Viral Infection of the Central Nervous System and Neuroinflammation Precede Blood-Brain Barrier Disruption during Japanese Encephalitis Virus Infection

Abstract

Japanese encephalitis is an acute zoonotic, mosquito-borne disease caused by Japanese encephalitis virus (JEV). Japanese encephalitis is characterized by extensive inflammation in the central nervous system (CNS) and disruption of the blood-brain barrier (BBB). However, the pathogenic mechanisms contributing to the BBB disruption are not known. Here, using a mouse model of intravenous JEV infection, we show that virus titers increased exponentially in the brain from 2 to 5 days postinfection. This was accompanied by an early, dramatic increase in the level of inflammatory cytokines and chemokines in the brain. Enhancement of BBB permeability, however, was not observed until day 4, suggesting that viral entry and the onset of inflammation in the CNS occurred prior to BBB damage. In vitro studies revealed that direct infection with JEV could not induce changes in the permeability of brain microvascular endothelial cell monolayers. However, brain extracts derived from symptomatic JEV-infected mice, but not from mock-infected mice, induced significant permeability of the endothelial monolayer. Consistent with a role for inflammatory mediators in BBB disruption, the administration of gamma interferon-neutralizing antibody ameliorated the enhancement of BBB permeability in JEV-infected mice. Taken together, our data suggest that JEV enters the CNS, propagates in neurons, and induces the production of inflammatory cytokines and chemokines, which result in the disruption of the BBB.

Importance: Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia, resulting in 70,000 cases each year, in which approximately 20 to 30% of cases are fatal, and a high proportion of patients survive with serious neurological and psychiatric sequelae. Pathologically, JEV infection causes an acute encephalopathy accompanied by BBB dysfunction; however, the mechanism is not clear. Thus, understanding the mechanisms of BBB disruption in JEV infection is important. Our data demonstrate that JEV gains entry into the CNS prior to BBB disruption. Furthermore, it is not JEV infection per se, but the inflammatory cytokines/chemokines induced by JEV infection that inhibit the expression of TJ proteins and ultimately result in the enhancement of BBB permeability. Neutralization of gamma interferon (IFN-γ) ameliorated the enhancement of BBB permeability in JEV-infected mice, suggesting that IFN-γ could be a potential therapeutic target. This study would lead to identification of potential therapeutic avenues for the treatment of JEV infection.

FIG 1

Survival, body weight, viral burden, and BBB permeability analysis in mice after JEV-P3 infection. (A) Eight-week-old age-matched C57BL/6 mice were intravenously injected with 10³ or 10⁵ PFU of JEV-P3. Kaplan-Meier curves show the survival distribution for mice over the 21-day period following challenge (n = 20). (B) Average percent body weight changes were monitored. Body weights were determined every other day, beginning on day 0 until day 14. Mock-infected group, n = 18; infection group, n = 180. (C) The C gene copies of JEV in the brains, spleens, lymph nodes, and livers of JEV-infected mice were measured by qRT-PCR. (D) Evans blue dye was injected intravenously into mice. Representative brains are shown from mock-infected mice or from mice showing mild and severe symptoms. The data are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

FIG 2

Viral loads and BBB permeability. Virus titers in the serum (A) and brain (B) were determined based on plaque formation in BHK-21 cells and are represented as geometric means (n = 5). The copy number of the JEV-C gene in the brain was monitored using qRT-PCR (C), and BBB permeability was determined with NaF uptake (D) for 7 consecutive days after injection with 10⁵ PFU of JEV-P3. At 45 min after intraperitoneal injection with sodium fluorescein, the sera and brains were processed for the measurement of fluorescence, and the brain/serum fluorescence ratios were calculated. Each data point represents a single animal, and the lines represent the group medians. *, P < 0.05; **, P < 0.01.

FIG 3

Pathological features of JEV infection in mouse brain. (A) Brain histopathological changes of JEV-infected mice with disease signs were examined by H&E staining at 5 dpi. c, perivascular cuff (black arrows); d, microglia nodule (red arrows). (B) Immunofluorescent triple staining of neuron infection (a to d), astrocyte activation (e to i), and microglial activation (m to t). Brain sections were collected from mock-infected and JEV-infected mice at 5 dpi. E, JEV envelope protein (red). MAP-2, GFAP, and IBA-1 indicate markers for neurons, astrocytes, and microglia, respectively (green). DAPI stains for nuclei (blue). Subpanels e to h and m to p show brain sections from mock-infected mice.

