Heat Stress Weakens Chicken Innate Immune Response By Inducing HSP70 To Inhibit NF?B Entering Nucleus

Heat stress affects the immunity of chickens,leading to tissue damage,thereby reducing production performance and causing serious economic losses to the livestock industry.Heat stress often causes high expression of HSP70 protein.Extracellular HSP70 promotes the immune response of mammalian NK cells and DC cells to antigens from T cells,but intracellular HSP70 inhibits TRAF6,IKK,JNK and NF?B in the innate immune response pathway of macrophages,thereby inhibiting Inflammatory signaling cascade.Because of the significant difference between the immune mechanism of poultry and the immune system of mammals,and there are no reports on how poultry heat stress proteins affect the innate immune response.Therefore,the purpose of this study is to elucidate the molecular mechanism of the effect of HSP70 on innate immunity of poultry under heat stress,and provide a theoretical basis for solving the problem of chicken immunity caused by heat stress during feeding and transportation,which has certain innovation and practical significance.In this study,the effects of heat stress on key organs and tissues of broilers were observed by tissue sectioning and q PCR.The peripheral blood mononuclear macrophages of chickens were used to express HSP70 gene,heat stress test and transcriptome sequencing analysis,to study how HSP70 affects chicken innate immune response under heat stress.The study found:(1)Chronic heat stress can cause damage and inflammation of the brain,heart and leg muscles of the chicken,lymphocytes of the thymus and bursa,and an enhanced immune response.The damage of tissues and organs of recessive white-feathered chickens(WRR),with poor heat tolerance,was more serious than that of Lingshan chickens(LS),local chickens,and the immune response was also more obvious than that of LS.When WRR is subjected to acute heat stress,the heat stress protein gene of spleen,thymus and bursa of Fabricius are rapidly expressed,and the innate immune genes expression of thymus and bursa of the bursa are increased,but the expression of innate immune-related genes in spleen are inhibited,and spleen heat should be suppressed.The heat stress response of the spleen is faster and more obvious than the innate immune response,and it is speculated that the former may have an inhibitory effect on the latter.The expression of HSP70 and HSP25 are higher,and the expression of HSP70 is inhibited by HSP25,but both of them can slow down the apoptosis.They may increase the resistance of cells in stressed tissues by inhibiting apoptosis.(2)At the transcriptional level,overexpression of HSP70 gene inhibits NF?B gene expression,which is related to influenza A pathway and MAPK signaling pathway.The heatstimulated macrophage supernatant culture medium was used to detect the release of HSP70,and it was found that heat stress-induced macrophage HSP70 protein was not released extracellularly to affect the immune response of other normal cells.Therefore,the effect of extracellular HSP70 on cells can be ruled out.Intracellular HSP70 was found to inhibit the expression of NF?B in the nucleus of the innate immune response.And it was found that HSP70 in the thymus and spleen of live SPF chickens also inhibited the expression of NF?B gene.It can be seen that the innate immune response of chicken macrophages is enhanced in the early stage of heat stress,but the innate immune response of macrophages is weakened in the late heat stress.How is this process regulated?(3)Based on the above problems,SPF chicken tissues and macrophages were treated with heat stress for different periods of time,and it was found that the immune responses of thymus,spleen and bursa were increased in the early stage of heat stress(1-2 h),and the innate immune response of macrophages increased,and various HSPs began to express high.In the late stage of heat stress(4-6 h),the immune responses of thymus,spleen and bursa were weakened,and the innate immune response of macrophages was weakened.During the whole heat stress process,the expression of HSP70 in the middle stage of heat stress(3 h)inhibited the expression of NF?B.In the late stage of heat stress,NF?B can still phosphorylate,but may not enter the promoter or enhancer of nuclear binding target gene,which can not enhance the innate immune response in late heat stress.At this time,phosphorylated NF?B may be misfolded due to misfolding.Phosphorylated HSP70 then binds to misfolded proteins to enhance the resistance of macrophages.In addition,whole transcriptome sequencing analysis further indicated that some lnc RNAs are involved in the MAPK signaling pathway by up-regulating NF?B in the early stage of heat stress,or that CTSK and IRF5 genes are up-regulated by multiple s RNA regulation,and circ RNA upregulates dynamin,Rab4,Rab11 and VBS37 genes enhancing the phagocytic ability of macrophages,resulting in the innate immune response that is enhanced in the early stage of heat stress.In the later stages of heat stress,some mi RNAs regulate the innate immunerelated genes TIR,TOLLIP,MAP3K8 and TLR1 LA,which may contribute to the decline of the innate immune response.In summary,heat stress damages chicken tissues and organs,but cells may attenuate inflammatory responses through apoptosis.HSP25 and HSP70 can attenuate apoptosis and enhance the body's resistance.In the early stage of heat stress,the enhancement of innate immune response may be achieved by lnc RNA regulating NF?B involved in MAPK signaling pathway,or CTSK and IRF5 genes are up-regulated by multiple s RNA regulation,or circ RNA up-regulates dynamin,Rab4,Rab11 and VBS37 genes to make macrophages Enhanced phagocytosis.In the middle stage of heat stress,the innate immune response begins to diminish,which is achieved by inhibiting the nucleus of NF?B into the nucleus by the peak of intracellular HSP70 overexpression,in which phosphorylated HSP70 may bind to cytoplasmic heat stress-induced misfolding of phosphorylated NF?B,thereby preventing transcription of inflammation factor genes.In the late stage of heat stress,mi RNA regulates TIR,TOLLIP,MAP3K8,TLR1 LA and other related immune genes to reduce the innate immune response of chicken macrophages.In conclusion,the inhibition of NF?B by HSP70 is an important factor in the attenuation of innate immune response during heat stress affecting the innate immune response in chickens.