Hemagglutinin-neuramidinase Protein Of Newcastle Disease Virus Upregulates Expression Of Trail Gene In Murine Natural Killer Cells Via NKp46 Pathway

The avian paramyxovirus Newcastle disease virus?NDV?possesses antitumor activity and is relatively safe for the host,promising an ideal candidate for tumor biotherapy.The antitumor mechanisms of NDV include apoptosis and autophagy in tumor cells and its ability to stimulate immunoreactive cells.Natural killer?NK?cells are the major cpmponents of innate immunity and the first line of defense against invading microbes and tumor cells.NK cells are required for the maintenance and renewal of tumor-specific T cell immunity.It was found that exposure of NK cells to NDV resulted in enhanced tumoricidal activity.That was mediated by up-regulated expression of the TNF-related apoptosis-inducing ligand gene?TRAIL?.TRAIL,also known as apoptosis-inducing ligand 2?Apo2L?,is recently identified as a critical effector of immune cells for tumoricidal activity.TRAIL-mediated apoptosis is triggered via the recognition of the proapoptotic membrane death receptor?DR?4/DR5,which are only expressed on tumor cells.Therefore the studies on TRAIL regulations are of great importance.NDV is able to induce expression of TRAIL in tumor microenvironment.However,the mechanisms that lead to this activity are unclear.We previously showed that exposure of NK cells to NDV resulted in enhanced tumoricidial activity that was mediated by upregulated expression of the TRAIL gene,via an interferon gamma?IFN-??-dependent pathway.Other pathways involved in the upregulated expression of TRAIL are yet to be identified.In the current study,the mechanisms and signal events were investigated based on IFN-?-independent manner in the NDV-stimulated NK cells.We here propose the direct recognition could mediate TRAIL upregulaion in an IFN-?-independent manner after NDV stimulation.NK cells cytotoxicity is mediated by the recognition of killer activatory receptors?KARs?and their responsible ligands expressed on the surface of tumor cells and virus-infected cells.NKp46isidentifiedasonememberofKARs.The hemagglutinin-neuraminidase?HN?of NDV is able to mediate the binding of the viral particle to host cells.It was found that HN expressed on the surface of NDV-infected tumor cells acts as a ligand for NKp46 receptor on human NK cells,directly activating NK cells and contributing to the antitumor effects of NDV mediated by upregulation of tumor necrosis factor??TNF-??.In the current study,the mechanisms and signal events were investigated based on IFN-?-independent manner in the NDV-stimulated NK cells.Our results provide evident for tumor immunotherapy based on NDV-NK stimulation.Objctive:To explore the recognition of HN and NKp46 mediate TRAIL upregulaion in an IFN-?-independent manner and the intracellular signaling cascades involved.Methods:1.Mice in which the IFN-?receptor gene was inactivated(IFN R^-/-)functionally were used in this study.NK cells from IFN R^+/+and IFN R^-/-mice were enriched by negative selection using immunomagnetic beads.The effects of NDV or IFN-?stimulation on the expression of membrane-type TRAIL?mTRAIL?and secretory TRAIL?sTRAIL?in NK cells were investigated using Western blotting and enzyme-linked immunosorbent assay?ELISA?,respectively.These effects on the proportion of TRAIL⁺NK cells were observed using flow cytometry?FCM?.2.The effects of recombinant HN and F protein stimulation on the expression of membrane-type TRAIL?mTRAIL?and secretory TRAIL?sTRAIL?in NK cells were investigated using Western blotting and enzyme-linked immunosorbent assay?ELISA?,respectively.These effects on the proportion of TRAIL⁺NK cells were observed using FCM.The possibility of direct binding of HN to NK cells were investigated using an ELISA after that coating of ELISA plates with NK cells.The abrogation effect of NDV or HN on binding ability of the NKp46 mAb to NK cells were investigated using both competition binding assays and competition binding inhibition assays.The blockade of recognition of NKp46 by HN was investigated using a mAb against HN or neuraminidase for desialylation.The effect of antibodies against HN and desialylation on IFN-?secretion was assessed by ELISA.3.The levels of phosphorylated Syk?pSyk?and phosphorylated I?B??pI?B??were assessed using Western blotting.The blockade of HN-NKp46 was assessed using Western blotting when NDV was treated with a neutralizing mAb against HN,while NK cells were treated with neuraminidase for desialylation.Blocking experiments using various inhibitors and the RNA interference method were conducted to investigate whether HN-induced upregulation of TRAIL was dependent on Syk and NF-?B activity.Results:1.The proportion of NK-enriched cells was>92%as determined by FCM using the CD49?DX5?