Regulatory Of TWEAK Expression On The Function Of NK Cells In Goat During PPRV Infection

Peste des Petits Ruminants virus?PPRV?is an important pathogen that seriously influences the productivity of small ruminants worldwide.PPRV is lymphotropic in nature and induce in the hosts a transient but severe immunosuppression,especially innate immunity.As the important immune cells in the natural immune system,NK cells generally become activity in an early phase of viral infection,and play an essential role in eliminating virus infected cells.Tumor necrosis factor-related weak inducer of apoptosis?TWEAK?is a secreted protein molecule which mainly expressed by leukocytes,including natural killer cells and dendritic cells.However,it remains largely unknown how NK cells respond and are regulated at the earliest time points after an acute viral PPRV infection in goats.In this study,we investigated the effect of PPRV infection on the expression of cytolytic effector molecules and cytokines by NK cells and the role of TWEAK in the regulation of NK cell activity.This study aimed to explore the role for TWEAK on the regulation of goat peripheral NK cells phenotype and immune function during PPRV infection.The main results obtained are as follows:1.Goat PBMCs were infected with PPRV Nigeria 75/1 vaccine virus strain for the indicated times in vitro,the role of multiple immune responses of NK cells and the kinetics of PPRV replication was determined.The flow cytometry analysis showed that the percentages of CD16+/CD14-NK cells in PBMCs significant increased at 24 hpi as compared with mock infected group.In contrast,our data revealed a reduced cytolysis activity and cytokines production of NK cells at early infection.The TCID₅₀0 assay and Western blot analysis showed that persistently increased virus production and replication was detected in a post infection time-dependent manner.Importantly,a significant virus titer was detected as early as 24 hpi in PPRV-infected goat PBMCs compared to mock infected cells,and it revealed a significantly inceased at 72 hpi than 24 hpi.Interestingly,PPRV infection increased the protein level of TWEAK in the cells as compared with mock infected cells2.To investigate the role of TWEAK in the regulation of NK cell activity and virus replication during PPRV infection,goat PBMCs were transfected with siRNA targeting TWEAK gene?si-TWEAK?,TWEAK overexpression Lentivirus,or corresponding controls.As expected,Western blot analysis showed that TWEAK overexpression suppressed NK cells cytotoxicity and cytokines expression during PPRV infection as compared to control group,while opposite effects were observed in cells transfected with siRNAs targeting TWEAK.The TCID₅₀0 assay and Western blot analysis showed that TWEAK overexpression facilitated the replication of PPRV in the goat NK cells,while downregulation of TWEAK inhibited the replication of virus.3.We identified differentially expressed miRNAs that were predicated by TargetScan algorithm and normalization of the Renilla luciferase signal to the firefly luciferase signal,we demonstrated that miR-1 can downregulate TWEAK expression through directly targeting its mRNA,and more importantly,there was an inverse correlation between the expression of miR-1 and TWEAK during PPRV infection in a viral dose-or post infection time-dependent manner.Collectively,the results which clearly showed that cellular miR-1 have negative effects on TWEAK expression and PPRV replication were confirmed by transfection of miR-1 mimic or miR-1 inhibitor and their corresponding control sequences in goat PBMCs.4.Furthermore,to elucidate whether the replication of PPRV is required for the inhibition of miR-1 levels,we further infected PBMCs with either replication-competent or UV-inactivated PPRV and then measured their effects on the miR-1 and TWEAK expression.The results suggested that active PPRV replication is required for miR-1 mediated TWEAK expression in PBMCs.To investigate the effect of PPRV viral proteins on miR-1 and TWEAK expression,goat PBMCs were transfected with PPRV-H,-N,and-V plasmid,and48 h later the cells were infected with PPRV at a MOI of 1.This suggested that non-structural V protein of PPRV plays an important role in miR-1 mediated TWEAK up regulation during PPRV infection.5.To study the expression of TWEAK pathway-related proteins after PPRV infection,TWEAK overexpression lentivirus,si-TWEAK targeting TWEAK gene?si-TWEAK?and corresponding controls were transfected into PBMCs,and 48 h later the cells were infected with PPRV at a MOI of 1.The findings suggested that the expression of TWEAK by PBMCs in response to PPRV infection suppress the immune functions of peripheral NK cells,and the underlying mechanisms may involve repression of STAT3 activation and promotion of MyD88-NF-?B p65 signaling in NK cells.In conclusion,the findings presented here suggest microRNA-1 negatively regulates peripheral NK Cells function via TWEAK signaling pathways during PPRV Infection.This study reveals a previously unrecognized role for TWEAK on the regulation of goat peripheral NK cells cytotoxicity and cytokines expression during acute PPRV infection.