Enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection

Abstract

Infection with laboratory-attenuated rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, which has been demonstrated to be an important factor for host survival, since it allows immune effectors to enter the central nervous system (CNS) and clear RABV. To probe the mechanism by which RABV infection enhances BBB permeability, the expression of tight junction (TJ) proteins in the CNS was investigated following intracranial inoculation with laboratory-attenuated or wild-type (wt) RABV. BBB permeability was significantly enhanced in mice infected with laboratory-attenuated, but not wt, RABV. The expression levels of TJ proteins (claudin-5, occludin, and zonula occludens-1) were decreased in mice infected with laboratory-attenuated, but not wt, RABV, suggesting that enhancement of BBB permeability is associated with the reduction of TJ protein expression in RABV infection. RABV neither infects the brain microvascular endothelial cells (BMECs) nor modulates the expression of TJ proteins in BMECs. However, brain extracts prepared from mice infected with laboratory-attenuated, but not wt, RABV reduced TJ protein expression in BMECs. It was found that brain extracts from mice infected with laboratory-attenuated RABV contained significantly higher levels of inflammatory chemokines/cytokines than those from mice infected with wt RABV. Pathway analysis indicates that gamma interferon (IFN-γ) is located in the center of the cytokine network in the RABV-infected mouse brain, and neutralization of IFN-γ reduced both the disruption of BBB permeability in vivo and the downregulation of TJ protein expression in vitro. These findings indicate that the enhancement of BBB permeability and the reduction of TJ protein expression are due not to RABV infection per se but to virus-induced inflammatory chemokines/cytokines.

Importance: Previous studies have shown that infection with only laboratory-attenuated, not wild-type, rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, allowing immune effectors to enter the central nervous system (CNS) and clear RABV from the CNS. This study investigated the mechanism by which RABV infection enhances BBB permeability. It was found that RABV infection enhances BBB permeability by downregulation of tight junction (TJ) protein expression in the brain microvasculature. It was further found that it is not RABV infection per se but the chemokines/cytokines induced by RABV infection that downregulate the expression of TJ proteins and enhance BBB permeability. Blocking some of these cytokines, such as IFN-γ, ameliorated both the disruption of BBB permeability and the downregulation of TJ protein expression. These studies may provide a foundation for developing therapeutics for clinical rabies, such as medication that could be used to enhance BBB permeability.

FIG 1

NaF uptake and inflammatory cell infiltration in the CNSs of mice infected with laboratory-attenuated CVS-B2c or wt DRV-Mexico. Female ICR mice were infected i.c. either with 1 or 10 LD₅₀ of CVS-B2c or with 10 or 100 LD₅₀ of DRV-Mexico. (A) At day 6 or 9 p.i., BBB permeability was assessed by NaF uptake in the cerebrum and the cerebellum. (B) CD3⁺ T cells were quantified and analyzed statistically. Data are means ± SEM of results from three independent experiments. Statistical analyses for panels A and B were performed using one-way ANOVA followed by Tukey's post hoc test. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (C) Harvested brains were subjected to HE staining for histopathology as well as to IHC for the detection of CD3⁺ T cells.

FIG 2

Expression of TJ proteins (claudin-5, occludin, and ZO-1) in the brains of mice infected with CVS-B2c or DRV-Mexico. Mice were infected i.c. either with 1 or 10 LD₅₀ of CVS-B2c or with 10 or 100 LD₅₀ of DRV-Mexico. (A) At day 6 or 9 p.i., animals were euthanized, and brains were harvested, fixed, and sectioned for measurement of the expression of TJ proteins using IHC with antibodies to the respective TJ proteins. (B) The expression levels of individual TJ proteins were estimated by quantifying IODs and were analyzed statistically. (C) The expression of TJ proteins in RABV-infected mouse brains was confirmed by Western blotting. (D) TJ protein expression as detected by Western blotting was quantitatively analyzed in brain extracts from mice infected with either virus (CVS-B2c or DRV-Mexico) or from sham-infected mice. Data are means ± SEM of results from three independent experiments. Statistical significance in panels B and D was assessed using one-way ANOVA followed by Tukey's post hoc test. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 3

Infection of human or mouse BMECs with RABVs and their effects on TJ protein expression. (A) Human or mouse BMECs were cocultured with CVS-B2c or DRV-Mexico at an MOI of 1, and cells were fixed at 120 h after inoculation to assess active viral replication by using FITC-conjugated anti-RABV N antibodies. mNA cells infected with each virus were included as positive controls. Phase-contrast microscopy was performed to ensure cell viability and structure. (B) Viral RNA from mouse or human BMECs infected with each virus was detected at 0, 24, 48, and 120 h by quantitative RT-PCR. (C) The expression of TJ proteins on BMECs cocultured with either virus was detected by using confocal microscopy and antibodies to TJ proteins. BMECs cocultured with the supernatant of mNA cells infected with rHEP-CXCL10 were included as a positive control.

