The Role Of MLKL Mediated Necroptosis In Neuronal Death And The Therapeutic Effect Of MSC On Neuroinflammation During JE In Mouse Models

Japanese encephalitis(JE)caused by Japanese encephalitis virus(JEV)is the most prevalent type of viral encephalitis in Asia which is characterized by acute overwhelming inflammation in the brain with high incidence of disability and fatality.Although the development of JE vaccine has markedly reduced the incidence,more than 68000 cases of JEV infection are reported annually worldwide according to WHO,among which approximately 20-30% are fatal and 30-50% suffer from permanent neuropsychiatric sequelae.Children are more susceptible to JEV,while there is an increasing occurrence in the old population.There are currently no effective methods to eliminate the virus or to cure the neuroinflammation but life-sustaining treatment leading to thousands of people succumbing to the devastating illness and the survivors often suffering from permanent neurological deficits which is devastated to patients and their families.JEV is neuro-invasive and neurons can be damaged by JEV directly and indirectly by inflammation mediated cytotoxicity.Meanwhile,JEV infection provokes microglia leading to increased activation of microglia accompanied with robust and uncontrolled production of pro-inflammatory cytokines as well as the breakdown of the blood brain barriers(BBB)followed by the invasion of inflammatory cells into the central nervous system(CNS).During JE,neuronal death,microglia activation and proinflammatory cytokines form the vicious cycle of inflammation in the CNS leading to progression and deterioration of JE.Thus,exploration of the mechanism of neuronal death as well as the effective treatment to inhibit the excessive neuroinflammation and damage is necessary.It has been demonstrated that apoptosis and autophagy are involved in JE and inhibiting the neuronal death can alleviate the progression.Necroptosis as the special way of necrosis is a newly discovered programmed cell death.Contrast to unregulated accidental cell disintegration,necroptosis was regulated mainly through the axis of TNF/RIPK1(receptor-interacting protein kinase 1)/RIPK3 /MLKL(mixed-lineage kinase domain-like protein).Meanwhile,TNF-? is one of the most important cytokines during JE accompanied with massive neuronal necrosis.Based on this,whether necroptosis is involved in the development of JE is worth exploring.The overactivation of inflammation is the main cause of neuropathology during JE.Inhibiting the inflammation during the progression of JE has been shown to reduce neuronal death and promote neurogenesis.Mesenchymal stem cell(MSC)is characterized with self-renewal capacity and multiple differentiation and has the capacity of immunoregulation and migration into the injury site which has been used as the drug-delivery carriers in many diseases contributing to tissue regeneration and immune homeostasis.It has been reported that transplantation of MSC can up-regulate the expression of brain-derived neurotrophic factor(BDNF)and nerve growth factor(NGF)and improve neurological recovery in many CNS diseases.MSC also controls local inflammation and maintains tissue homeostasis by enhancing the innate and adaptive immune response as well as regulating the activation and function of microglia.In addition,MSC regulates BBB integrity by promoting the expression of vascular endothelial growth factor and angiogenesis.MSC has been reported therapeutically effective in many CNS diseases.The effect of MSC on JE is worth exploring.Meanwhile,growth arrest-specific protein 6(Gas6)participates in the maintainence of homeostasis of CNS.Gas6 is an important immunoregulator which plays a great role in regulating the activation of glia and other immune cells during inflammation.Meanwhile,Gas6 regulates the function of vascular endothelial cells as well as vascular remolding to keep the integrity of BBB.Moreover,Gas6 can improve the function of oligodendrocytes contributing to the myelination and recovery of CNS.In this experiment,the gas6 gene modified MSC was established successfully through lentiviral vector to combine the therapeutic effect of both MSC and Gas6 during JE.The main procedures and results are presented here.1.MLKL mediated necroptosis accelerated JE and death in miceJEV was propagated in the mosquito cell C6/36 and concentrated by extra-high speed centrifuge and titrated by plaque assay as 4×108 PFU/ml.Then 5×107 PFU JEV in 200 ?l PBS was injected into female C57BL/6 mice(4-6 w)intraperitoneally then neuroethology,JEV antigen and the level