Study On The Adjuvant Effect And Mechanism Of Recombined Agrocybe Aegerita Lectin(rAAL)

Agrocybe aegerita lectin（AAL）which is extracted from Agrocybe aegerita has antiviral activity,and can promote the proliferation of lymphocyte,increase the antibody level of immunized mice as adjuvant for inactivated virus vaccine.Avian influenza,especially the highly pathogenic avian influenza is still a major threat to the poultry industry and can cause a huge economic burden.Vaccination can be a useful tool for control of avian influenza.In past study we found that recombined AAL by prokaryotic expression（r AAL）has the effects of strengthening immune reaction.In order to determine the adjuvant effect and mechanism of r AAL,the following experiments were carried out.Experiment 1.The adjuvant effect of r AAL on the inactivated avian influenza H9N2 virus.50 healthy BALB/c male mice weighing 20±2g were randomly allotted into 5 groups（n=10/group）,each containing 10 mice: blank group,adjuvant free group,Alum adjuvant-treated group and 2 r AAL adjuvant-treated groups.The last two groups were immunized with inactivated H9N2 virus associated with r AAL（0.5mg r AAL/kg BW and 2.5mg/ r AAL/kg BW respectively）by subcutaneous injection.PBS and Alum were respectively used in the adjuvant free and Alum adjuvant-treated groups.The mice were immunized 4 times with a 7-day interval.Before the last immunization,spleen lymphocytes were separated from 5 mice of each group to analyze the proliferation of lymphocyte and CD4+T/CD8+T ratio.7 days after the last immunization the serum of mice were separated to analyze the level of H9N2-specific Ig G.Spleen lymphocytes from normal mice were incubated in 24 holes cell culture plate by RPMI 1640.The lymphocytes were stimulated with sterile r AAL（final concentration 0,10,25 and 50μg/ml,with 3 replicates per treatment）.After 24 h incubation,the expression level of cytokine gene of lymphocyte was detected by realtime PCR.Results: Compared with adjuvant free group,the H9N2-specific Ig G titer of Alum-treated group was extremely significantly increased（P < 0.001）and that of 2.5mg r AAL /kg BW group significantly increased（P < 0.05）,whereas no significant change was detected in that of 0.5mg r AAL /kg BW group.Compared with blank group,the proliferation of spleen lymphocyte of adjuvant free group,Alum-treated group and r AAL（0.5mg r AAL /kg BW）group was extremely significantly promoted（P < 0.01）,and that of r AAL（2.5mg r AAL /kg BW）group significantly promoted（P < 0.05）.Compared with adjuvant free group,the proliferation of spleen lymphocyte of r AAL（0.5mg r AAL /kg BW）group was significantly promoted（P < 0.05）.Compared with blank group,the percentages of CD4+T and CD8+T in spleen lympthocyte of all adjuvant-treated groups showed an increasing tendency（P > 0.05）and the CD4+T/CD8+T ratio of r AAL（0.5mg r AAL /kg BW）group declined（P > 0.05）.The transcription IFN-γ gene was significantly promoted by r AAL（10μg/ml and 25μg/ml）（P < 0.05）and extremely significantly promoted by r AAL（50μg/ml）（P < 0.01）.The transcription IL-10 gene was extremely significantly promoted by both r AAL（10μg/ml and 25μg/ml）（P < 0.001）and r AAL（50μg/ml）（P < 0.01）,and was dose-dependent.Conclusion: Recombined AAL by prokaryotic expression（r AAL）can promote the secretion of cytokine,regulate the differentiation of lymphocyte and enhance the antigenicity of inactivated H9N2 virus.Experiment 2: The change of spleen transcriptome of mice injected with r AAL.60 healthy mice weighing 20±2g were alloted into 6 groups,each containing 10 mice: control group（0.2ml PBS）,adjuvant free group（0.1ml inactivated virus+0.1ml PBS）,r AAL adjuvant group 1（0.1ml inactivated virus +0.1ml 0.2mg/ml r AAL,1mg /kg BW）,r AAL adjuvant group 2（0.1ml inactivated virus+0.1ml 0.5mg/ml r AAL,2.5mg /kg BW）,only r AAL group 1（0.1ml PBS+0.1ml 0.2mg/ml r AAL,1mg /kg BW）and only r AAL group 2（0.1ml PBS+0.1ml 0.5mg/ml r AAL,2.5mg /kg BW）.The mice were immunized by subcutaneous injection four times at a 7-day interval.3 days after the second immunization,5 mice were killed to extract the spleen m RNA.7 days after the fourth immunization,the HI titer in serum were analyzed.The r AAL adjuvant group with highest HI titer was chosen to analyze the change of transcriptom.Results: The HI titer in serum of adjuvant free group,r AAL（1mg rAAL/kg BW）adjuvant group,and r AAL（2.5mg r AAL/kg BW）adjuvant group were respectively 5.3±0.83,7.2±1.25 and 7.4±0.42.Compared with adjuvant free group,the HI titer was significantly improved by r AAL（1mg r AAL/kg BW）（P < 0.05）and r AAL（2.5mg r AAL/kg BW）（P < 