylation of Histones,can change the charge of histones which subsequently affect the structure of chromatin to upregulate or downregulate gene expression.For example,histone acetylation,the most common alteration of histone,is catalyzed by HATs and HDACs.The influence of histone acetylation has been reported in inflammatory response and fibrosis,and histone acetylation has revealed its capacity of regulation in PQ induced parkinson.Therefore,we wonder if the histone acetylation plays an important role in PQ induced pulmonary fibrosis and acute inflammation,and how to play its role?As a RNA-guided transcriptional activator system,CRISPR-ON is a novel and powerful tool that can effectively induce spe-cific gene expression.The CRISPR-ON consists of three components:a nucleolytically inactive Cas9-VP64 fusion,a single guide RNA(sgRNA)incorporating two MS2 RNA aptamers at the tetraloop and stem-loop,and the MS2-P65-HSF1 activation helper protein.With the ability to robustly activate coding andlincRNA,CRISPR-ON could be used to regulate gene expression epigenetically.Therefore,the aim of this study was to determine the impact of IL-6 in PQ-induced pulmonary fibrosis and to explore whether the epigenetic regulators play a role in the transcriptional regula-tion of IL-6.Part ?:The role of IL-6 in paraquat induced pulmonary fibrosisMethods:1.Build and verify animal model.2.Screen abnormal expression genes by testing their mRNA and protein expression level.3.Locate the source of the high expression IL-6.4.Determine the functional role of IL-6 in PQ-induced pulmonary fibrosis by blocking IL-6 signaling pathway.5.Explore the mechanism through which IL-6 influence pulmonary fibrosis.Results:1.We examined the effect of PQ-induced acute lung injury and chronic pulmonary fibrosis by performing H&E and Masson's trichrome staining in the lung sections from PQ treated mice for 3 days and 1 month.2.We found that PQ potently increases the mRNA and protein expression of IL-6 in a dose-dependent manner in the lung and serum.While we did not observed a significant change in the level of IL-1?,IL-8,COX-2,MMP9,TNF-?,and VC AM1 gene expression.3.PQ significantly increased the mRNA and protein expression of IL-6 in a dose-dependent and time-dependent manner in macrophages.However,the level of IL-6 remains unchanged in human lung fibroblast and epithelial cells 16HBE.4.We next blocked IL-6 trans-signaling with recombinant soluble Gp130Fc.PQ exposure significantly increased the expression level of TGF-?,?-SMA,GREM1,and FN1 in mouse lung,which were reduced in the presence of recombinant Gp130Fc.In addition,in vivo neutralization of IL-6 trans-signaling attenuated pulmonary fibrosis phenotype.However,the fibrotic genes were unchanged in mice fibroblasts with recombinant IL-6 protein which indicated IL-6 did not influence fibrosis and inflammation via paracrine actions.5.We treated 16HBE with PQ-treated macrophage lysates in the presence or absence of recombinant Gp130Fc.The data showed that the expression of EMT-related genes were elevated in 16HBE following PQ-treated macrophage lysates but reduced in the presence of recombinant Gp130Fc.Conclusions:We built PQ-induced acute and chronic lung injury mouse model and found that PQ incresed IL-6 expression.By functional analysis,we found that PQ-induced IL-6 expression in macrophages plays a central role in pulmonary fibrosis through enhanced EMT transition.Part II:The regulation mechanism of IL-6 in PQ-inducedpulmonary fibrosisMethods:1.HD AC inhibition and HAT inhibition were performed in vitro and in vivo to explore the impact of histone acetylation on IL-6.2.HDAC inhibition and HAT inhibition were performed in vitro and in vivo to explore the impact of histone acetylation on pulmonary acute inflammationand pulmonary fibrosis.3.Determine the role of HDACs in PQ-induced pulmonaryfibrosis by examine the expression of all subtype of HDAC.4.Using IL-6 promoter-luciferase reporter plasmids,ChIP analysis and CRISPR-ON strategy to determine epigenetic regulation mechanism of IL-6.Results:1.We measured IL-6 mRNA and protein expression in the lung,serum,and macrophages to find that PQ-induced upregulation of IL-6 mRNA and protein levels and they were further enhanced in the presence of VPA but reduced in the presence of Anacardic acid.2.PQ-induced pulmonary acute inflammation was aggravated in the presence of VPA and mitigated in the presence of the Anacardic acid.For 1 month treatment,Masson's trichrome and COL1? IF in lung sections indicated that Anacardic acid reversed PQ-induced pulmonary fibrosis.The data are consistent to the observation that the mRNA and protein level of EMT-related genes were decreased in the lung tissues from PQ-treated mice in the presence of Anacardic acid.3.We found decreased the expression of HDAC3 in PQ-treated lungs in a dose-dependent manner.In macrophages treated with PQ,we also observed the loss of HDAC3 expression in a dose-dependent and time-dependent manner.4.The relative IL-6 promoter was enhanced in the presence of VPA,which remains unchanged in the presence of Anacardic acid and PQ.CRISPR-ON targeted to the IL-6 promoter regions increased the expression of IL-6 The ability of CRISPR-ON to drive IL-6 gene transcription was increased in the presence of VPA while attenuated in the presence of Anacardic acid.ChIP-qPCR experiments shown that marks of open chromatin structures including H3K4me3 and H3K9ac binding at the IL-6 promoter region were significantly increased in the presence of HD AC inhibitor,scriptaid.Conclusions:Here,we showed that acetylation of histones through the increased binding of HAT p300 to the promoter regions of IL-6 elevates gene tran-scription.HAT inhibitor reduced IL-6 transcription.In addition,HAT inhibitor reduced the expression of fibrosis-related genes and mitigated the degree of PQ-induced pulmonary fibrosis.Therefore,histone acetylation potently regulates the expression of IL-6 gene in macrophages which may be of significance in the treatment of diseases that result from the overproduction of IL-6,such as PQ-induced pulmonary fibrosis.ConclusionsOverexpression of IL-6 has been proposed to contribute to pulmonary fibrosis and other fibrotic diseases.However,the regulatory mechanisms and the role of IL-6 in fibrosis remain poorly understood.Epigenetics refers to alterations of gene expression without changes in the DNA sequence.Alternation of chromatin accessibility by histone acetylation acts as a critical epigenetic mechanism to regulate various gene transcriptions.The goal of this study was to determine the impact of IL-6 in PQ-induced pulmonary fibrosis and to explore whether the epigenetic regulations may play a role in transcriptional regulation of IL-6.In PQ-treated lungs and macrophages,we found that the mRNA and protein expression of IL-6 was robustly increased in a time-dependent and a dose-dependent manner.Our data demonstrated that PQ-induced IL-6 expression in macrophages plays a central role in pulmonary fibrosis through enhanced EMT.IL-6 expression and its role to enhance PQ-induced pulmonary fibrosis were increased by HDAC inhibition and prevented by histone acetyltransferase HAT inhibition.In addition,the ability of CRISPR-ON to promote transcription of IL-6 was enhanced by HDAC inhibitor and blocked by HAT inhibitor.Chromatin immu-noprecipitation experiments revealed that HDAC inhibitor increased histones activation marks H3K4me3 and H3K9ac at IL-6 promoter regions.In conclusion,IL-6 functioning through EMT in PQ-induced pulmonary fibrosis was regulated dynamically by HDAC and HAT both in vitro and in vivo via epigenetically regulating chromatin accessibility.