| BackgroundViral encephalitis is one of the diseases that pose great threat to human beings.Japanese Encephalitis Virus（JEV）is the leading pathogen causing viral encephalitis in Asia.Globally,60,000 to 70,000 JEV infected patients are reported annually among which 10 to 15%are lethal and 30 to 50%of the survivors suffer from permanent neurological sequelae due to the lack of effective therapeutic treatment.China is the main epidemic area of Japanese Encephalitis（JE）.Even though the incidence of JE has decreased significantly after the immunization with JEV vaccine for children,the local outbreaks and sporadic infections often occur.Because of the high mortality and morbidity,JE is still one of the important public health issues in China.Skin bitten by Culex tritaeniorhynchus carrying JEV is the main route of infection.After that,JEV replicates in the biting site,leading to transient viremia.Finally,JEV invades the central nervous system（CNS）and targets to neurons in which the neuroinflammation was caused by the activation of glia,breakdown of blood brain barrier（BBB）and the infiltration of immune cells leading to neuronal death and injury.However,the detailed molecular mechanisms are still unclear.During the JEV infection,how the pro-inflammatory factors are activated or protective factors are inhibited,and how to reduce neuroinflammation or promote recovery of the nervous system remain to be answered.In our study,we found the expression of RIPK3（Receptor interacting serine/threonine-protein kinase 3）was increased while gas6（growth arrest specific gene 6）was decreased in brains after JEV infection.According to the histopathological examination,massive neuronal necrosis occurred in the JEV infected brains.In our study,it has been demonstrated that phosphorylated MLKL（p MLKL）which is a classical executor of necroptosis mediated the death of JEV infected neurons and deletion of MLKL alleviated JE progression in mouse models.As an upstream molecule of MLKL,RIPK3 played more complicated roles in apoptosis,inflammasome formation and inflammatory factors production besides necroptosis.Meanwhile,RIPK3 also modulated the propagation of virus in the host cells.While,the role of RIPK3 in inflammation and virus proliferation during JEV infection is unclear.Meanwhile,neuronal death and brain injury furtherly broke the CNS microenvironment,leading to exacerbated neuroinflammation.Gas6promotes CNS homeostasis by increasing the neuronal viability,down-regulating activated microglia,increasing remyelination and enhancing the integrity of BBB through binding to TAM receptors.The neuronal death and brain damage induced by JEV infection led to decreased level of gas6 in the CNS.While,there is no reports about the gas6/TAM in JE.Mesenchymal stem cells（MSCs）have been employed in the anti-inflammation and drug transport because of the immunoregulation and targeted migration which is mediated by chemokines/chemokine receptors（CRs）.While,the expression of CRs on MSCs decreases during culture in vitro.CRs modified MSCs show improved homing ability.The expression of CCL2 was increased significantly in the mouse brains during JE.Thus,ccr2 modified MSCs were used as a carrier of gas6 to improve the level of gas6 in CNS.And the ccr2 and gas6 modified MSCs were construct and the biological functions were assessed in this study.Objective1.To identify the effects and mechanisms of increased RIPK3 induced by JEV infection on neuronal viability,neuroinflammation and virus propagation;2.To demonstrate the impact of reduced gas6 due to JEV induced neuronal death and brain damage on the progression of encephalitis as well as the role of the gas6 during JEV infection.3.To assess the feasibility of gas6/ccr2 modified MSCs to improve the expression of gas6 in the CNS.Methods and ResultsHere,we clarified the role of RIPK3 and gas6 in the progression of JE based on the RIPK3 and AXL（one of the gas6 receptors）knockout mice,gene expression profiling,and gene expression regulation technology and et al.And the gas6 and ccr2 modified MSCs were constructed to explore effective treatments for JE.The main methods and results are as follows:1.The role and mechanism of RIPK3 in the JEV infected CNSIn this part,we found that the progression of JE was alleviated in RIPK3-/-mice with increased survival time compared with WT mice.In order to clarify the effect of RIPK3on neuronal survival and microglia activation,the RIPK3 downregulated neuro2a cells and N9 cells were constructed.The results showed that the