0.01）.Afterwards,rAAL（2.5mg rAAL/kg BW）adjuvant group,rAAL（2.5mg rAAL/kg BW）group,adjuvant free group and control group were chosen to study the change of spleen trancriptome.In the following part,we called adjuvant free group “H9N2” group,and r AAL adjuvant group “H9N2+r AAL” group,and the other two groups were “r AAL” and “control”,respectively.The transcriptome results: We got 48 million clean reads from each of the four groups.86% reads of each group were mapped to mouse genome and the percentages of genes with high coverage in each sample were high,indicating the high quality and reliablity of sequencing results.Gene Ontology（GO）enrichment analysis and pathway enrichment analysis were performed on the different expressed gene（DEGs）between control group and r AAL group（Control-vs-r AAL）and DEGs between H9N2 group and H9N2+r AAL group（H9N2-vs-H9N2+r AAL）.Through GO analysis,we found the DEGs of Control-vs-r AAL were significantly enriched in terms of extracellular region part and secretory granule.In biological process of GO,the DEGs were significantly enriched in 3 biological processes respectively related with immunization,stimulation and stress,and metabolism（P < 0.05）.There are 6 terms directly related with immunization including defense response,immune system process,response to bacterium,response to molecule of bacterial origin,inflammatory response,leukocyte migration,and cell activation.The DEGs of Control-vs-r AAL were extremely significantly enriched in defense response and immune system process（P < 0.0001）.Analysis found that a total of 41 DGEs were enriched in multiple immune processes,only 1（IL-1β）enriched in six immune term at the same time.We analyzed the network of the six terms directly related with immunization,and found that inflammatory response and leukocyte migration were located at the tail end of the network.This indicated that r AAL might have influenced the process of inflammatory response and leukocyte migration.In the pathway enrichment analysis of KEGG of DEGs of Control-vs-rAAL,we found that the DEGs were significantly enriched in 7 pathways,5 of which(Cytokine-cytokine receptor interaction,Hematopoietic cell lineage,Malaria,African trypanosomiasis,Staphylococcus aureus infection were related with immunization.There were 21 DEGs significantly enriched in Cytokine-cytokine receptor interaction.There were 12 DEGs significantly enriched in Hematopoietic cell lineage.Combining the results of GO and Pathway enrichment analysis,we found 14 interesting DEGs including 11 up-regulated genes [IL-1β,Itgam（CR3）,Itgb2 l,MMP-9,PF-4,Ppbp,CXCL5,Cxcr1,Cxcr2,Selp and G-CSFR] and 3 down-regulated genes（Ccl3,Ccl4 and Ccl8）.The 14 genes participated in the primary adherence,lymphocyte activation,secondary adherence and transmembrane migration of leukocyte migration.Interesting,DEGs between H9N2+r AAL-vs-H9N2 and r AAL-vs-H9N2 participated in the primary adherence,lymphocyte activation,secondary adherence and transmembrane migration of leukocyte migration,too.Thus,we deduced that r AAL could improve the immunity by increasing the number of neutrophil leucocyte in circulation,promoting the chemotactic activity and improving the innate immunity.In GO enrichment analysis,DEGs of H9N2+r AAL-vs-H9N2 were significantly enriched in terms related with extracellular region of molecular component group（extracellular region and extracellular region part）.In pathway enrichment analysis,DEGs of H9N2+r AAL-vs-H9N2 were significantly enriched in 8 pathways,1 of which（African trypanosomiasis）was directly related with immunization and 6 of which with metabolism.We analyzed all DEGs of H9N2+r AAL-vs-H9N2 enriched in immunization-related terms and found that 8 DEGs appeared in these terms repeatedly.They were Ido1（15930）,Cfd（11537）,2m（232345）,Adipoq（11450）,Cd209b（69165）,Id1（15901）,Itgb4（192897）and Neil（72774）,and they were all up-regulated.These genes are important in leukocyte migration and T cell differentiation.Conclusion: r AAL as the adjuvant to inactivated avian influenza H9N2 virus can increase the H9N2-specific antibody.This transcriptome study reveals that r AAL can improve the innate immunity by increasing the number of leukocyte,promoting the migration of leukocyte and T cell differentiation.Experiment 3: The change of spleen mi RNA of mice injected with r AAL.The same samples（the mice spleen of 4 treatment groups）in Experiment 2 were use in mi RNA sequencing to explore the effect of r AAL on the expression of mi RNA.The sequencing results were: in control group the percentage of clean reads in total reads was 96.94%,in r AAL