Study On The Role Of Vimentin In The Activation Of NF-?B Signaling In Enterovirus 71 Infection

HFMD caused predominantly by EV71 can be complicated by neurological manifestations,including ataxia,tremor,myoclonus,polio-like paralysis,encephalomyelitis,cardiopulmonary failure,and death.Unfortunately,there is still very little information concerning EV71 caused severe HFMD.It is important to study the mechanism of severe HFMD caused EV71 infection.Recently vimentin has been reported as an attachment receptor for Enterovirus 71.Nuclear factor-?B(NF-?B)is a ubiquitously expressed transcription factor that plays a key role in regulating the expression of genes involved in a variety of cellular processes,including innate and adaptive immune responses.Besides vimentin as an attachment receptor for Enterovirus 71,it is whether involved in the process of the activation of NF-?B signaling,leading to the production of inflammatory mediators.In this study,we would study the regulatory role of vimentin in NF-?B activation in vitro cell and mouse model with EV71 infection.In this study,The isolated EV71 strain from clinical swab specimens can infected astrocyte cells(U251 cell).To examine EV71 infection whether activate phosphorylation of ERK and the activation of NF-?B signaling,U251 cells infected with EV71 were collected.Western blot results showed that the levels of P65 had increased dramatically in EV71 infected U251 cells when compared to uninfected cells,and the levels of the 1KB ? decreased in U251,and the levels of phospho-ERK1/2 had increased dramatically in EV71 infected U251 cells when compared to uninfected cells.The result indicated that EV71 infection can activate phosphorylation of ERK and the activation of NF-?B signaling.The further study indicated that the activation of NF-?B signaling is dependent on the VP1 of EV71.We have successfully constructed vimentin knockdown U251(VK-U251)cell line by CRISPR/Cas9 technique.The vimentin knockdown U251 cells(VK-U251)infected with EV71 were collected and subjected to western blot.The results showed that the levels of phospho-ERK1/2 and P65 were no change in EV71 infected VK-U251 cells when compared to uninfected VK-U251 cells.Taken together,these studies suggest that phosphorylation of ERK and the activation of NF-?B signaling with EV71 infect U251 cell was depended on vimentin.To determine whether ERK mediated activeation of of NF-?B signaling in U251 cell with EV71 infection,U251 cell were incubated with PD098059(inhibitors of ERK)for 60 min before infection with EV71,Western blot showed that the levels of P65 was no change in U251 cells after pre-incubated with PD098059,So the results indicted that ERK mediated activation of NF-?B signaling in U251 cell with EV71 infection.To investigate the role of vimentin in vivo after EV71 infection,4 day-old A129 mice and vimentin KO mice were infected via intracranial injection route with EV71 clinical strain at a dose of 107 to 108PFU/mouse.We find that higher lethality rate of WT mice compared to vimentin KO mice after intracranial injection,WT mice all died(10/10)at days 3-12 after infection,whereas 60%(6/10)KO mice died.Approximately 40%(4/10)developed hind limb paralysis upon EV71 infection,Only 10%(1/10)KO mice that display little hind limb paralysis upon EV71 infection.Those data showed that the death and paralysis rates for the WT mice model are generally higher than those for the vimentin KO model.The quantitative PCR analysis using primer pairs specific to IL-6?IL-1??TNF??CXCL10?CCL3 cytokines were performed to detect the lever of mRNA in brain tissues.After quantitative PCR,we found expression of IL-6,CXCL10 and IL-1? were upregulated in the brain tissues of WT mice,compared to infected KO mice,while we found that the expression of TNFa and CCL3 have no change in WT and KO mice.The different Organs and tissues were harvested and paraffin embedded,sectioned and stained with Hematoxylin and Eosin(H&E).The brain slides were incubated with the EV71 antibody(biorbyt)for immunohistochemical analysis.The HE result showed that the lesions in intestines and lungs of WT mice were more severe than those of KO mice.The immunohistochemical result indicted that VP1 protein of EV71 virus in WT mice was more widespread distribution than that in vimentin KO mice.These results suggest that EV71 virus pathogenesis was different in WT mice compared to vimentin KO mice.Taken together,we examine EV71 infection can activate NF-?B signaling mediated by vimentin for the first time,the NF-?B signaling may mediate by VP1-vimentin-ERK complex.The vimentin may be involved in the process of the pathogenesis of EV71 infection in mice..The cytokine storm in severe HFMD may cause by activation of NF-?B signaling through vimentin.