neuronal death as well as microglia activation and IL-1βproduction was decreased when the expression of RIPK3was downregulated.Meanwhile,JEV proliferation was inhibited in RIPK3-/-brains and neurons.Then,the experiments on RIPK3 complementation/overexpression demonstrated that RIPK3 promoted JEV propagation in neurons.Furtherly,RNA-sequencing of brain tissues showed that the level of the IFN-induced protein 44-like gene（IFI44L）was significantly increased in JEV-infected RIPK3-/-mouse brains,RIPK3-/-neurons and RIPK3-RNAi-neuro2a cells.Then,it was demonstrated that the propagation of JEV was inhibited in IFI44L-overexpressing neuro2a cells and enhanced in IFI44L and RIPK3 double knockdown neuro2a cells.Taken together,our results showed that the increased expression of RIPK3 following JEV infection played complicated roles.On the one hand,RIPK3 participated in neuroinflammation and neuronal death during JEV infection.On the other hand,RIPK3 inhibited the expression of IFI44L to some extent,leading to the propagation of JEV in neurons.2.The role of gas6/AXL signal in the development of JENeurons and neural progenitors are the main source of gas6 in CNS.JEV infection led to neuronal death in the RIPK3-dependent and non-dependent manners resulting in decreased level of gas6 in the CNS.To explore the role of gas6 in JEV infection,AXL-/-,AXL/Mertk-/-(AM-/-)and WT mice were infected with JEV via foot pad injection.The susceptibility and mortality in JEV infected AXL-/-and AM-/-mice was increased significantly.Meanwhile,the neuroinflammation and viral load in AXL-/-mouse brains were higher than that of WT mice.What’s more,we found that the blockage of gas6/AXL impaired the integrity of BBB which promoted invasion of peripheral JEV into CNS according to the level of JEV proliferation in the CNS as well as the peripheral system in AXL-/-,AM-/-and WT mice and the changes of BBB permeability.To verify the role of gas6/AXL in the BBB structure,BBB in vitro model was constructed.it demonstrated that recombinant protein gas6 increased the expression of tight junction proteins in the brain microvascular endothelial cells and the integrity of BBB.Furtherly,to explore the role of gas6 during JE development in mouse models,Recombinant AAV-gas6 virus was constructed and intracerebroventricular injection of AAV-gas6 in WT mice was performed.Then,it was found that mice with gas6 overexpression showed reduced morbidity and mortality after JEV infection accompanying with alleviated neuroinflammation and BBB destruction.The above results indicated that the gas6/AXL signal was helpful to maintain BBB integrity and inhibitneuroinflammation.Exogenous gas6 deliveryto brain might be a strategy to alleviate JE progression.3.Construction of gas6/ccr2 modified MSCs and its impact on JE progressionMSCs are featured with capacity of immunoregulation and directional migration.To deliver gas6 into the CNS effectively and safely,gas6 and ccr2 modified MSCs was constructed through lentivirus mediated infection and identified via trilineage-induced differentiation experiments.Cell proliferation assay and chemotaxis experiment showed that gas6/ccr2-MSCs showed higher proliferation activity and chemotaxis than MSCs in vitro.Furtherly,the small animal in vivo imaging results showed that the recruitment of gas6/ccr2-MSCs into the CNS was increased in the JE models after transplantation via tail vein injection.Meanwhile,the JEV infected mice transplanted with gas6/ccr2-MSCs showed increased survival rate compared with MSCs treated mice.ConclusionJEV infection induced the expression of RIPK3 which played complicated roles in the CNS during JEV infection.On the one hand,the increased RIPK3 in the neurons inhibited the expression of IFI44L leading to JEV evasion from the cellular innate immune.On the other hand,increased RIPK3 mediated neuronal death and neuroinflammation.Massive death of neuronal cells contributed to decreased production of gas6.The impaired gas6/AXL resulted in reduced expression of tight junction protein and BBB breakdown which led to invasion of JEV and inflammatory cells into CNS.Mice supplemented with exogenous gas6 in the CNS showed increased resistance to JEV infection and decreased viral load as well as neuroinflammation.Then,gas6/ccr2-MSCs were constructed to delivery gas6 into CNS.And the JEV models treated with gas6/ccr2-MSCs showed improved survival rate than other groups.Our experiments provided experimental data for further exploration of effective treatments to JE. |
