The Role Of PD-1 Pathway In The Process Of BVDV Inhibiting Proliferation Of Bovine Peripheral Blood T Lymphocytes And Inducing Apoptosis And Its Mechanism

Bovine viral diarrhea-mucosal disease(BVD-MD)is an important infectious disease that can cause extensive economic losses in cattle industry worldwide,and also a key infectious disease in cattle farm and international trade quarantin.Acute infection with BVDV results in peripheral blood lymphopenia,apoptosis and immunosuppression.In the process of chronic viral infection,PD-1 signaling pathway can mediate co-inhibitory signals,inhibit the activation and proliferation of T lymphocytes,and induce apoptosis.However,the regulatory effect of PD-1pathway on T cells in acute viral infection is not clear.In particular,it is not clear whether PD-1pathway is involved in the regulation of peripheral blood lymphopenia and apoptosis in the acute infection of BVDV and promotes the formation of immunosuppression.To determine whether the PD-1 pathway is associated with bovine peripheral blood lymphopenia and apoptosis caused by acute BVDV infection.Firstly,peripheral blood mononuclear cells(PBMC)were isolated from neck venous blood of cattle acutely infected with BVDV by using density gradient centrifugation.The peripheral blood lymphocytes(PBL)and CD14+ peripheral blood mononuclear cells(PBM)were sorted by magnetic cell separation.The expression levels of PD-1 and PD-L1 were detected by q RT-PCR and Western blot.The apoptosis and proliferation of PBL were detected respectively by flow cytometry and CCK-8.Secondly,CP BVDV-1 NADL strain and NCP BVDV-1 KD strain were selected to infect healthy bovine PBMC in vitro,and PBL and CD14+ PBM were selected to detect the expression levels of PD-1 and PD-L1,as well as the apoptosis and proliferation of PBL.The results showed that the expression levels of PD-1 m RNA(p < 0.001)and protein(p < 0.01)were significantly up-regulated in PBL from cattle acutely infected with BVDV compared with healthy cattle,the expression levels of PD-L1 m RNA(p < 0.01)and protein(p < 0.01)were significantly up-regulated in CD14+ PBM.Meanwhile the PBL apoptosis was significantly increased(p <0.001)and proliferation was significantly decreased.In addition,both CP and NCP BVDV infection induced significant upregulation of the m RNA and protein expression of PD-1 and PD-L1.The expression levels of PD-1 and PD-L1 m RNA gradually increased with the extension of infection time.The upregulation of PD-1 and PD-L1 was accompanied by a significant increase in PBL apoptosis(p < 0.05)and a significant decrease in proliferation.To determine the effect of blocking PD-1 pathway on PBL apoptosis and proliferation.The CP BVDV-1 NADL strain and NCP BVDV-1 KD strain were selected to infect PBMC from healthy bovine in vitro,respectively.Monoclonal antibody(m Ab)was used to block the PD-1pathway,followed by flow cytometry to detect PBL apoptosis,CCk-8 method to detect PBL proliferation,ELISA to detect cytokine secretion,and q RT-PCR to detect viral load.The results showed that PD-1 blockade could significantly reduce the apoptosis of PBL induced by BVDV infection in vitro,restore the proliferation of PBL(CP BVDV,p < 0.05),reduce the viral load(p< 0.05),and increase the secretion levels of IFN-?(CP BVDV,p < 0.01)and IL-2(p < 0.05).To determine the effect of blocking PD-1 pathway on the apoptosis and proliferation of PBL subsets during BVDV infection in vitro.Healthy bovine PBMC was infected with CP BVDV-1NADL strain and NCP BVDV-1 KD strain,respectively,and PBL subsets such as CD4+ T cells,CD8+ T cells and CD21+ B cells were selected by magnetic cell separation to detect the expression level of PD-1 protein on PBL subsets.The effect of PD-1 blockade on the apoptosis and proliferation of PBL subsets with high expression of PD-1 was detected by blocking test with PD-1 m Ab.The results showed that there was a significant increase in PD-1 expression on CD4+(CP BVDV,p < 0.01;NCP BVDV,p < 0.001)and CD8+(CP BVDV,p < 0.001;NCP BVDV,p < 0.05)T cells,but not on CD21+ B cells after CP and NCP BVDV infection in vitro.In addition,PD-1 blockade can significantly reduce the apoptosis of CD4+ and CD8+ T cells caused by CP and NCP BVDV infection.In CP BVDV infection,PD-1 blockade can significantly restore the proliferation of CD4+ and CD8+ T cells.However,in NCP BVDV infection,PD-1 blockade only significantly restored the proliferation of CD4+ T cells.To determine the molecular mechanism by which PD-1 pathway regulates apoptosis and proliferation of CD4+ and CD8+ T cells during BVDV infection in vitro.The protein abundance and phosphorylation levels of PD-1 downstream signaling molecules regulating apoptosis and proliferation were detected by Western blot after blocking the PD-1 pathway in vitro.In the CP BVDV infected CD4+ and CD8+ T cells,the expression levels of p-PI3 K,p-Akt,p-m TOR and p-ERK were significantly upregulated by PD-1 blockade.Meanwhile,the expression levels of cleaved-caspase 9 and cleaved-caspase 3 were significantly downregulated.Likewise,In the NCP BVDV infected CD4+ and CD8+ T cells,we observed a significant increase in p-PI3 K and p-Akt,and a significant decrease in cleaved-caspase 9 and cleaved-caspase 3 by PD-1 blockade.Remarkably,the expression level of p-m TOR was significantly upregulated in the NCP BVDV infected CD4+ T cells(p < 0.01)and not significantly changed in the NCP BVDV infected CD8+T cells by PD-1 blockade.In addition,PD-1 blockade had no significant effect on p-ERK expression in the NCP BVDV infected CD4+ and CD8+ T cellsIn conclusion,CP BVDV infection can inhibit the activation and proliferation of CD4+ and CD8+ T cells and induct apoptosis by inhibiting the PD-1 downstream PI3K/Akt/m TOR and ERK pathway and activating the caspase 9/caspase 3-mediated apoptotic signal pathway.NCP BVDV infection inhibits the CD4+ T cells proliferation by PI3K/Akt/m TOR pathway inhibited by PD-1,induces the apoptosis of CD4+ and CD8+ T cells by activating caspase 9/caspase 3pathway,and can not inhibit the CD8+ T cells proliferation by PI3K/Akt/m TOR and ERK pathway.We presume that NCP BVDV infection may inhibit the activation and proliferation of CD8+ T cells by negative immune regulatory signals mediated by co-inhibitory molecules other than PD-1 or multiple co-inhibitory molecules.Our findings provide a scientific basis for exploring the molecular mechanism of immunosuppression caused by acute BVDV infection and anti-PD-1 therapy to control BVDV infection.