Dissecting The Immune Activation Function Of EZH2 Inhibitor In Porcine Alveolar Macrophages

Alveolar macrophages are the first defence line against various respiratory pathogens,and their immune response status has a significant impact on the consequences of respiratory tract infection.Previous studies have shown that epigenetic inhibitors against the core Poly comb group protein EZH2 can be used for tumor therapy by promoting immune activation.Can EZH2 inhibitors also affect the immune activation of alveolar macrophages?And what are their effects on virus infection?Currently,these questions remain unclear.This study aims at addressing these questions.By using porcine 3D4/21 alveolar macrophages as a model and integrating transcriptomic and epigenomic techniques,this study revealed the immune activation function of EZH2 inhibitor GSK126 on porcine alveolar macrophages,and preliminarily explored its mechanism.Further experiments showed that the infection of swine influenza virus can be significantly inhibited by GSK126 treatment,suggesting that EZH2 inhibitors play an antiviral role by promoting innate immune response.The major findings of this study are summarized below:Firstly,Western blot confirmed that GSK126 remarkably reduced the global protein level of H3K27me3,comparative analysies of gene expression profiles of GSK126-treated porcine 3D4/21 alveolar macrophages were performed by using RNA-seq.The results demonstrate that inhibition of EZH2 caused the increased expression of hundreds of genes,and there were substantial differences in the upregulated genes after different time periods of EZH2 treatment.Functional enrichment analysis indicates that upregulated genes are closely related to innate immune response.In addition,data visualization and qRT-PCR experiments further validated the activation of numerous innate immune response genes.These results support the function of EZH2 inhibitors in promoting the innate immune activation of alveolar macrophages.Secondly,comparative analysis of ChIP seq data of H3K27me3,revealed that most of the activated immune genes after GSK126 treatment were located outside the H3K27me3 domains.Further given that EZH2 participated in epigenetic repression mainly through the catalyzing H3K27me3,it indicates that GSK126 tends to promote immune activation through an indirect mechanism.Interestingly,many retrotransposon families were activated after GSK126 treatment,and the corresponding retransposon families are highly enriched in the H3K27me3 domains.Taken together,it is hypothesised that through a "viral mimicry"mechanism,EZH2 inhibitors cause abnormal expression of retrotransposons which produce double-stranded RNA molecules that are subsequently recognized by the RIG-I/MDA5MAVS pathway,and finally leading to innate immune activation of porcine alveolar macrophages.Finally,to investigate the potential antiviral function of EZH2 inhibitors,the infection of swine influenza viruses in GSK126-pretreated porcine 3D4/21 alveolar macrophages was investigated.Comparative analysis of the transcription level of the M gene and NP protein abundance of the swine influenza virus,showed that GSK126 treatment can significantly reduce the infection level of the swine influenza viruses.In addition,GSK126 could significantly reduce the infection of porcine epidemic diarrhea virus on pig intestinal epithelial cells.Overall,these results indicate that EZH2 inhibitors have the effect of promoting the antiviral defence of porcine alveolar macrophages,and their antiviral effect may be broadspectrum in other cell types.In summary,this study for the first time revealed that EZH2 can promote the innate immune activation of porcine alveolar macrophages and plays a role in enhancing antiviral defence.These findings give new insights for understanding the immune regulation of porcine alveolar macrophages and for the development of novel antiviral strategies.