FIG 4

Expression of TJ proteins (occludin, claudin-5, and ZO-1) in the brains of JEV-infected mice. (A) Mice were infected intravenously with either 10⁵ PFU of JEV-P3 or PBS. At 5 dpi, the animals were euthanized, and the brains were harvested, fixed, and sectioned to measure the expression of TJ proteins using IHC with antibodies to the respective TJ proteins. Black arrows indicate continuous seals that TJ formed around the inner sides of microvessels. Red arrows point to the continuity disruption of occludin, claudin-5, and ZO-1. Inset boxes in subpanels d, e, and f show high-magnification images of regions demarcated by dashed lines, respectively. (B) The expression of TJ proteins in JEV-infected mouse brains was detected by Western blotting. The infected group was divided into asymptomatic, mild, severe, or moribund. (C) Results of panel B were normalized with β-actin and densimetrically quantified as the fold change over that for mock-infected controls. The samples from asymptomatic, mild, severe, and moribund mice were quantified as the infected group. The data are means ± SEM of results from three independent experiments. The statistical significance in panel C was assessed using a two-tailed Student t test. Asterisks indicate statistical significance (*, P < 0.05).

FIG 5

Expression of AM proteins (PECAM-1, VCAM-1, and ICAM-1) in the brains of JEV-infected mice. (A) At 5 dpi, animals were euthanized, and the brains were then harvested and homogenized. After centrifugation, the supernatants from brain homogenates were analyzed by Western blotting for the expression of AM proteins. (B) The results of panel A were normalized with β-actin and densimetrically quantified as the fold change over that of mock-infected controls. The samples from asymptomatic, mild, severe, and moribund mice were quantified as the infected group. The data are means ± SEM of results from three independent experiments. Statistical significance was assessed by using a two-tailed Student t test. Asterisks indicate statistical significance (*, P < 0.05).

FIG 6

Measurement of cytokines by using a Luminex assay in the brain extracts from mice infected with JEV-P3. C57BL/6 mice were infected intravenously with either 10⁵ PFU of JEV-P3 or PBS. Each day from day 0 to 7 dpi, animals were euthanized, blood was collected, and the brains were harvested and homogenized. After centrifugation, the sera and supernatants from brain homogenates were used for measurement of the indicated cytokines using a Luminex assay. The expression levels of 6/13 cytokines/chemokines are shown in serum (A) and brain extracts (B). Pentagrams indicate the expression levels of cytokines beyond the upper detection limit. The data are shown as means ± SEM (n = 4) in each experimental condition. Statistical analyses were performed with two-tailed Student t test. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.005).

FIG 7

Effects of brain extracts on endothelial barrier permeability. (A) After b.End3 monolayers were grown to confluence on transwell membranes, the cells were treated with brain extracts, UV-inactivated brain extracts, JEV, or UV-inactivated JEV for 24 h or 48 h. FITC-dextran-10000 was applied apically at 1 mg/ml for 30 min, and then the permeability was measured from the samples of lower chamber with a fluorometer (excitation, 492 nm; emission, 520 nm). (B) Mouse BMECs were treated as described above, and then protein samples were collected and subjected to Western blotting for the TJ proteins occludin, claudin-5, and ZO-1. β-Actin was used as a loading control. (C) The results of panel B were normalized with β-actin and densimetrically quantified as the fold change versus control brain extracts (control BE). Means ± SEM of three independent experiments were shown. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.005).

FIG 8

Effects of anti-IFN-γ neutralizing antibody on the BBB permeability of JEV-infected mice. (A) Eight-week-old age-matched C57BL/6 mice were intravenously injected with 10⁵ PFU of JEV-P3. At 0, 1, 2, and 3 dpi, the mice were intraperitoneally injected with anti-IFN-γ neutralizing antibody or isotype control. At 5 dpi, the BBB permeability was determined with NaF uptake. (B) At 5 dpi, brain tissues were collected to measure the expression of TJ proteins by Western blotting. (C) The results of panel B were normalized with β-actin and densimetrically quantified as the fold change versus a mock-infected control. (D) At 5 dpi, viral loads in the brain were determined with a plaque assay after antibody administration. The data are means ± SEM of the results from three independent experiments. The statistical significance values in panel C were assessed by using two-tailed Student t test. Asterisks indicate the statistical significance (*, P < 0.05; **, P < 0.01).

FIG 9

Schematic representation of possible mechanisms regulating BBB permeability during JEV infection. During infection, JEV particles penetrate the BBB from the periphery into the CNS without changing BBB permeability. Neurons are vulnerable to JEV infection. Once neurons are infected, the virus begins multiplying and replicating in neurons, which causes the first round of neuronal injury accompanied by the production of cytokines or chemokines. These cytokines/chemokines activate microglia and astrocytes, which in turn stimulates more production of proinflammatory cytokines/chemokines and contributes to further neuronal injury. Cytokines and chemokines can activate immune cells inside the brain that initiate and/or potentiate BBB dysfunction and alter the architecture of tight junctions and adhesion molecules on BBB. Furthermore, transendothelial migration of leukocytes causes acute neuronal tissue damage. Consequently, BBB permeability increases.