Mechanism Of Vimentin-mediated Activation Of Inflammasome In The Central Nervous System Infected With EV71

BACKGROUND AND OBJECTIVESEnterovirus 71(EV71)is a neurotropic virus that is one of the major pathogens causing HFMD and viral angina in infants and young children.Severe ill children may also have serious central nervous system diseases,such as aseptic meningitis,encephalitis and poliomyelitis-like paralysis,etc.Some critically ill children,the disease progresses rapidly leading to death.In recent years,more and more researches show that innate immune recognition system plays an important role in the body against viral infection and inflammation,and host cells can induce inflammatory response through specific receptor proteins and activation of cell signaling pathways.NLRP3,the inflammasome,is thought to have a close relationship with the pathogenesis of the CNS.It is a complex of multiple proteins that regulates the activation of caspase-1,The cleavage of pro-IL-1β and pro-IL-18,precursors to pro-IL-18,plays an important role in the functioning of the innate immune system.Vimentin(VIM),which is considered as a receptor of pathogens infected host early binding to pathogens,activates signal factor NF-κB,which is transferred into the nucleus to stimulate cells to produce a large number of inflammatory cytokines.So,in severe EV71 infection,whether VIM activates NF-κB signaling is involved in the regulation of NLRP3 inflammasome is the focus of this study.METHODS1.Isolation of EV71 virus strains from positive pharyngeal swab specimensThe extracted clinical isolate EV71 was selected by the Guangzhou Children’s Hospital Central Laboratory from a 3 years old severe hand,foot and mouth disease patients.We take three positive swab specimens for screening culture.The swab swab preservation solution was filtered through a 0.45 μm filter and 200μl of the preservation solution was used to inoculate a 6-well plate that had been covered with a single layer of human rhabdomyosarcoma(RD).The presence of a characteristic virus-induced cytopathic effect was observed with an inverted microscope everyday,and whole RNA was extracted and then specifically detected with an EV71-specific virus fluorescence kit.2.Detection of vimentin and inflammasome activation in vivo by western blotIn vitro infection of astrocytoma(U251)wild type(WT)and VIM knockout(VIMKD).The two cells were subcultured and expanded to a sufficient number and divided into experimental group and blank control group.The virus was infected with the MOI=5 ratio for 6 h and 12 h.The control group was only added equal volume of PBS.Then extract the whole cell protein.Western blot technique was used to compare the activation of NLRP3 inflammasome and its downstream products in both groups of cells.3.To investigate the role of this signal factor in the activation of inflammasome bodies with NF-κB signaling blockers.U251 cell VIMKD and WT strains were expanded,counted and plated in cell culture dishes.The NF-κB signal blocker Caffeic acid phenethyl ester(CAPE)was added at a concentration of 30 μM per dish,and placed in a cell culture incubator for 30 min.Then EV71 was added in an amount of MOI=5,and the cell-treated group and the untreated group were infected for 12 hours.Western blot was used to detect the activation of NLRP3 inflammasome and its downstream products in both groups of cells.4.Mouse animal model to investigate the regulation of vimentin on inflammatory bodiesThe 3 to 5 days old VIM knockout mice(VIM-/-)and wild type(WT)suckling mice were randomly divided into EV71 infection experimental group and control group.In the experiment group,each suckling mouse was injected intraperitoneally with 10 μL of a virus solution having a concentration of 1×108 half tissue culture infective dose(TCIDso),and the control group was injected with an equal volume of PBS per mouse.The infection status of mice was observed for one week,and the central nervous system injury and inflammatory body activation were detected by Western blot,ELISA,RT-PCR and immunohistochemistry.5.Statistical methodsData analysis and graph production were performed using SPSS 20.0 statistical software and GraphPad Prism 5.0 analysis software,respectively.WB stripe gray analysis was performed using Image J analysis software.The t test was used for comparison between the two groups.One-way ANOVA and measurement data were all mean±standard deviation(Mean±SD),All experiments were repeated three times and averaged.The statistical test level was a=0.05.RESULTS1.Specific identification of EV71After isolated and expanded cultured,EV71 virus strains were infected to the U251 cells as descri-bed above,whole RNAs were extracted and detected by specific virus fluorescence kits.It was found that compared with WT cells,The content of virual RNA in the VIMKD cells was significantly less than the WT group.In the time gradient of EV71-infected U251,the detection amount of EV71 RNA also increased with the increase of action time,demonstrating that EV71 can infect U251 and replicate proliferation.It also suggests that the virus proliferates more rapidly in wild cells.2.Vimentin participates in regulating the activation of inflammasomeIn vitro experiments,Western blot analysis revealed that after EV71 infected VIMKD cell line,compared with WT group,VIMKD group cells had no significant activation of NLRP3 inflammasome and production of caspase-1,while wild type(WT)With the increase of the infection time,the content of NLRP3 and caspase-1 in the group cells also increased(p<0.05).In vivo experiments,the activation of inflammasomes in the CNS of mice was detected by Western blot,ELISA,and RT-PCR techniques.It was found that the levels of NLRP3 inflammasome,IL-1β and Caspase-1 in CSF of wild mice after infection with EV71 were significantly increased,while the contents of NLRP3,IL-1β and Caspase-1 in the CSF of VIM knockout mice were significantly lower(P<0.05);The mRNA copy number of IL-1β and Caspase-1 was also significantly lower than that of wild mice(P<0.05).3.NF-κB signaling pathway is involved in activation of inflammasome in vivoAfter treating the U251 with CAPE,the infection with EV71 was also performed.Compared with common U251 without CAPE,VIMKD group and CAPE treatment group had lower levels of inflammatory body activation(P<0.05),and almost no caspase-1 production.This suggests that the regulatory role of VIM in the inflammatory body is likely to be accomplished through the NF-κB signaling pathway.4.EV71 infection activates the inflammasome and causes damage to the CNS in miceIn this study,immunohistochemical technique was used to detect the brain tissue of mice in the experimental group and the control group.It was found that the degree of brain damage in the knockout mice infected with EV71 was significantly lower than that in the wild type mice(P<0.05).It was shown that EV71 infection differs in the activation of inflammasomes in wild-type and knockout mice.CONCLUSION1.This subject confirms that U251 is a susceptible host for EV71,and EV71 can infect astrocytes and proliferate;2.Both in vitro and in vivo experiments confirmed that EV71 can activate inflammasome;3.Activation of Inflammasomes in infected host cells of U251 may relate to vimentin proteins4.VIM regulates the activation of inflammatory bodies by activating the NF-κB signaling pathway.After the blocker CAPE of NF-κB signaling pathway was acted on by the WT U251 cells,the activation of inflammasomes was significantly reduced compared with untreated WT strains.5.The injury of CNS caused by EV71 in mice may be related to the activation of VIM-regulated inflammatory bodies.The above findings may suggest the possible mechanism of severe central and nervous system injury caused by hand,foot and mouth disease.