Immune Regulation Mechanism Of Chicken Peripheral Blood Lymphocyte Infected With Reticuloendotheliosis Virus

Reticuloendotheliosis virus(REV)infection is one of the three major infectious diseases in the poultry industry.The REV infection rate of the broiler breeding industry in China has reached 20%-30%,and there is not yet an effective vaccine.At present,although some studies of REV-infected fibroblasts and spleen tissues have been carried out on the molecular level,the role and regulatory mechanism of the main immune cells,lymphocytes,in chickens infected with REV are still unclear.This paper focuses on the immune system of chickens,and an REV infection model was prepared both in vivo and in vitro.A series of studies were carried out on the expression level of differentially expressed genes in lymphocytes after REV infection,the mediation of the signal pathway,and the regulation of the expression of key genes,the purpose of which was to elucidate the immune regulation mechanism and the related molecular regulatory networks of lymphocytes after REV infection in chickens.The results of this study will provide a theoretical basis and development ideas for the prevention,treatment,and breeding of REV infection in chickens,and will ultimately promote the development and improve the economic benefits of the broiler industry.The main results of this paper are as follows:1.Establishment of chicken REV infection modelThis experiment is based on the REV infection model of peripheral blood lymphocytes both in vivo and in vitro.Therefore,the validity of the REV infection model in vivo and in vitro is the key to either the success or failure of this experiment.Two hundred 1-day-old SPF chickens and isolated peripheral blood lymphocytes were used to prepare the REV infection model.One week after REV infection in vivo,or 36 hours after infection of lymphocytes in vitro,qRT-PCR detection showed that there was a higher gene expression level of LTR in the peripheral blood lymphocytes of both the in vivo and in vitro REV models,but there was no LTR in the control group.Morphological changes of the livers and spleens of chickens at 1,2,3,4,and 5 weeks after infection with REV demonstrated that the liver and spleen tissues of the infected group and the control group developed with age.However,from the 2nd week to the 4th week after infection,the livers and spleens of the infected group began to enlarge significantly compared with the control group,and had the pathological features of REV infection.Compared with the control group,the spleen immune index first decreased with the development,and then increased significantly from the second week after inoculation in the REV-infected group.Conversely,the thymus and bursal immune indexes decreased with development;the spleen and bursal immune indexes were significantly lower in the REV infected group than in the control group.The spleen,thymus,and bursal immune indexes exhibited significant differences between the two treatment groups at the 2nd and 3rd weeks(P<0.05 or P<0.01).After 3 weeks of REV infection in chickens or 60 hours of in vitro lymphocyte REV infection,The MTT assay indicated that the number of lymphocytes in the REV-infected group was significantly lower than that in the live chickens or lymphocytes infected with REV.In the uninfected group,the TNFa and IL-8 levels were significantly higher in the two treatment groups than in the corresponding control group(P<0.05 or P<0.01).The levels of IFNy,IL-2,and IL-18 were significantly lower in the two treatment groups than in the corresponding control group(P<0.05 or P<0.01).In conclusion,the experimental results showed that the REV infection models were successfully established both in vivo and in vitro,which also laid a foundation for the progress of this experiment.In addition,the pathological features of REV-infected 1-day-old chickens were the most obvious at the 2nd week.Samples at this time point were selected for subsequent validation tests.2.Analysis of immune regulation mechanism of chicken peripheral blood lymphocytes infected with REVIn order to eliminate interference factors in the in vivo model of REV,this chapter is based on the established model of REV infection in vitro,and peripheral blood lymphocytes were used as the entry point to perform RNA-Seq sequencing.The mechanism of the immune regulation of lymphocytes infected with REV was analyzed systematically from the aspects of gene expression change after REV infection and signal pathway mediation,and the model of REV infection in vivo was simultaneously verified.Using | log2(Fold change)|≥ 2.0 and Padj<0.05 as screening criteria,2977 differentially expressed genes(DEGs),including 996 up-regulated genes and 