Newcastle Disease Virus Activates The NLRP3 Inflammasome And Its Role In Virus Infection

Newcastle disease virus(NDV) is a member of the new genus avulavirus within the family Paramyxoviridae. NDV is a single-stranded,non-segmented,negative-sense RNA virus that causes a highly contagious disease in poultry. While NDV is not pathogenic to humans,it has potential utility in cancer therapies. Understanding oncolytic mechanisms in NDV-induced tumour cell death is crucial for further improvement of NDV-based oncolytic virotherapy. It is believed that the antitumor effects of NDV are based on virus-mediated cell lysis and the stimulation of the innate and adaptive immune system. Pathogen-induced cell death is caused by a variety of complex mechanisms.Generally,cell death may have significant consequences in terms of the ensuing response to the dead cell by modulating inflammation or influencing the immune response,cell death is includes dichotomously apoptosis or necrosis, many studies have found that NDV induced apoptosis and autophagy; Apoptosis is mediated by a subset of Caspases,morphology change includes nuclear and cytoplasmic condensation and formation of membrane-bound cellular fragments or apoptotic bodies,apoptosis does not stimulate inflammation.Autophagy degrade cellular components within the dying cell in autophagic vacuoles. Moreover,NDV-induced autophagy promoted viral replication by blocking cancer cells from Caspase-dependent apoptosis. Thus, we hypothesized that other form of cell deaths involved the oncolytic effect of NDV, and the mechanism of activataion of immunity response caused by NDV infection remains unclear, which are the major focus of this study.The innate immune system is the first line of host defence against pathogens,and plays a important role in antitumor effects.The inflammasome is a supramolecular signalling complex that activates the proinflammatory cytokines pro-IL-1β and pro-IL-18,which triggers a form of programmed necrosis known as pyroptosis,IL-1β and IL-18 secretion requires two distinct signalling pathways. The first is pro-IL-1β and pro-IL-18 expression induced by initiation of nuclear-initiated NF-κB signalling pathway through pattern recognition receptors(PRRs),such as Toll-like receptors(TLRs). The second signal is inflammasome-mediated and leads to auto-catalytic cleavage of Caspase-1. Active Caspase-1 converts pro-IL-1β and pro-IL-18 into their active forms; Pyroptosis a Caspase-1 dependent programmed cell death and has a discrete morphology compared to other forms of cell death with the hallmarks of hidden pathogen exposure and induction of pathological inflammation. Whether NDV infection cause pyroptosisf tumor cells by activing inflammasome remain unclear, in this study, NDV and THP-1 cells were used as model to address this question, the present results indicated that:(1)NDV induces IL-1β in PMA-stimulated THP-1 cells,we differentiated human monocytic THP-1 cells into macrophage-like cells using PMA stimulation. The highly virulent NDV Herts/33 strain induced IL-1β secretion from PMA-stimulated THP-1 cells after 12 h of infection and induced the maturation of IL-1β from the precursor form. The extent of IL-1β secretion was dependent on both MOI and time. UV-irradiated NDV failed to induce IL-1β secretion indicating that NDV replication is required for inflammasome activation. In general,pro-IL-1β is cleaved by Caspase-1,and the catalytic activity of Caspase-1 is tightly regulated by inflammasomes. Z-YVAD-FMK is a specific peptide inhibitor of Caspase-1 that inhibited both NDV infection maturation and secretion of IL-1β. Furthermore,western blot analysis demonstrated Caspase-1 was activated during NDV infection. These data indicate that PMA stimulation of THP-1 cells infected with NDV causes Caspase-1-dependent IL-1β secretion.(2) NDV activates the NLRP3 inflammasome,We generated THP-1 cells stably expressing short hairpin RNA(shRNA) against human NLRP3(THP-1-shNLRP3) and confirmed knockdown of NLRP3 by western blots. Previous reports have shown that ATP can activate the NLRP3 inflammasome. We treated THP-1 cells with LPS and ATP and found that IL-1β secretion was reduced in NLRP3 knockdown cells. NLRP3 knockdown cells also failed to support maximal IL-1β secretion after NDV infection. We also constructed THP-1 cells stably expressing shRNA against ASC or pro-Caspase-1 and found that IL-1β secretion after NDV infection was also reduced in these knockdown cells compared with control cells. We also verified the results in LPS-primed bone marrow-derived macrophages(BMDMs) from NLRP3-deficient and wild type mice. These data suggest that IL-1β caused by NDV infection is dependent upon NLRP3,ASC,and Caspase-1.(3) Inflammasome activated by NDV infection result in pyroptosis,The inflammasome can activate a Caspase-1-dependent cell death pathway termed pyroptosis. To compare the cytotoxic effects of NDV in THP-1-shNLRP3,THP-1-shASC,THP-1-shCaspase1 and THP-1-shNC,all cell lines were infected with Herts/33 at an MOI of 1. LDH release was detected at 18 h post infection. Knockdown of NLRP3,ASC,or Caspase-1 reduced the extent of cell death normally induced by NDV infection,as measured using LDH release assays. Treating cells with a Caspase-1,Caspase-3,or pan-Caspase inhibitor also reduced LDH release after NDV infection,Caspase-1 is the effector of pyroptosis,and Caspase-3 activation leads to apoptosis. Furthermore,pan-Caspase inhibitors can irreversibly inhibit Caspase activity,and NDV infection induced cleavage of GSDMD in THP-1and HeLa cell,Thus,inflammasome activation may induce cell death through pyroptosis.(4) Activated inflammasome suppress NDV replication; THP-1 cell infected with 0.01 MOI Herts/33, after 60 h post infection, NDV titres were significantly higher in NLRP3, ASC, and Caspase-1 knockdown cell lines compared with the control cell line. Virus replication may benefit from cellular resistance to pyroptosis. To confirm this result, PMA-stimulated THP-1 cells were treated with Caspase-1,Caspase-3,or pan-Caspase inhibitors. NDV titres were also higher under these conditions. Thus, the NLRP3 inflammasome is important for controlling NDV infection.(5) NDV P and V protein inhibits NLRP3 inflammasome and reduce IL-1β secretion; We established THP-1 cell stably expressing the P,V,W and NP protein of NDV, the cells expressing P and V secreted significantly smaller amounts of IL-1β in response to LPS plus ATP than control cells, forthermore, 293 T cells are deficient in endogenous ASC and NLRP3, so we transiently transfected 293 T with plasmids encoding components of the NLRP3 inflammasome: NLRP3, ASC, proCaspase-1 and pro-IL-1β,we examined the effect of the virus proteins on IL-1β secretion of the cells, the P and V protein inhibited IL-1β secretion. Next, we examined intracellular localizations of the virus proteins and NLRP3 in 293 T cells, NLRP3 was colocalized with P and V protein.In conclusion, we found that NDV infection induces activation of the NLRP3 inflammasome in THP-1 cells, and induces pyroptosis; Knockdown of inflammasome components or chemical inhibition of Caspase-1 activity shows that cell survival was augmented and benefited NDV replication; and the P and V proteins of NDV suppresses NLRP3 inflammasome-mediated IL-1β secretion; This study shows that NLRP3 inflammasome activation is an innate cellular response to NDV infection and offers insights into the oncolytic specificity of NDV.