Brain microvascular endothelial-astrocyte cell responses following Japanese encephalitis virus infection in an in vitro human blood-brain barrier model

Adjanie Patabendige,Benedict D Michael,Alister G Craig,Tom Solomon

doi:10.1016/j.mcn.2018.04.002

Abstract

Japanese encephalitis virus (JEV) remains a leading cause of encephalitis, globally, which continues to grow in importance despite the availability of vaccines. Viral entry into the brain can occur via the blood-brain barrier (BBB), and inflammation at the BBB is a common final pathway in many brain infections. However, the role of the BBB during JEV infection and the contribution of the endothelial and astrocytic cell inflammation in facilitating virus entry into the brain are incompletely understood. We established a BBB model using human brain endothelial cells (HBECs) and human astrocytes. HBECs are polarised, and therefore the model was inoculated by JEV from the apical side to simulate the in vivo situation. The effects of JEV on the BBB permeability and release of inflammatory mediators from both apical and basolateral sides, representing the blood and the brain side respectively were investigated. JEV infected HBECs with limited active virus production, before crossing the BBB and infecting astrocytes. Control of JEV production by HBECs was associated with a significant increase in permeability, and with elevation of many host mediators, including cytokines, chemokines, cellular adhesion molecules, and matrix metalloproteases. When compared to the controls, significantly higher amounts of mediators were released from the apical side as opposed to the basolateral side. The increased release of mediators over time also correlated with increased BBB permeability. Treatment with dexamethasone led to a significant reduction in the release of interleukin 6 (IL6), C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10) from the apical side with a reduction in BBB disruption and no change in JEV production. The results are consistent with the hypothesis that JEV infection of the BBB triggers the production of a range of host mediators from both endothelial cells and astrocytes, which control JEV production but disrupt BBB integrity thus allowing virus entry into the brain. Dexamethasone treatment controlled the host response and limited BBB disruption in the model without increasing JEV production, supporting a re-investigation of its use therapeutically.

Highlights

Encephalitis is inflammation and swelling of the brain, which can be caused by a range of viruses, often with devastating results (Solomon et al, 2014)
Infection of the co-culture human blood-brain barrier (BBB) model with Japanese encephalitis virus (JEV) from the apical side led to a significant drop in transendothelial electrical resistance (TEER) 2 dpi (p < 0.001), and remained at this level (Fig. 2B), indicating that BBB integrity is lost early during infection, and the BBB permeability is not increased despite further viral production within the endothelium
We found the limited production of JEV when infected with at an multiplicity of infection (MOI) of 1 within human brain endothelial cells (HBECs) is associated with upregulation of host inflammatory markers and increased permeability of the BBB model, as demonstrated by a significant drop in TEER at 2 dpi

Summary

Introduction

Encephalitis is inflammation and swelling of the brain, which can be caused by a range of viruses, often with devastating results (Solomon et al, 2014). JEV is one of the most important causes. Largely confined to South and East Asia, this mosquito-borne flavivirus (genus Flavivirus, family Flaviviridae) has spread to the northern territories of Australia and potentially to European countries with the recent discovery of JEV RNA in mosquitoes in northern Italy (Ravanini et al, 2012). The virus is closely related to West Nile virus (WNV) and Zika virus; the former causes encephalitis in a small proportion of patients; the latter has caused large outbreaks of febrile Corticosteroids such as hydrocortisone and dexamethasone in low doses (nM range) have been shown to improve BBB integrity in vivo (Salvador et al, 2014) and in vitro (Deli et al, 2005)

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