The Effect And Mechanism Of IL10 In Regulating Bovine Parainfluenza Virus Type 3 Infection

Bovine parainfluenza virus type 3(BPIV3)belongs to the genus of respiratory viruses in the Paramyxoviridae family,which is one of the important pathogens that cause acute respiratory infections in calves.The seropositivity rate of cattle is usually very high in the endemic areas,ranging from 50%to 90%.BPIV3 infects lung epithelial cells and alveolar macrophages,leading to tissue damage and immunosuppression,and subsequent secondary bacterial infections lead to severe bronchial pneumonia.IL10 is one of the few anti-inflammatory cytokines with powerful negative regulatory functions.Epithelial cells,monocytes and macrophages are all target cells for IL10 inhibition.Many viruses rely on multiple ways to induce IL10 to down-regulate the host immune response after infection,and some viruses have evolved to encode IL10 in their own genome.Studies have shown that IL10plays an important role in the immune pathogenesis of HIV,FMDV,LCMV,PCV2,PRRSV and other viruses.The epidemic strain of BPIV3 was first isoloated and its pathogenic characteristics was analyzed in this study,which was confirmed that the epidemic strain of BPIV3can infect epithelial cells and mononuclear macrophages of cattle and mice.Then a mouse respiratory tract infection model and a mouse primary macrophage infection model were established,and the high-throughput transcriptome data of mice and primary macrophages confirmed that BPIV3 may use IL10 and its important downstream product SOCS3(Suppressor of cytokine signaling 3,SOCS3)to down-regulate the host's anti-infection immune response.Subsequently,studies on the biological activity and mechanism of bovine IL10,IL10R1,and SOCS3 were carried out,which was confirmed that bovine IL10 binds to IL10 receptors to transduce signals,and activates the JAK-STAT3 pathway to rapidly and highly express SOCS3on both mouse and bovine cells.Finally,we confirmed that the relevant mechanism of BPIV3 mainly relies on My D88,p38 MAPK and NF-?B signaling pathways to express IL10,negatively regulate the host antiviral response and improve the replication ability of BPIV3.This study laid a theoretical foundation for elucidating the pathogenic role of IL10/STAT3/SOCS3 signal axis in BPIV3 infection and inhibiting the IL10 pathway to prevent bovine respiratory infectious diseases.First,a survey of BPIV3 antibodies was carried out with 1793 sera of calf pneumonia in Heilongjiang Province,Hebei Province and other places in 2017,which showed that nearly 70%of calf respiratory diseases can be partly attributable to the infection of BPIV3 involved.Subsequently,a strain of BPIV3 was isolated from a diseased bovine nasal swab,which can infect MDBK,BHK21,BBEC,BL,BTB,BT,ML,PBMC,BMDM.The pathogenicity of the isolated BPIV3 was analyzed by infecting the lungs of mice through the respiratory tract,which revealed the lungs of the infected mice had pathological changes,and the virus in the lungs could not be completely eliminated within 12 days,and the expression of a variety of inflammatory factors was detected in the infected lungs,and the important regulatory anti-inflammatory factor IL-10 was persistence within 12 days after infection,except for the 5th days,the transcription of other time points were significantly higher than that of the control.Through the analysis of high-throughput transcriptome sequencing,differential expression analysis,gene enrichment and protein interaction network,the change patterns of important host immunity and regulatory molecules were studied in mice and primary macrophages before and after BPIV3 infection.After BPIV3 infects mice and infects macrophages,the expression of IL10 and SOCS3 was significantly increased in the early stage of infection,and SOCS3 was significantly activated in almost all groups.And the key anti-infection immune genes such as Tyk2,STAT1,STAT2,IRF7,ISG15,Mx1,RSAD2,and PML were significantly down-regulated in most groups,IL10 and SOCS3 may participate in the suppression of host antiviral response after BPIV3 infection.Furthermore,the bovine IL10,bovine IL10R1 and SOCS3 genes were cloned and their biological information and evolutionary conservation were analyzed to explore the signal transduction of bovine IL10 and whether it can whether it can exert negative immune regulation.The IL10,IL10R1 extracellular domain and SOCS3were expressed and purified,and the polyclonal antibodies against IL10,IL10R1 and SOCS3 were prepared.Bovine IL10 and IL10R1 extracellular domain were expressed with the adenovirus system,which was confirmed that the recombinant IL10 can rapidly and strongly induce the expression of SOCS3 on bovine and mouse cells,and this effect can be inhibited by IL10R1 extracellular domain.Both IL10 and SOCS3have the function of inhibiting the expression of type I interferon and its downstream molecules.That is,bovine IL10 and SOCS3 can be used in the study of negative immune regulation on cattle and mouse cells,and IL10R1 extracellular domain can be used as a blocker of IL10 pathway.The relevant mechanism of IL10 involved in negative regulation in BPIV3infection was explored,studies have shown that BPIV3 induces the rapidly up-regulation of IL10 m RNA and secretion of IL10 protein at the very early stage of infection on primary macrophages and lung epithelial cells in a time-and dose-dependent manner.And the expression of TNF-?,type I interferon and its downstream protein Mx1 was obviously suppressed.The production of IL10 in primary macrophages is much higher than that of epithelial cells,both exogenous and endogenous IL10 can increase the titer of BPIV3.IL10R1 or inhibitor can reduce the replication ability of BPIV3 by blocking the pathway.The expression of IL10/SOCS3was significantly reduced in BMDM^{My D88-/-}cells and BMDM cells treated with My D88 inhibitor,indicating that pattern receptor recognition and signal transduction that depend on My D88 signal transduction played an important role in inducing IL10during BPIV3 infection.In addition,the application of pathway inhibitors proved that NF-?B and p38 MAPK pathway are important signaling pathways of IL10 induction with BPIV3 infection.Many molecules in the JAK-STAT pathway also have complex regulatory effects with IL10 and BPIV3.In summary,BPIV3 is one of the main pathogens of infectious pneumonia in calves in China,which can significantly up-regulate the expression of IL10 and SOCS3,and cause host immunosuppression after infection.It mainly down-regulates the antiviral immune response with type I interferon and its downstream molecules,this negative regulation mediated by IL10 depends on the My D88,NF-?B and p38MAPK signaling pathways.This study can provide a theoretical basis not only for further elucidating the molecular pathogenesis of BPIV3,but also for the development of therapeutic immune prevention and control targeting the elimination of IL10immune negative regulation.