Japanese encephalitis is a neuropathological disorder caused by Japanese encephalitis virus (JEV), which is characterized by severe pathological neuroinflammation and damage to the blood–brain barrier (BBB). Inflammatory cytokines/chemokines can regulate the expression of tight junction (TJ) proteins and are believed to be a leading cause of BBB disruption, but the specific mechanisms remain unclear. IP-10 is the most abundant chemokine produced in the early stage of JEV infection, but its role in BBB disruption is unknown. The administration of IP-10-neutralizing antibody ameliorated the decrease in TJ proteins and restored BBB integrity in JEV-infected mice. In vitro study showed IP-10 and JEV treatment did not directly alter the permeability of the monolayers of endothelial cells. However, IP-10 treatment promoted tumor necrosis factor alpha (TNF-α) production and IP-10-neutralizing antibody significantly reduced the production of TNF-α. Thus, TNF-α could be a downstream cytokine of IP-10, which decreased TJ proteins and damaged BBB integrity. Further study indicated that JEV infection can stimulate upregulation of the IP-10 receptor CXCR3 on astrocytes, resulting in TNF-α production through the JNK-c-Jun signaling pathway. Consequently, TNF-α affected the expression and cellular distribution of TJs in brain microvascular endothelial cells and led to BBB damage during JEV infection. Regarding regulation of the BBB, the IP-10/TNF-α cytokine axis could be considered a potential target for the development of novel therapeutics in BBB-related neurological diseases.

Resolution of viral infections requires activation of innate cells to initiate and maintain adaptive immune responses. In this study, we examined Japanese encephalitis virus (JEV) infection leading to acute encephalopathy depending on suppression of the adaptive immune responses mediated by innate cells. Infection with P3 strains of JEV enhanced myeloid-derived suppressor cell (MDSC) populations, and the survival rate of JEV-infected mice improved after MDSC depletion. Mechanically, P3-induced MDSCs suppressed CD4 T cell immune responses, especially responses of T follicular helper (Tfh) cells, leading to decreased splenic B cells (CD19) and blood plasma cells (CD19CD138) and reduced levels of total IgM and JEV-specific neutralizing Abs. Upon depleting P3-induced MDSCs in vivo, the Tfh cell population, B cells, plasma cells, and Ab production recovered. These findings provide unique insights regarding MDSC functions in mediating immune suppression via inhibiting Tfh cell responses and further impairing humoral immunity, which facilitate the progression of infection.

Infection with Japanese encephalitis virus (JEV) induces high morbidity and mortality, including potentially permanent neurological sequelae. However, the mechanisms by which viruses cross the blood-brain barrier (BBB) and invade into the central nervous system (CNS) remain unclear. Here, we show that extracellular HMGB1 facilitates immune cell transmigration. Furthermore, the migration of immune cells into the CNS dramatically increases during JEV infection which may enhance viral clearance, but paradoxically expedite the onset of Japanese encephalitis (JE). In this study, brain microvascular endothelial cells (BMECs) were utilized for the detection of HMGB1 release, and leucocyte, adhesion, and the integrity of the BBB in vitro. Genetically modified JEV-expressing EGFP (EGFP-JEV) and the BBB model were established to trace JEV-infected immune cell transmigration, which mimics the process of viral neuroinfection. We find that JEV causes HMGB1 release from BMECs while increasing adhesion molecules. Recombinant HMGB1 enhances leukocyte-endothelium adhesion, facilitating JEV-infected monocyte transmigration across endothelia. Thus, JEV successfully utilizes infected monocytes to spread into the brain, expanding inside of the brain, and leading to the acceleration of JE onset, which was facilitated by HMGB1. HMGB1-promoted monocyte transmigration may represent the mechanism of JEV neuroinvasion, revealing potential therapeutic targets.

Background: Infection with the Japanese encephalitis virus (JEV) induced high morbidity and mortality, even caused permanent neurological sequelae. However, the pathways and mechanisms of JEV invasion into the central nervous system (CNS) remain elusive. It is confirmed that extracellular HMGB1 facilitates immune cells transendothelial migration. Furthermore, it is observed that the migration of immune cells into the CNS dramatically increased during JEV infection which may benefit to viral clearance, but paradoxically accompanied by the expedite onset of Japanese encephalitis (JE) in advance. Thus, exploration of JEV neuroinvasion pathways is important for pathogenesis and prevention of JE.Methods: Brain microvascular endothelial cells were utilized for the detection of HMGB1 release in vitro. The blood-brain barrier (BBB) monolayer model (brain microvascular endothelial cells) and recombinant HMGB1 were applied for the measurement of endothelial cell activation and cells adhesion, the integrity of the BBB and the interaction with the immune cells. A genetically modified JEV expressing EGFP (EGFP-JEV) was used to trace the transmigration of JEV-infected immune cells crossing the BBB to mimic the process of neuroinfection.Results: JEV has the characteristic of neurotropism, causing HMGB1 released from BMEC and increasing adhesion molecules. BEMC-derived HMGB1 enhances leukocyte-endothelium adhesion, facilitating the transendothelial migration of JEV-infected monocytes across the BBB entry into the CNS. Thus, JEV successfully utilized the monocyte as a “Trojan horse” to spread the virus to the brain, expanding the brain infection, leading the acceleration of JE onset.Conclusion: JEV-infected monocytes, acting as “Trojan horse”, migrate to the brain, which was facilitated by BMEC-derived HMGB1, contributing to JEV neuroinvasion, and leading neuroinflammation and pathological changes of JE.

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