Effects Of DUSP1 And HSPA5 On Chicken Antigen Presentation Pathway

High temperature is one of the most important external stressors,which mainly affects the immunity and production performance of chicken.Under high temperature conditions in the summer,chickenswill reduce immunity and disease resistance,reduce food-intake,slow down the growth and development,endocrine dysfunction and increase mortality,whichseriously impact on chickenintensive production and causeeconomic losses.Therefore,thefinding of chickens with strong disease resistance under heat stress has become a problem for chicken breeders to solve.In this study,SPF chickens were used as experimental materials,the not immune and notheat stress groups,the immune and not heat stress groups,the immune and heat stress groups were set.The spleen and bursa of fabricius were collected from each group for RNA-seq.Through analyzing,we find the key genes that influence the antigen presentation in chickens under heat stress and their functions were verified at the cellular level.The main results are as follows:1.The level of antibodyafter immunization in chicken was significantly higher than that of non-immune(P <0.01).The high temperature will make the chicken show obvious shortness of breath,H/L values and the cortisone levelwere significantly increased(P <0.001),and CD4+/CD8+ were significantly decreased(P <0.001),indicating that chicken immune and heat treatment success,and heat stress will decrease chicken immunity.2.The analysis of GO enrichment showed that there were 6differential expression genes(DEGs)(FGG,IGSF11,ALDOB,etc.)related to immune after immunizationin spleen;there were 15DEGs(GZMA,DUSP1,HSPA5,etc.)associated with immune and heat stress proteins after heat stressin spleen.KEGG pathway analysis showed that the DEGs were significantly enriched in the complement and coagulation cascade pathways after immunizationinspleen,and the DEGs were significantly enriched inthe chemokine signaling pathway and complement and coagulation cascade pathways after heat stress.These results show that the immunoreactivity and heat stress-related DEGs change inversely in the spleen after immunization and heat stress,indicating that chicken immune and heat stress in the opposite effect.The expression of DUSP1 and HSPA5 in heat stress was significantly lower than that in non-stress group.Two genes are associated with immunity,suggesting that the differential expression of DUSP1 and HSPA5 may be related to post-heat-stress immunity.3.The analysis of GO enrichment showed that there was1DEG(CD69)related to immune after immunization in bursa of Fabricius;there were 3 DEGs(COL21A1,CD69 and HSP90AB1)associated with immune and heat stress proteins after heat stress in bursa of Fabricius.KEGG pathway analysis showed that the DEGswere not enriched in any pathways after immunizationin bursa of Fabricius,and the DEGs were significantly enriched insynaptic vesicle cycle and protein digestion and absorption pathways after heat stress.There were few genes associated with immune response and heat stress in bursa of Fabricius after immunization and heat stress,indicating that the bursa of Fabricius is not the main place to deal with the vaccine and heat stress is not sensitive.4.HD11 can respond to LPS and cause changes in the expression of genes associated with antigen presentation.The expression of IL1 B,IL6,CD1 C,CD80,CD86,TLR4,MHC-I and MHC-II in LPS-stimulated group was significantly higher than that in control group,indicating that HD11 has an antigen presenting function.The expression of MHC-II antibody on LPS-stimulated HD11 was significantly higher than that of HD11,indicating that LPS can present antigens through the MHC-II pathway.5.Chicken overexpression vector DUSP1 was transfected into HD11,the mRNA and protein expression level of DUSP1 was increased significantly and the high expression level of DUSP1 did not cause HD11 immunoreaction.After overexpression of DUSP1,the ability of HD11 to recognize LPS was enhanced,and the efficiency of HD11 to LPS was improved and the duration of LPS was reduced.Interference fragment of chicken DUSP1 was transfected into HD11,and the mRNA and protein expression level of DUSP1 were significantly decreased.Interference with DUSP1 inhibited the ability of HD11 to present LPS.6.Chicken overexpression vector HSPA5 was transfected into HD11,and the mRNA and protein expression level of HSPA5 was increased significantly and the high expression level of HSPA5 did not cause HD11 immunoreaction.After overexpression of HSPA5,the ability of HD11 to recognize LPS was enhanced,and the efficiency of HD11 to LPS was improved and the duration of LPS was reduced.Interference fragment of chicken HSPA5 was transfected into HD11,and the mRNA and protein expression level of HSPA5 were significantly decreased.Interference with HSPA5 inhibited the ability of HD11 to present LPS.In summary,the low expression of DUSP1 and HSPA5 inhibited the antigen presentation,which may be the two key genes that cause chicken immunity to decline.