Novel strategy for treatment of viral central nervous system infection by using a cell-permeating inhibitor of c-Jun N-terminal kinase

Abstract

Viral encephalitis is a major cause of morbidity and mortality worldwide, yet there is no proven efficacious therapy for most viral infections of the central nervous system (CNS). Many of the viruses that cause encephalitis induce apoptosis and activate c-Jun N-terminal kinase (JNK) following infection. We have previously shown that reovirus infection of epithelial cell lines activates JNK-dependent apoptosis. We now show that reovirus infection resulted in activation of JNK and caspase-3 in the CNS. Treatment of reovirus-infected mice with a cell-permeating peptide that competitively inhibits JNK activity resulted in significantly prolonged survival of intracerebrally infected mice following an otherwise lethal challenge with T3D (100 x 50% lethal dose). Protection correlated with reduced CNS injury, reduced neuronal apoptosis, and reduced c-Jun activation without altering the viral titer or viral antigen distribution. Given the efficacy of the inhibitor in protecting mice from viral encephalitis, JNK inhibition represents a promising and novel treatment strategy for viral encephalitis.

FIG. 1.

Reovirus-induced activation of pS73 c-Jun in primary cortical neuronal culture is inhibited with D-JNKI-1. Representative photographs are shown from 48 h postinfection. Primary MCCs were mock infected, T3A infected, or treated with D-JNKI-1 prior to T3A infection. (A to C) Primary MCCs were colabeled with antibodies to pS73 c-Jun (red) and neuron-specific antibodies to MAP2 (green). (D to F) Primary MCCs were colabeled with antibodies to pS73 c-Jun activation (red) and antibodies to σ3 reovirus antigen (green). (G) Graphic display of the percentage of primary MCCs that stained positive for pS73 c-Jun. Blinded cell counts from three replicates are shown of mock-infected or T3A-infected primary MCCs treated with D-JNKI-1, TAT control, or vehicle (PBS). Vertical bars indicate standard errors of the means. *, P ≤ 0.05. Original magnification of images shown in panel A, ×400.

FIG. 2.

T3A-induced JNK phosphorylation in brains of neonatal mice. Two-day-old Swiss Webster mice were mock or T3A infected and sacrificed when moribund. Tissue was processed for whole-brain lysates and subjected to immunoblotting for evidence of phosphorylated JNK and total JNK. Each band represents an individual.

FIG. 3.

T3A-induced activation of c-Jun in brains of neonatal mice. Two-day-old Swiss Webster mice were mock or T3A infected and sacrificed when moribund. (A) Histological tissue sections from T3A-infected mice were evaluated using fluorescence immunohistochemistry for pS73 c-Jun (green) in the cingulate cortex, hippocampus, and thalamus. Images (original magnification, ×400) provide representative observations of the 12 animals sampled per group. (B) Whole-brain lysate samples were evaluated for evidence of activated (pS63) c-Jun. Each band represents an individual. Images are representative of eight animals sampled per group.

FIG. 4.

D-JNKI-1 treatment protects from T3A-induced CNS injury. Mice were treated with D-JNKI-1 or vehicle control, followed by viral challenge with T3A. (A) Representative brain tissue samples were stained with hematoxylin and eosin in order to evaluate for evidence of histological damage. Samples represent four replicates of the experiment consisting of six mice per treatment group per replicate. Original magnification of images shown in panel A,×200. (B) Blind neuropathology scoring of histologic sections show significantly decreased injury in the D-JNKI-1-treated mice compared to untreated, T3A-infected mice in areas typical of reovirus histologic injury: the cingulate cortex (*, P = 0.005), hippocampus (*, P = 0.02), and thalamus (*, P = 0.006). Histopathology scoring: 0, no lesions; 1, <10% of total section shows CNS injury; 2, 10 to 40%; 3, 40 to 75%; 4, >75%.

FIG. 5.

D-JNKI-1 treatment results in significantly prolonged survival for both T3A- and T3D-infected mice. (A) Mice were treated with D-JNKI-1, challenged with T3A 6 h later, and sacrificed when moribund. T3A-infected, untreated mice had an MDD of 10.6 (standard deviation [SD], ±0.6), and T3A-infected, D-JNKI-1 treated mice had an MDD of 14 (SD, ±1.9; P = 0.006). (B) Mice were challenged with T3D and treated 24 h later with vehicle control, TAT control peptide, or D-JNKI-1. Survival studies revealed 38% long-term survival in the T3D-infected, D-JNKI-1-treated group. Of the individuals that died, the MDD was prolonged for T3D-infected, D-JNKI-1-treated mice compared to T3D-infected, untreated mice (13.2; SD, ±1.42; and 9.8; SD, ±2.3 respectively; P < 0.00001). Survival of TAT peptide-treated T3D-infected mice did not significantly differ from that of vehicle control, T3D-infected mice (data not shown).

FIG. 6.

D-JNKI-1 treatment of T3A- or T3D-infected mice decreases activation of c-Jun in vivo. (A) Representative immunohistochemical staining of brain sections of mock-, T3A-, or T3D-infected mice treated with vehicle (PBS), TAT control peptide, or D-JNKI-1 (DJNKI). Successive sections were stained with antibodies to pS73 c-Jun (PcJun) (green) and colabeled with either antibody to reovirus σ3 protein (red) or antibody to MAP2 neuron-specific antigen (red). Images are provided from the cingulate cortex (T3A) or the hippocampus (T3D); original magnification, ×400. (B) Representative immunoblots of whole-brain lysate are shown following mock or T3A infection and treatment with vehicle (PBS) or D-JNKI-1. Blots were probed for pS63 c-Jun and actin. Densitometry studies represent values obtained from individual blots from three mice per treatment group normalized to corresponding actin densitometry values. Vertical bars indicate standard errors of the means.

FIG. 7.

D-JNKI-1 treatment of T3D-infected mice decreases cleaved caspase-3. Adjacent tissue sections from mock- or T3D-infected mice treated with TAT control peptide or D-JNKI-1 were evaluated with dual-label fluorescence immunohistochemistry using antibody to σ3 reovirus protein (Reo Ag) (green) and cleavage specific (activated) caspase-3 (Casp) (red). Representative images are shown (original magnification, ×400) from the hippocampus of the T3D-infected groups. T3A data are not shown.

FIG. 8.

D-JNKI-1 treatment does not alter viral growth in the CNS. Two-day-old Swiss Webster mice were infected with T3A or T3D and treated with vehicle control or D-JNKI-1. Six hours following D-JNKI-1 or vehicle control treatment, mice were challenged with T3A and sacrificed on day 10 postinfection. Mice infected with T3D were treated with D-JNKI-1 or vehicle control 24 h postinfection and were sacrificed at day 8 postinfection. Each treatment group represents three replicates with six mice. Vertical bars indicate standard errors of the means.