1981 down-regulated genes,were screened by RNA-Seq sequencing.The cluster analysis based on the 2977 DEGs showed that the REV-infected group was completely separated from the control group,and that the three repeats in the group were clustered together,indicating that the RNA-Seq data had high accuracy.The expression of partial DEGs was verified by qRT-PCR.The results indicated that the results obtained by RNA-Seq and qRT-PCR were consistent.The correlation analysis showed a high positive correlation(r=0.9014,P<0.01),which further supported the reliability of the RNA-Seq data.GO functional enrichment analyses demonstrated that GO terms,such as defense response to bacterium,inflammatory response,and cell proliferation,were significantly enriched.KEGG Pathway analysis showed that MAPK-AP1 immune response-related pathways(Toll-like receptor,NOD-like receptor,and salmonella infection)were included in REV infection.Cell proliferation and apoptosis-related pathways(FOXO and p53)were significantly enriched(P<0.05).130 immune-related DEGs,56 cell cycle-related DEGs,and 55 adipose metabolism-related DEGs were screened.Combined analysis found that the expression levels of the IL-8,IL-18,TLR2A,TLR4,TLR7,TRAF3,TRAF5,and TRAF6 genes enriched in immune-related signaling pathways were significantly down-regulated in lymphocytes after REV infection,while CD40,CD80,NOD1,and MYD88 were found to be up-regulated by the screening of related signaling pathways and related DEGs.It is concluded that these genes are the key genes that regulate the immune response of lymphocytes infected with REV in vitro.The three signaling pathways of the Toll-like receptor,NOD-like receptor,and salmonella infection were simultaneously gathered in the MAPK-AP1 pathway.By changing the expression of immune-related functional genes in the pathway,the expression levels of IL-8 and IL-18 are directly down-regulated.DEGs,CCNB2,CCNB3,CCND1,CCND2,CCND3,CCNG2,CDKN1B,and GADD45A,which are associated with cell proliferation,were enriched in the FOXO signaling pathway,and GADD45A,CCNB2,CCNB3,CCND1,CCND2,CCND3,CCNE2,and CDK6 were enriched in the p53 signaling pathway.In addition,the CCNG2,SIAH1,and WIPI2 genes were enriched in the p53 negative feedback of the p53 pathway.The expression of SMAD3,FOXO1,FOXO3,FOXO4,CCND1,CCND2,CCNE2,CDKN1B,and other genes were down-regulated by qRT-PCR(P<0.05,P<0.01).However,the expression levels of TGFB,TGFBR2,IL10,CCNG1,GADD45A,CCNA2,CCNB2,CCND3,and CDK6 were up-regulated significantly(P<0.05,P<0.01).Therefore,it is inferred that the above genes are the key candidate genes for the regulation of cell proliferation after REV infection in vitro.After REV infection,the peripheral blood lymphocytes of chickens are infected.Down-regulating the expression of cell cycle-related genes by inhibiting the FOXO and p53 signaling pathways ultimately inhibits the proliferation of lymphocytes.In addition,55 DEGs related to fat metabolism were screened.Among them,it was found that the expressions of ACSL3,ACSL6,and DGAT2(a gene related to fatty acid metabolism and fat synthesis)were significantly lower in REV-infected lymphocytes than in control cells(P<0.01).The expressions of the PPARG,FABP3,FABP4,LPL,and PLIN2 genes related to fat decomposition and fatty acid transport were significantly higher in lymphocytes infected with REV than in control cells(P<0.01).It is suggested that the ability of utilizing exogenous fatty acids in chicken lymphocytes infected with REV may be enhanced by ingesting more exogenous fatty acids in order to provide the energy which is needed for the pathogenic process of REV.3.Effect of MAPK-AP1 pathway on chicken immune response to REV infectionCombined with the results of the REV infection model of lymphocytes in vitro and based on the cell level study,the specific inhibitors and activators of the MAPK-AP1 signaling pathway were respectively used to verify the mediated effect of MAPK-AP1 on REV infection both in vivo and in vitro.The key genes involved in the regulation of the MAPK pathway were further identified.Furthermore,the phosphorylated activities of JNK and p38MAPK in normal lymphocytes were inhibited separately.The results indicated that after the JNK and p38MAPK signaling pathways were inhibited,the level of API protein was significantly down-regulated,and the number of lymphocytes was significantly decreased The secretion of IL-8 and IL-18 decreased significantly.On the contrary,after activating the phosphorylated activity of JNK and p38MAPK in lymphocytes infected with REV alone,the level of API protein was obviously up-regulated,and the number of lymphocytes and the secretion of immune factors IL-8 and IL-18 were significantly increased.After both the JNK