MiRNA Expression Profiles Of Chicken Macrophages Stimulated By BP7 And BP9 And Application In Promoting Immune Response Of Avian Influenza Vaccine

Vaccination has become a key measure to prevent diseases and protect the health of livestock and poultry,which is an important prerequisite for the development of animal husbandry.Except for the protective antigen,the appropriate adjuvants are important for vaccine preparations to promote the effective immune response.Bursa of Fabricius(BF)is a recognized humoral central immune organ,which provides an ideal research model for the study on the functional mechanism of human and mammalian central humoral organs and vaccine development.Clinical application showed that the immune effect of the vaccine containing bursa of Fabricius was much higher than that of the corresponding chicken embryo and cell vaccine,suggesting that there were active molecules involved in promoting the immune response of the vaccine.In recent studies,two kinds of bursal polypeptides bp7 and BP9 were isolated from bursa of Fabricius extract,and they have been proved to have the function of promoting vaccine immune response in mouse immune model.However,the molecular basis of regulating antigen presentation and its clinical application potential of promoting chicken vaccine immune have not been reported.MicroRNAs(miRNAs)play the important roles in tissue development,cell differentiation,proliferation and apoptosis,gene expression,as well as virus replication.However,the mechanism of miRNA on regulation of immune response by immunoactive peptides is not clear.This article first explored the expression of immune active molecules in chicken macrophages stimulated by BP7(GGCDGAA)and BP9(LMTFRNEGT),then analyzes the miRNA expression profile of chicken macrophages stimulated by two peptides with high-throughput sequencing technology,and finally studies the clinical application potential of two active peptides in promoting chicken vaccine immunity.Mainly divided into the following parts:1.BP7 and BP9 stimulated the expression of immune active molecules and microRNA expression profile of chicken macrophagesMacrophages have the function of antigen-presenting cells,which are important objects of cellular and molecular immunology.In order to study the regulatory mechanism of immune active peptide derived from BF on antigen presentation,the mRNA expressions of IL-1β,IL-6,iNOS,IFN-α and IL-10 in chicken macrophage HD11 cells were detected by quantitative PCR.The results showed that the expressions of IL-1β,IL-6,iNOS and IFN-α in HD11 cells stimulated by 100ng/mL bp7 for 48h were significantly higher than that of control;After stimulated by 100 ng/mL BP9 for 24 hours,the cytokines of HD11 cells changed significantly,in which the levels of IL-6 and iNOS were highest.In order to study the mechanism of BP7 and BP9 on chicken macrophages,the miRNA expression profile was analyzed by high-throughput sequencing,and the pathway interaction and gene function analysis of the differentially expressed miRNA were carried out.Pathway interaction analysis showed that the differentially expressed genes involved in many physiological and signal transduction pathways in BP7-treated HD11 cells,including IgA intestinal immune network,fructose and mannose metabolism and endocytosis.Also,BP9 stimulated Notch signaling pathway,herpes simplex infection,RNA transport and gene replication.GO functional analysis showed that BP7 and BP9 mainly affected cytokines,chemokines,T,B cell immune process,Toll like receptor pathway and MHC antigen presentation.The results laid a foundation for the subsequent screening of key microRNAs and the further study of the immunological function mechanism of peptides,and provided experimental basis for the regulation of chicken macrophage function mechanism by bursal active peptide.2.Functional identification of key miRNAs regulated by BP7 and BP9 in chicken macrophagesIn order to find the key microRNAs for the regulation of immune response by bursal active peptide,the miRNA expression profile of chicken macrophages was analyzed.Compared with the control group,BP7 stimulated 58 up-regulated miRNAs and 61 down-regulated miRNAs,while BP9 stimulated 9 up-regulated miRNAs and 20 down-regulated miRNAs.In addition,we randomly selected up and down-regulated miRNAs for quantitative PCR verification,and analyzed the GO and KEGG signal pathways of miRNAs to screen the key miRNAs that caused polypeptides to play a role.BP7 regulated gga-mir-132a-5p to participate in lysosome and ribosome synthesis process.and regulated gga-mir-193a-5p to mainly participate in cell macromolecule generation.BP9 induced gga-mir-210a-3p mainly involved in the adrenal function of cardiomyocytes,and regulated gga-mir-218-5p involved in the metabolic pathway.This study laid a molecular foundation for further study on the functional mechanism of polypeptide regulating antigen presentation and immune response.3.BP7 and BP9 promoted antibody responses of chicken avian influenza vaccineClinical animal immunity is an ideal model to verify and develop the clinical application of vaccines and vaccine adjuvants,which can better reflect the real situation of clinical animals.In order to further study the clinical application potential of bursal-derived peptides as adjuvant,we used chicken as the animal model to be immunized with AIV antigen and BP7 or BP9 respectively,and collected serum to detect antibody level and cytokine level,and finally isolated spleen cells for lymphocyte proliferation.The results showed that compared with that of the antigen alone control group,the antibody levels of the BP7 combined antigen immunization group was increased.The levels of cytokines IL-4 and IFN-y of BP7 immunized groups were higher than the antigen control group,and the lymphocyte viability was higher than that of the antigen control group.The HI antibody levels of chickens immunized with three doses of BP9 and AIV vaccine groups were significantly higher than the vaccine control,in which HI antibody levels of chickens immunized with AIV vaccine combined with 0.05 mg/mL BP9 were the highest in all experimental groups.The cell viability was significantly higher than the vaccine control group.These results show that BP7 and BP9 could promote the immune response of immunized chickens,which showed that these two peptides could be used as potential new vaccine adjuvants for the clinical application of avian influenza vaccine,which provided an important basis for the development of new vaccine adjuvants and vaccine improvement,and provided the strong technical support for the prevention and control of avian influenza epidemic.