| Avian influenza is an infectious disease of the respiratory tract caused by avian influenza virus(AIV)that invades the host primarily through the mucous membranes of the respiratory tract.There are a large number of dendritic cells(DCs)distributed in the submucosa of the respiratory tract,which is the most powerful immune cell with the ability to present antigens,and dendritic cells play an irreplaceable role in the recognition and presentation of antigens.AIV can infect dendritic cells and impair the function of DCs by influencing its antigen-presenting ability.Therefore,it is important to study the mechanism by which AIV affects DCs’ function.Previous research in our laboratory showed that inactivated AIV had better stimulation effect on DCs than alive virus.The difference between an inactivated virus and alive virus is that the living virus produces non-structural proteins in addition to nucleic acid activity during replication.The proteins encoded by the virus are divided into structural proteins and non-structural proteins,in which non-structural proteins are not assembled in mature viral particles and play a crucial role in virus replication.Therefore,the effects of AIV non-structural proteins M42,NS2 and PB1-F2 on DCs were investigated in this study.The effects of AIV non-structural proteins on DCs’ function was explored from the changes of dendritic phenotype,antigen presentation,cytokine secretion,signal pathway activation and so on.The effects of AIV H9N2 subtype and non-structural protein NS2 on chicken’s bone marrow derived dendritic cell miRNA and LncRNA were investigated by high-throughput sequencing.Finally,the molecular mechanism of AIV non-structural protein NS2 inhibiting dendritic cells’ antigen presentation by regulating miRNA synthetases,IRF3,Mettl3 were preliminarily elucidated.This study was divided into the following parts:1.The Effect of AIV Nonstructural Protein on the Function of Dendritic CellsThree non-structural proteins M42,NS2 and PB1-F2 of AIV were constructed and their effects on DCs’ function were studied.First,treatment of mouse DCs with nonstructural protein NS2 with LPS significantly inhibited the upregulation of the DCs’phenotype induced by LPS(CD40(p<0.05),CD86,MHC Ⅱ(p<0.01)),and NS2 was found to modulate the secretion of cytokine IL12,but had no significant effect on the secretion of other cell cytokines(TNF-α,IFN-β,and CCL20).Further study showed that NS2 could significantly inhibit the expression of TBK/IRF3 in DCs stimulated by NS2(p<0.05).Subsequently,antigen-presenting assays showed that non-structural proteins M42 and NS2 significantly inhibited antigen-presenting capacity of DCs and reduced proliferation of naive T cells(p<0.01).To further explore whether non-structural proteins have a significant inhibitory effect on dendritic cells,this study further examined the effects of non-structural proteins on chicken bone marrow derived DCs activated by LPS.The results showed that NS2 could significantly decrease the expression of MHC Ⅱ in LPS-activated chicken DCs.It was also found that the non-structural protein M42 could significantly promote the secretion of cytokines IL10 and IFN-β(p<0.05),but had no significant effect on the secretion of TNF-α.The results showed that the non-structural protein M42,NS2 and PB1-F2 did not affect the secretion of inflammatory factors in the DCs of chicken.2.Effect of Nonstructural Protein NS2 on Expression of miRNA and LncRNA in Chicken Dendritic CellsNon-structural protein NS2 was transfected into chicken dendritic cells for 24 h to screen the differentially expressed miRNA and LncRNA.Target gene prediction and bioinformatics analysis were performed for differentially expressed miRNA and LncRNA.First,sequencing of the miRNA revealed that there were no differentially expressed miRNA after stimulation of chicken dendritic cells with AIV non-structural protein NS2.Secondly,sequencing of LncRNA showed that after stimulation of chicken dendritic cells with AIV non-structural protein NS2,1057 LncRNA were up-regulated and 1129 LncRNA were down-regulated,then GO analysis showed that the differentially expressed LncRNA target genes mainly focused on the regulation of cell process,while KEGG classification analysis showed that AIV non-structural protein NS2 mainly regulated MAPK signaling pathway,the regulation of actin cytoskeleton and tight junctions.In addition,the increased miRNA-674 induced by NS2 inhibits the antigen presentation ability of dendritic cells by targeting TAPBPR mRNA,a gene related to antigen presentation.The results showed that non-structural protein NS2 could affect the function of dendritic cells by affecting the skeleton and proliferation of dendritic cells.3.Molecular Mechanism of Nonstructural Protein NS2 Regulating Antigen Presentation of Dendritic CellsAs a result of previous studies,no miRNA changes were found in DCs stimulated with NS2.Therefore,we speculate that NS2 may modulate the role of miRNA synthesis pathways in the antigen-presenting capacity of DCs.In this study,QRT-PCR was used to detect the expression of Drosha,Exportin5,Ago2 and Dicer in the process of miRNA production.The results showed that NS2 could significantly reduce the expression of Dicer.Subsequently,we showed that the non-structural protein NS2 can interact with Exportin5 by immunoprecipitation experiments.In addition to miRNA synthase,previous studies have shown that stimulation of NS2 significantly reduces IRF3 expression,but does not affect IFN-β secretion.Therefore,we conjecture whether IRF3 can modulate the antigen presentation of DCs.First,we found that IRF3 inhibitors did not affect the maturation of LPS-activated DCs.Subsequently,the antigen-presenting experiment showed that the inhibitor of IRF3 could significantly inhibit the antigen-presenting of dendritic cells,and then inhibit the proliferation of CD4+T cells(p<0.01).Finally,we found that NS2 and IRF3 were co-localized in the cytoplasm of HEK293T cells using confocal experiments.The coimmunoprecipitation experiments showed that NS2 can interact with IRF3.Knockout of M6A methyltransferase Mettl3 can significantly affect T cell activation and DCs’ phenotype.In this study,the changes of m6A-related enzymes in DCs stimulated by NS2 were detected by QRT-PCR.The results showed that NS2 significantly down-regulated the gene expression of methyltransferase Mettl3 and reading protein YTHDF.Subsequently,further studies have shown that NS2 stimulates DCs to inhibit Mettl3 protein expression.Finally,coimmunoprecipitation experiments showed that NS2 may interact with Mettl3. |