and p38MAPK signaling pathways were inhibited or activated simultaneously,the number of lymphocytes and the secretion levels of IL-8 and IL-18 were significantly higher than those in the control group.In addition,the number of lymphocytes and the secretion of immune factors(IL-8 and IL-18)decreased significantly in normal peripheral blood lymphocytes after treatment with specific inhibitors of the AP1 protein.This confirmed that JNK and p38 MAPK(a two-branch signaling pathway)were involved in the immunomodulation of peripheral blood lymphocytes after REV infection.The peripheral blood lymphocytes of REV-infected chickens therefore inhibited the JNK/p38MAPK-AP1 signaling pathway.Furthermore,the proliferation of lymphocytes and the secretion of immune factors IL-8 and IL-18 were inhibited.Finally,based on the inhibition or activation of JNK or p38MAPK-AP1 activity alone or in combination in vitro,and the number of cells and the secretion of immune factors IL-8 and IL-18 after inhibiting API activity,it was found that there was a significant positive correlation between them.Combined with the down-regulation of IL-8 and IL-18 expression in peripheral blood lymphocytes after REV infection,it is suggested that the decrease in the secretion of immune factors IL-8 and IL-18 in peripheral blood lymphocytes after REV infection is due to the down-regulation of gene expression in lymphocytes and the decrease in the number of lymphocytes.4.Regulation of LncRNA on the expression of key genes and their pathways in lymphocyte response to REV infectionLncRNA can alter the expression of target genes through epigenetic regulation,transcriptional regulation,and post-transcriptional regulation,and can be used as precursor molecules of small RNAs,such as miRNA,piRNA,and endogenous siRNA.Therefore,in order to further understand the molecular regulation mechanism of immune response to REV in infected chickens,the key regulatory genes and signaling pathways that have been screened in Chapter 3 are studied from the view of LncRNA-regulated genes.134 differentially expressed LncRNAs were detected from the RNA-Seq data(REV-infected group vs.without infection group).In combination with the immune-related signaling pathways screened in Chapter 3,and by analyzing 134 target genes expressing LncRNA differentially,it was determined that two LncRNAs(L11530 and L09863)might regulate the expression of NOD1 and TRAF5 genes in the NOD-like receptor signaling pathway.The results of gene structure analysis showed that these two LncRNA candidates regulated the expression of corresponding target genes by cis-regulation.qRT-PCR analysis demonstrated that the expression level of L1 1530 and its target gene NOD1 in lymphocytes after REV infection in vitro was significantly up-regulated(P<0.05 and P<0.01).However,the expression level of L09863 and its target gene TRAF5 was significantly down-regulated(P<0.05 and P<0.01).Similarly,the same expression changes occurred in the peripheral blood lymphocytes of chickens infected with REV.Considering the important role of the NOD-like receptor signaling pathway in the immune response of chickens infected with REV,these results suggest that LncRNAs L11530 and L09863 may be involved in the immune regulation of chickens infected with REV by targeting the expression of NOD1 and TRAF5 in the peripheral blood lymphocytes.In conclusion,in this study,the molecular regulation mechanism of lymphocyte immune response after REV infection was analyzed in terms of signal pathway mediation and key gene expression regulation.It was found that the peripheral blood lymphocytes decreased cell proliferation by inhibiting the FOXO and p53 signaling pathways after REV infection.At the same time,by inhibiting the MAPK(JNK/p38MAPK)-AP1 signaling pathway,the secretion of immune factors IL-8 and IL-18 was reduced,and the effect of immune regulation was achieved.In this immune regulation process,the down-regulated genes IL8L1,IL18,TLR2A,TLR4,TLR7,TRAF3,TRAF5,and TRAF6,and the up-regulated genes CD40,CD80,NOD1,MYD88,were found to be the key genes of immune regulation in lymphocytes after REV infection.LncRNA participates in the immune regulation of chickens infected with REV by targeting the expression of NODI and TRAF5.In addition,the down-regulated expression genes SMAD3,FOXO1,FOXO3,FOXO4,CCND1,CCND2,CCNE2,and CDKN1B,and the up-regulated expression genes TGFB,TGFBR2,IL10,CCNG1,GADD45A,CCNA2,CCNB2,CCND3,and CDK6,were found to be the key genes for the regulation of the proliferation of lymphocytes.Based on the results,a molecular regulatory network was constructed,and provides new evidence for revealing the potential molecular mechanism of the immune response of chicken lymphocytes to REV.