The Mechanism Of Blood-brain Barrier Breakdown During Japanese Encephalitis Virus Infection

Japanese encephalitis(JE)is caused by Japanese encephalitis virus(JEV)which is a mosquito-borne zoonosis.The main features of JE is the extensive neuroinflammation in the central nervous system(CNS),accompanied with blood-brain barrier(BBB)disruption.Although JEV infection leads to an acute encephalitis and BBB breakdown,the specific mechanisms remain unclear.In this study,JEV infected mouse model was used to investigate the mechanism of BBB disruption.As shown in the results,just a small number of residual viruses were detected at day 1-2 post JEV infection and disappeared subsequently,indicating that the virus was cleared soon in the periphery;but in the CNS,viruses could be detected from day 2,which increased dramatically at day 4 and reached its peak at day 5.The NaF uptake was significantly elevated at day 4 post JEV infection.These results indicate that JEV could not replicate massively in periphery,but some virus invaded into CNS via a certain way without BBB disruption.Once sneaking into the CNS,the virus started to multiply and replicate,leading to BBB breakdown and the death of mice.In the CNS,IP-10,IFN-?,IL-6 and CCL4 were remarkably upregulated at day 1 post JEV infection,but CCL2 and CCL3 significantly upregulated at day 3-4.What's attractive was that the expression of CCL5 increased about 15-20-fold at day 3 post JEV infection and TNF-? level markedly elevated at day 4 post JEV infection.Using the monolayer of bEnd.3 endothelial cell as an in vitro BBB model,the integrity of BBB was damaged by the brain supernatant from mouse brain homogenates,even viruses were inactivated with UV,demonstrating it is the inflammatory factors rather than JEV per se that augments BBB permeability.We firstly performed IFN-? neutralization assay to investigate the function of IFN-? on BBB disruption in JEV-infected mouse model.As shown in results,neutralizing IFN-? obviously ameliorated the downregulation of tight junction proteins during JEV infection,indicating that IFN-? played a significant role on BBB damage.Further study showed that neutralization of IFN-? ameliorated the down-regulation of TJ proteins to maintain the integrity of BBB.Given the high expression of IP-10 at day 1 post JEV infection and subsequent increased,the expression was the earliest and the change was the most significant in the CNS.After IP-10 neutralization assay,we found neutralizing IP-10 could also ameliorate the down-regulation of tight junction(TJ)proteins and the disruption of BBB.To verify the source of IP-10,we detected the expression of IP-10 on CNS.We found IP-10 primarily expressed on astrocytes,with a little expression on neuron and microglia.IP-10 had no effect on BBB permeability in mice brain microvascular endothelial cell line bEnd.3,indicating that IP-10 might regulate the expression of other inflammatory factors and then damage the integrity of BBB.High level of TNF-? on astrocytes supernatant was detected after IP-10 and/or JEV treatment.Once neutralization of IP-10 with neutralizing antibody,TNF-? expression was remarkably down-regulated.These results indicated that IP-10 acted on astrocytes via autocrine or paracrine manner and stimulated TNF-? production.TNF-? regulatd TJ proteins expression and translocation,leading to the augment of transendothelial permeability.JEV infection also up-regulated the IP-10 receptor CXCR3 on primary astrocytes.Once binding to its receptor CXCR3,IP-10 mainly activated and phosphorylated c-Jun N-terminal kinase(JNK)and downstream immediate early transcription factor c-Jun,regulating TNF-? expression.TNF-? could disrupt BBB directly,leading to the invasion of peripheral immune cells to CNS and the activation of CNS glial cells.Severe and broad neuroinflammation caused the death of mice.In summary,JEV infection induced substantial inflammatory factors,which was the leading cause of BBB disruption and animal death.IFN-? induced by JEV infection could directly augment BBB permeability,while IP-10 affected the BBB integrity via an indirect manner.By inducing downstream inflammatory factor,IP-10 could regulate BBB permeability.What's more,astrocytes were the primary source of IP-10 in the CNS.After combining with CXCR3,IP-10 activated JNK-c-Jun signal cascade and induced TNF-? expression.TNF-? changed the expression and location of endothelial cell TJ proteins to regulate BBB permeability.Elucidating the BBB breakdown induced by IP-10/TNF-? cytokines network provides a reference for viral induced encephalitis of other Flaviviridae viruses as well as provides a potential therapeutic target for BBB associated CNS diseases.