FIG 4

Effects of brain extracts derived from mice infected with CVS-B2c or DRV-Mexico on the expression of TJ proteins in BMECs. (A) Mouse BMECs were cocultured with brain extracts from mice infected with different doses of CVS-B2c or DRV-Mexico. After 24 h, BMECs were fixed and were stained with DAPI and an anti-claudin-5 antibody or with DAPI and an anti-occludin antibody. The staining was visualized by confocal microscopy. (B) MFI for the ROI drawn around the cells and statistical analysis. (C) The expression of TJ proteins in BMECs after coculturing with brain extracts was also detected using Western blotting. (D) TJ protein expression as detected by WB was quantitatively analyzed in BMECs treated with brain extracts from mice infected with either virus (CVS-B2c or DRV-Mexico). bEnd.3 cells treated with brain extracts from sham-infected mice were included as controls. Data are means ± SEM of results from three independent experiments. Statistical analyses in panels B and D were performed with one-way ANOVA followed by Tukey's post hoc test. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Three samples were collected from three replicates in each group for statistical analysis.

FIG 5

Measurement by Luminex assay of cytokines in brain extracts from mice infected with CVS-B2c or DRV-Mexico. ICR mice were infected i.c. either with 1 or 10 LD₅₀ of CVS-B2c or with 10 or 100 LD₅₀ of DRV-Mexico. At day 6 or 9 p.i., animals were euthanized, and brains were harvested and homogenized. After centrifugation, the supernatants were loaded in order to measure the concentrations of the indicated cytokines by using the Luminex assay. Shown are expression levels of chemokines (A), interleukins, TNF-α, and IFN-γ (B), and growth factors (C). Experiments were performed with three replicates for each time point and condition. Hash tags indicate expression levels of cytokines beyond the upper detection range. Data are means ± SEM of results from three independent experiments. Statistical analyses were performed with one-way ANOVA followed by Tukey's post hoc test. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 6

Ingenuity pathway analysis of the immune response, regulatory networks, and pathways mediated by infection with CVS-B2c or DRV-Mexico. Data generated by the Luminex assay were analyzed with IPA software. One network of genes expressed in the brains of mice infected with CVS-B2c (A) and one network of genes expressed in the CNSs of mice infected with DRV-Mexico (B) were established. Nodes represent genes; their shapes represent the functional classes of the gene products (C); and arrows indicate the biological relationships between the nodes. The intensity of the node color indicates the degree of upregulation (red) in mice inoculated with either RABV. White (noncolored) nodes are nonfocus genes that are biologically relevant to the pathways but were not identified as differentially expressed by our Luminex analysis.

FIG 7

(A and B) IFN-γ neutralization ameliorates the enhancement of BBB permeability in RABV-infected mice (A) and the downregulation of TJ protein expression in BMECs treated with brain extracts from RABV-infected mice (B). (A) Female ICR mice infected i.c. with 10 LD₅₀ of CVS-B2c were injected i.p. with 100 μg of an anti-IFN-γ neutralizing antibody or an isotype control antibody in PBS at 0, 2, and 4 days p.i. At day 6 p.i., BBB permeability was assessed by NaF uptake in the cerebrum and the cerebellum. Statistical analysis was performed with Student's t test. (B) Mouse BMECs were cocultured with brain extracts (treated either with DMEM, with 0.3 μg/ml of an anti-IFN-γ antibody, or with an isotype control antibody) from mice infected with 10 LD₅₀ of CVS-B2c. After 24 h, BMECs were fixed and were stained either with DAPI and an anti-claudin-5 antibody or with DAPI and an anti-occludin antibody. The staining was visualized by confocal microscopy. (C) The MFIs at the ROI drawn around the cells under the different conditions were compared and analyzed statistically using one-way ANOVA followed by Tukey's post hoc test. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 8

Schematic model for the mechanism(s) by which infection with laboratory-attenuated RABV induces the reduction of TJ protein expression and the enhancement of BBB permeability.