group 98.11%,in H9N2 group 98.11%,and in H9N2 +r AAl group 97.47%.22 nt small RNA is the main type of small RNAs in all samples,which was complied with the standard of sequencing.The results indicated that the data of mi RNA sequencing with Solexa method was reliable.There were 73 differently expressed mi RNA between r AAL group and control group（r AAL-vs-control）.Compared with control group,there were 60 significantly up-regulated mi RNA and 13 significantly down-regulated mi RNA in r AAL group.Mi R-124-3p was down-regulated significantly（P < 0.01）,mi R-3473 e up-regulated significantly（P < 0.01）.29978 target genes of differently expressed mi RNA of r AAL-vs-control were predicted by RNAhybrid.GO and pathway enrichment analysis revealed that these target genes were not enriched significantly in any GO term or KEGG pathway,but in the top 20 enriched pathways 7 were directly related with immunization: Bacterial invasion of epithelial cells,Staphylococcus aureus infection,NF-kappa B signaling pathway,Intestinal immune network for Ig A production,Cell adhesion molecules（CAMs）,Epithelial cell signaling in Helicobacter pylori infection and Complement and coagulation cascade.The target genes of the differently expressed mi RNA were widely expressed on the surface of antigen present cell,T cell and cytotoxic T cell.There are 57 differently expressed mi RNA between H9N2+rAAL group and H9N2 group（r AAL-vs-control）.Compared with H9N2 group,there were 19 up-regulated mi RNA and 38 down-regulated mi RNA in r AAL group.Sample analysis revealed that r AAL significantly up-regulated the expression of mi R-3473e（P < 0.01）and down-regulated the expression of mi R-124-3p.298876 target genes of differently expressed mi RNA of H9N2+r AAL-vs-H9N2 were predicted by RNAhybrid.GO and pathway enrichment analysis revealed that these target genes were not enriched significantly in any GO term or KEGG pathway,but in the top 20 enriched pathways 6 were directly related with immunization: Bacterial invasion of epithelial cells,Jak-STAT signaling pathway,Staphylococcus aureus infection,NF-kappa B signaling pathway,Cell adhesion molecules（CAMs）,Epithelial cell signaling in Helicobacter pylori infection and Complement and coagulation cascade.In the molecular adherence process,the target genes of the differently expressed mi RNA the same as those in r AAL-vs-control were widely expressed on the surface of antigen present cell,T cell and cytotoxic T cell.We found that r AAL significantly down-regulated the expression of mi R-124-3p（P < 0.01）and up-regulated the expression of mi R-3473e（P < 0.01）.MiRanda,RNAhybrid and argetscan were used respectively to predict the target genes of mi R-124-3p.1800 target genes were predicted by the three methods simultaneously.GO analysis of these target genes revealed that the target genes were enriched in 22 terms in biological process.The significantly enriched（P < 0.05）12 terms were related with metabolism of amine and lipid and histomorphology.Target genes were also enriched in T cell differentiation and T cell activation with low P-value.The target genes enriched in 3 terms of molecular function were related with the activity of oxydoreductase and transmembrane migration of kumetallic ion.Pathway enrichment analysis showed that the target genes significantly enriched in 4 pathways and directly related with immunization were pulmonary tuberculosis metabolism,influenza A metabolism,RIG-I like receptor signal pathway and salmonella infection signal pathway.Genes appearing simultaneously in the 4 pathways were NM₀01168508,NM₀01168513,NM₀01168514,NM₀11951 and NM₀09045.Searching the gene names of these gene IDs from NCBI,we found that the first four gene IDs were the same gene P38（Mapk 14,mitogen-activated protein kinase 14）and NM₀09045 was P65（Rela,v-rel reticuloendotheliosis viral oncogene homolog A）.These two genes were related in MAPK signal pathway and NF-κB signal pathway.In the transcriptom analysis of r AAL-vs-control,the expression of p38 in r AAL group was significantly up-regulated.Through a comprehensive analysis of transcriptom and the function of target gene of mi R-124-3p,we surmised that r AAL mediated the immune response by activating the MAPK and NF-κB signal pathway.Conclusion: r AAL has an adjuvant function and enhances the antigenicity of inactivated H9N2 virus.The possible mechanism of its adjuvant function is that r AAL stimulates the secretion of IL-1β,down-regulates the expression of mi RNA-124-3p,activates MAPK and NF-κB signal pathway,and promotes the migration of leukocyte and T cell differentiation.