The Regulation And Mechanism Of Microglia In The Neurological Injury Induced By Porcine Hemagglutinating Encephalomyelitis Virus

Porcine hemagglutinating encephalomyelitis is an acute and highly contagious disease in pigs,caused by porcine hemagglutinating encephalomyelitis virus(PHEV).PHEV impacts piglets and causes acute death characterised by remarkable neurological symptoms including convulsions,squeals,vomiting,suggesting that PHEV mainly attacks the central nervous system(CNS).However,how CNS innate immunity affects PHEV infection has not been clarified.Microglia,as the specialized immune cell colonized in CNS,is an important component participating CNS innate immunity.In CNS diseases,like infection,degenerative disease,trauma,ischemia,etc.,different stimuli and changes in the CNS microenvironment induce variety microglia-mediated immune responses.How microglia are activated,participate and influence PHEV infection is still not well understood.Therefore,we focus on microglia and study their role and activation mechanism during PHEV infection.Here,PHEV infection model was firstly established,we screened an infection model with stable clinical manifestations and disease progression,concluding that PHEV causes typical viral non-suppurative encephalitis damage,include vascular sheath,neuron degeneration and necrosis,neurophilic change and glial cell nodular.In cellular level,PHEV infection activates microglia and astrocytes,destroys the blood-brain barrier(BBB),and recruits peripheral monocytes in CNS for immune response.In cellular level,PHEV infection activates microglia and astrocytes,destroys the blood-brain barrier(BBB),and recruits peripheral monocytes in CNS for immune response.In molecular level,PHEV infection induces strong CNS innate immune response,characterised by severe pro-inflammatory immune response and significantly upregulated type I interferon signal,but lack of T cell immunity and ability to mediate Th2 immune response.In this study,PHEV animal infection model,in vitro brain slice culture model and neuron-glia co-culture model was used to investigate characteristics of microglial activation during PHEV infection.The temporal and spatial distribution of activated microglia was found closely related to PHEV m RNA replication,and microglial activation could be divided into two status during PHEV infection: moderate activated status and hyper activated status.In PHEV animal infection model and in vitro brain slice culture model,it was found that microglia could be activated and mediated immune response independently of peripheral immunity during PHEV infection,yet the hyper microglial activation at late PHEV infection was closely associated with BBB destruction,peripheral monocyte infiltration,and pro-inflammatory cytokine expression.in in vitro brain slice culture model and neuron-glia co-culture model,Microglial activation was found to be the main effector of early CNS immune response.Microglial activation displays multiple biological roles during CNS immunity and injury process.In this section,the role of microglial activation in PHEV infection and interaction with PHEV replication,BBB destruction and monocyte infiltration was fully studied by Minocycline,PLX3397,Clodronate Liposome treatment in PHEV i.n.and i.g.infection model.Inhibition of microglial activation by Minocycline demonstrated that microglial moderate activation restricts PHEV replication during early PHEV CNS infection,while microglial hyper-activation intensifies immunopathological damage and promotes PHEV replication in late PHEV infection.Microglial depletion by PLX3397 in PHEV infection model,further demonstrated that microglia have strong proliferation and activation ability during PHEV infection and are closely related to peripheral monocyte infiltration.Peripheral monocyte depletion by Clodronate liposome during PHEV infection showed that monocyte infiltration leads to microglial hyper-activation,together with PHEV replication and BBB destruction form a vicious circle trap,which aggravates PHEV infection and immunopathological damage.Microglia express abundant receptors for micro-environmental signal recognition,mediating their activation and responses in physiological or pathological processes.In this study,eight candidate receptor genes,including P2X4 R,IFN?R1,TNF?R2,TLR3,TLR7,RIG ? and MDA5,were screened based on microarray results of early and late infection mice brain cortex,gene abundance and cell expression specificity.The co-culture model of PHEV infected N2 a and Bv2 showed that N2 a supernatant without moderate activated microglia,and P2X4 R was identified as a key receptor signaling molecule for moderate activation of microglia by P2X4 R and IFN?R inhibitor assay.The co-culture model of PHEV infected N2 a and monocyte cell line J774 A.1 showed that J774 A.1 could be infected by PHEV and activated to secrete high levels of pro-inflammatory cytokines and IFN-?.Activated J774 A.1 supernatant stimulated significant microglial actication,which was regarded as microglial hyper-activation.IFN?R,IFN?R and TNF?R were identified as key receptor signaling molecules of microglial hyper-activation by IFN?R,IFN?R and TNF?R inhibitor assay.Finally,PHEV infected N2 a were incubated with moderate microglial activation media and hyper microglial activation midia in vitro.The results showed that moderate microglial activation mediated the protective effect,while hyper microglial activation mediated neural immuno-pathologic injury of PHEV infected N2 a cells.In summary,our finding summarize the characteristics of innate immunity and microglial activation during PHEV infection,and firstly reveal the effect and influence of distinct microglial activated status during PHEV infection,and liminarily studied the microglial activation mechanism: Microglial activation are closely involved in CNS immune and injury processes during PHEV infection.On one hand,microglial moderate activation mediated by P2X4 R signal can inhibit PHEV replication and mediate neuroprotective effects.On the other hand,microglial hyper activation mediated by IFN?R,IFN?R,TNF?R can promote PHEV replication and exacerbate immunopathological damage during PHEV infection.This study not only provide animal model data supporting the study of CNS innate immunity in PHEV infection,but also provide a new perspective thinking about the immune and pathogenic mechanisms of microglia involved in PHEV infection,which is scientifically important.