-FITC mAb.In IFN-?receptor normal NK cells,TRAIL induction as well as a marked increase in the number of TRAIL⁺NK cells were observed after treatment by IFN-?or NDV.In IFN-?receptor deficient NK cells,TRAIL induction was observed after treatment by NDV.IFN-?had no effect.These findings were confirmed by in-vivo experiments using intraperitoneal injection with NDV.No difference in the levels of IFN-?secreted by IFN R^+/+and IFN R^-/-NK cells following stimulation with NDV in vitro.However,the lower level of systemic IFN-?production was observed in IFN R^-/-mice in comparison with that in IFN R^+/+mice.2.An increased numbers of TRAIL⁺NK cells were observed when NDV or HN was used as stimuli.When F protein was used to stimulate NK cells,the numbers of TRAIL⁺NK cells was unchanged.Western blot and ELISA ananlysis confirmed these findings.We speculate that HN may stimulate TRAIL production possibly via NKp46.To investigate the possibility of direct binding of HN to NK cells,we performed an ELISA after that coating of ELISA plates with NK cells.HN or NDV bound to NK cells-coated ELISA plates.This binding could be efficiently blocked by HN mAbs.Consistent with the recognition of sialic acid moieties by the viral HN,the binding of NDV was lost after desialylation.The binding ability of NKp46 mAbs was abrogated when NK cells were pre-incubated with NDV or HN.F protein has no effect.NKp46 was necessary for does-dependent HN binding to NK cells in vitro on the early phase.When NK cells were pre-incubated with a mixture of antibodies against HN and NDV,the binding ability of NKp46 mAbs was partially abrogated.The upregulated TRAIL expression was then efficiently blocked when NDV was treated with a mAb against HN,or mice NK cells were also treated with neuraminidase for desialylation.However,the portion of the HN protein that participates in the molecular interactin between NDV and NK cells remains unknown.A marked decrease level of IFN-?was observed with the effect of antibodies against HN and desialylation.3.The increase in the levels of pSyk and pI?B?was observed after treatment with HN.We observed an increase in pSyk level 30 min after treatment with HN,peaking after 60 min,and then returning to the basal level.There were no differences in Syk levels of IFN-R^-/-NK cells at various time points after stimulating with HN.We found that pI?B?began to increase 30 min after treatment with HN,peaking after 60 min,and then slowly declining in 90 min.To confirm Syk and nuclear factor kappa B?NF-?B?activation by KARs,NDV was treated with a neutralizing mAb against HN,while NK cells were treated with neuraminidase for desialylation.We found that the levels of pSyk and pI?B?were clearly reduced following these treatments,compared with those in controls.IFN R^-/-NK cells were pre-incubated with Syk inhibitor herbimycin A or NF-?B inhibitor PDTC following stimulation with HN.These two inhibitors partly blocked HN-induced TRAIL expression.Silencing of Syk or P65 in IFN R^-/-NK cells partly impair HN-induced TRAIL expression.A clear decrease in phosphorylation levels of I?B?was observed for HN-stimulated NK cells pretreated with Syk inhibitor herbimycin A and silencing of Syk compared with those in controls.Conclusions:We have provided evidence that NDV HN triggers the upregulation of TRAIL in murine NK cells via an IFN-?-independent pathway.Base on this finding,some preliminary discussions about the downstream intracellular signaling cascades involving TRAIL expression were further made.Both Syk and NF-?B pathways play potential roles in the regulation of TRAIL expression in HN-stimulated NK cells.These findings indicate that the interaction between HN and NKp46 induces the upregulation of TRAIL via the modulation of Syk and NF-?B activity.Our results highlight the immunostimulatory capabilities of NDV upon oncolytic activity and innate immune response mediated by NK cells.Our results provide insights into tumor immunotherapy based on NDV-NK stimulation.