of TNF-? in the brain were tested to construct and identify the JE models.Propidium Iodide(PI)staining in vivo and electron microscopy were used to identify the necrosis in brains during JE.Immunochemistry combined with immune-electron microscopy of JEV infected mouse brains demonstrated that the expression of MLKL was increased in the cytoplasm and tended to localize along the cellular membrane after JEV infection.Western blot of the total protein in mouse brains showed that the protein level of MLKL and pMLKL was upregulated after JEV infection.And the qRT-PCR showed increased expression of MLKL in mRNA.Meanwhile,the level of MLKL in mRNA was correlated with the severity of JEV infected mice.In vitro,Neuro2 a cells were infected with JEV,and the level of MLKL and pMLKL was detected by western blot.MLKL and pMLKL was increased as the increase of infection MOI and time.Then MLKL-/-mice and WT mice were used to constructed JE models.MLKL-/-mice showed alleviated progression of JE and decreased level of inflammatory cytokines.2.MSC alleviated JEV-induced neuroinflammation and mortality.The mouse bone marrow-derived MSC was isolated by the adhesive screening method and analyzed by flow cytometry and multi-differentiation evaluation.In vivo,8-to 10-week-old mice were infected intraperitoneally with JEV and syngeneic bone marrow MSC was administered through the caudal vein at first and third day post infection(dpi).The mortality,body weight and behavior were monitored daily.Brains from each group were harvested at 6 dpi for hematoxylin–eosin staining,immunohistochemical observation,flow cytometric analysis,TUNEL staining,western-blot,qRT-PCR and BBB permeability assays.MSC treatment reduced JEV-induced mortality and improved the recovery from JE in our mouse models.The histopathological lesion and inflammatory cytokines in MSC treated mice were alleviated compared with JEV infected mice without MSC transplantation.The microglia activation and neuronal loss were significantly decreased in the MSC-treated group.The permeability of BBB was alleviated in MSC treated mice accompanied with increased expression of ZO-1.The viral load(VL)in the brains was also decreased in MSC treated mice.In vitro,coculture and mixed culture experiments of MSC with either microglia or neurons were performed and then the activation state of microglia and survival rate of neurons were tested 48 hours post infection.In coculture experiments,MSC reprogrammed M1-to-M2 switching in microglia and improved neurons survival.Additionally,the VL was decreased in Neuro2 a cells in the presence of MSC accompanied by increased expression of immunoregulatory cytokines TSG-6 as well as TGF-? and interferons including IFN-?/?.3.Construction and identification of gas6-MSCIn this part,lentivirus expressing Fluc was constructed then Fluc gene modified MSC was built.BALB/c mice(female,6-8w)were infected with JEV and then Fluc-MSC was transplantated through vein at 1 and 3 dpi.At 6 dpi,Fluc in the brains was greatly increased after Fluc-MSC transplantation compared with control which showed that MSC migrated into brain when injected through vein in JE models.Meanwhile,gas6 gene was amplified and p Lenti-gas6-GFP-zeocin was constructed,then the gas6-lentivirus was packaged and concentrated.Gas6-MSC was built through infection and purified with zeocin screening.At 1 and 3 dpi,the JEV infected mice were transplanted with MSC,gas6-MSC respectively.The primary therapeutic effect of gas6-MSC on JE was evaluated.Compared with MSC,gas6-MSC treated group showed increased survival rate and alleviated symptoms.In summary,JEV is the main pathogen of viral encephalitis with high disability and fatality.The neuronal death and neuroinflammation are the key pathogenesis of JE.Whereas,the mechanism of neuronal death and the treatment of inflammation in CNS caused by JEV infection are still unclear.In this study,the role of MLKL mediated necroptosis in JE and the effect of MSC on neuroinflammation during JE were researched.This study provided evidence for the participation of necroptosis in the pathogenesis of JEV infection.The deletion of MLKL alleviated the inflammation and progression of JE.Targeting necroptosis will hopefully lead to the development of novel therapeutic strategies for the treatment of JE.MSC treatment alleviated JEV induced inflammation and mortality in mice and at the same time inhibited the JEV proliferation to some extent.Meanwhile,gas6-MSC has greater improvement in the treatment of JE.Our study laid the foundation to the further exploration of the mechanism and treatment of JE.