Newcastle Disease Virus Regulates The Expression Of IL-12 In Dendritic Cells Thereby Inhibiting Antigen Presentation

Newcastle disease(ND)is a highly contagious disease transmitted via the respiratory tract and spread of ND results in great economic losses to the poultry industry worldwide.The causative agent of ND is Newcastle disease virus(NDV),a member of the family Paramyxoviridae,genus Avulavirus.It has been reported that NDV suppresses the innate immune responses through several mechanisms like other Paramyxoviruses.Generally,Paramyxoviruses can inhibit the activation and proliferation of T cells induced by antigen presentation of dendritic cells(DCs).However,the full impact of NDV on DCs,particularly on induction of adaptive immunity,has not been extensively evaluated.DCs are professional antigen presenting cells(APCs)and function as messengers between innate and adaptive responses(APC to T cells).It has been shown that NDV vectored vaccines were efficient to induce high levels of humoral and cellular immune responses.Although,infection with NDV triggers a strong innate immune response from DCs,it causes high proportions of apoptotic cells which suppresses antigen presentation.Therefore,exploring the role of NDV infected DCs in regulating the nature of innate-adaptive crosstalk would provide better understandings of the immune evasion of NDV and improvement of NDV vectored vaccines.This study took advantages of NDV lentogenic LaSota strain and DCs of mice to explore the antigen presentation and pathogenic mechanisms of NDV in vivo and in vitro.The main research contents are as follows:1.DCs were matured when infected with NDV:In order to explore whether DCs can be activated by LaSota,recombinant cytokines IL-4 and GM-CSF were used to simulate the mouse bone marrow monocytes into DCs in vitro.Then,cells were infected with r NDV expressing EGFP(r NDV-EGFP)and the results showed that NDV could infect DCs and T cells.Then the detection of surface molecular markers revealed a variety of surface molecules of DCs upregulated during infection,including MHC-I,MHC-II,CD40 and costimulatory molecules CD80 and CD86.Moreover,infection induced the secretion of pro-inflammatory cytokines TNF-?,IFN-?,IFN-?and IFN-?,but not IL-12p70.FITC-Dextran capture assay showed that antigen uptake capability of DCs was markedly reduced upon infection.At the same time,cell migration assay indicated a intact migratory capacity of DCs when infected with NDV.These results suggested that DCs could be activated by NDV,showing phenotypic maturation of DCs.2.Infection of NDV inhibited antigen presentation of DCs and induced Th2 type immune responses in vivo and in vitro:To clarify how NDV influences antigen presentation,CCK-8 was added to test the proliferation of T cells in co-cultures,and the results showed that NDV treated DCs were weaker in priming T cells than PBS treated DCs.Moreover,co-cultured with NDV-infected DCs resulted in a significant increase of Th2 responses.Release of Th2 immunosuppressive cytokine IL-10increased in the supernatant of infected DCs and co-cultures,indicating that infection of NDV inhibited the antigen presentation of DCs and induced CD4~+Th0 cells differentiated into Th2 cells.For in vivo assay,r NDV r L-Luc expressing firefly luciferase gene was rescued.After identification,the recombinant virus was injected intramuscularly or intraperitoneally into mice,and in vivo imaging was performed at indicated times.The results showed that the fluorescence was the most obvious at 12 h after infection,and then depleted,finally it was weak at 72 hpi.Then,mice were injected with NDV intramuscularly and spleens were separated at indicated times.Analysis of splenic populations showed that CD11c~+MHC-II~+DCs were depleted to a nadir early after infection,increased and reached a peak at 12 h,but stabilized thereafter at 72 h.Compared to this marked diminution of T cells,the proportion of B cells(CD19~+)in spleens rose strikingly as well as a predominant activation of Th2 cells(CD3~+CD4~+IL-4~+T cells).This section showed that NDV inhibited antigen presentation of DCs and induced B cells biased proliferation and predominant Th2 differentiation.3.Virus infection inhibits the secretion of IL-12p70 of DCs:Since we confirmed that NDV lentogenic strain suppressed proliferation of T cells,we next explored the immunosuppressive mechanisms.DCs were pretreated with NDV for 12 h and activated by LPS.Infection resulted in downregulation of transcriptional and protein levels of IL-12p40 thereby inhibiting secretion of IL-12p70.Western blot and p-p65 ELISA analysis of key proteins in the p38 MAPK pathway(IL-12 productive pathway)showed that NDV infection did not affect the total amount of key proteins in the pathway,but reduced the phosphorylation levels of p38 and p65 in cytoplasm and nucleus.Accordingly,IL-12p70 downstream cytokines IFN-?,TNF-?and IL-6 decreased in co-cultures.Therefore,NDV inhibits the production of IL-12p40 by inhibiting the phosphorylation of the p38 and p65 to inhibit secretion of IL-12p70,thereby suppressing the proliferation of T cells and the secretion of cytokines.4.NDV enhances viral infection through inhibiting the secretion of DCs IL-12p70:In order to explore the effects of inhibition of IL-12p70 on viral replication,we first infected DCs with r NDV-EGFP and then co-cultured to prove that NDV could infect T cells through DCs.Next,T cells were pretreated with NDV,and then stimulated with PMA.After 48 h,we found NDV infection inhibited the production of IFN-?and TNF-?stimulated by PMA.We next co-cultured different combinations of infected DCs and T cells which showed the decrease of IL-12p70 was the main reason for the inhibition of T cell proliferation and cytokines secretion in co-cultures.Then,DCs and T cells were pretreated with exogenous cytokines,and then infected with r NDV-EGFP.Several suppressed cytokines could effectively inhibit the infection and transmission of NDV from DCs to T cells.Finally,DCs were pretreated with different NDVs and the detection showed that various NDVs could inhibit the expression of IL-12p,IFN-?,TNF-?,IL-6as well as antigen presentation.In conclusion,NDV inhibits the secretion of IL-12,IFN-?,TNF-?and IL-6 to enhance viral infection.To summarize,this study proved that NDV lentogenic strain LaSota can activate DCs,but suppress the expression of IL-12 via inhibiting the phosphorylation of p-p65.Thus,antigen presentation of DCs was dampened and Th2-type immune responses were induced in vivo and in vitro by NDV to enhance the efficiency of infection.Moreover,this inhibition of IL-12 is a common mechanism of NDV to suppress immune responses.