Upregulation of RANTES gene expression in neuroglia by Japanese encephalitis virus infection

Abstract

Infection with Japanese encephalitis virus (JEV) causes cerebral inflammation and stimulates inflammatory cytokine expression. Glial cells orchestrate immunocyte recruitment to focal sites of viral infection within the central nervous system (CNS) and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since immune cell infiltration is prominent, we investigated the production of a responding chemoattractant, RANTES (regulated upon activation, normal T-cell expressed and secreted), in response to JEV infection of glial cells. Infection with JEV was found to elicit the production of RANTES from primary neurons/glia, mixed glia, microglia, and astrocytes but not from neuron cultures. The production of RANTES did not seem to be directly responsible for JEV-induced neuronal death but instead contributed to the recruitment of immune cells. RANTES expression required viral replication and the activation of extracellular signal-regulated kinase (ERK) as well as transcription factors, including nuclear factor kappa B (NF-kappaB) and nuclear factor IL-6 (NF-IL-6). The induction of RANTES expression by JEV infection in glial cells needed the coordinate activation of NF-kappaB and NF-IL-6. Using enzymatic inhibitors, we demonstrated a strong correlation between the ERK signaling pathway and RANTES expression. However, JEV replication was not dependent on the activation of ERK, NF-kappaB, and NF-IL-6. Altogether, these results demonstrated that infection of glial cells by JEV provided the early ERK-, NF-kappaB-, and NF-IL-6-mediated signals that directly activated RANTES expression, which might be involved in the initiation and amplification of inflammatory responses in the CNS.

FIG. 1.

Induction of RANTES expression in JEV-infected neuron/glia cultures. After 12 days in vitro, neuron/glia cultures were subjected to immunocytochemistry with antibodies against MAP-2, GFAP, and ED-1. Neuron/glia cultures before harvest were also photographed under phase contrast. Scale bar, 50 μm (A). Neuron/glia cultures were mock-infected or infected with JEV (NT113) over time. Total cellular RNAs were harvested and subjected to RT-PCR for the detection of RANTES and GAPDH (B). Culture supernatants were collected, and the concentration of RANTES was determined by enzyme-linked immunosorbent assay (n = 4) (C).

FIG. 2.

Neuronal toxicity of JEV. Neuron/glia cultures were mock infected or infected with JEV (NT113) over time. The integrity of neurons was determined by immunocytochemistry with antibody against MAP-2 (A), and the levels of JEV RNA and β-actin mRNA were determined by RT-PCR (C). The integrity of neurons 24 h after mock or JEV (NT113) infection was also assessed in neuron-enriched cultures by immunocytochemistry with antibody against MAP-2 (B). Scale bar, 50 μm.

FIG. 3.

Cell specificity of RANTES production. Neuron/glia, mixed glia, neuron, microglia, and astrocyte cultures were mock infected or infected with JEV (NT113) for 24 h. The production of RANTES and expression of RANTES mRNA were determined by enzyme-linked immunosorbent assay (n = 4) and RT-PCR, respectively (A). The supernatants of mock- and JEV-infected microglia and astrocytes (24 h) were collected and mixed with an equal volume of fresh medium. Microglia-derived conditioned medium was added to new astrocyte cultures and vice versa. The level of RANTES in supernatants before and 24 h after addition was determined by enzyme-linked immunosorbent assay (n = 3) (B).

FIG. 4.

Glial infectivity of JEV. Microglia and astrocyte cultures were infected with JEV (NT113) for 24 h. The morphological characteristics of microglia and astrocytes were assessed by immunocytochemistry with antibodies against ED-1 (microglia) and GFAP (astrocytes). The infected cells in each culture were assessed by immunocytochemistry with antibody against JEV NS3. Scale bar, 50 μm.

FIG. 5.

Vial protein and RANTES immunoreactivity in mixed glia. Mixed glia were mock infected or infected with JEV (NT113) for 24 h, and the immunoreactivity of viral protein and RANTES was examined by immunocytochemistry with antibodies against JEV NS3 and RANTES. JEV-infected cultures were photographed before harvest under phase contrast. The immunoreactive cells marked by arrows and asterisks are representatives of astrocytes and microglia, respectively. by their morphological characteristics. Scale bar, 50 μm.

FIG. 6.

Production of RANTES depends on viral replication. Mixed glia were mock infected or infected with JEV (NT113), UV-inactivated JEV (NT113), or boiling-inactivated JEV (NT113) for 24 h. The production of RANTES and expression of RANTES mRNA were determined by enzyme-linked immunosorbent assay (n = 3) and RT-PCR, respectively (A). Mixed glia were mock infected or infected with other available JEV strains (Peking-1, Nakayama, and JaGar) for 24 h. The production of RANTES and expression of RANTES mRNA were determined by enzyme-linked immunosorbent assay (n = 3) and RT-PCR, respectively (B).

FIG. 7.

Requirement of ERK activation for RANTES expression. After mock or JEV (NT113) infection, mixed glia were cultivated in medium alone or in the presence of U0126 (10 μM) or NAC (5 mM). The level of RANTES in the supernatants (24 h postinfection) was determined by enzyme-linked immunosorbent assay (n = 4) (A). The levels of phosphorylated ERK (8 h postinfection), total ERK (8 h postinfection), and JEV NS3 protein (24 h postinfection) were determined by Western blot analysis. One of three separate experiments is shown (B).

FIG. 8.

Activation of RANTES promoter activity in JEV-infected cells. C6 glioma cells were transiently cotransfected with the three RANTES promoter reporters (pGL-A, pGL-B, and pGL-C) and the Renilla luciferase reporter gene. Twenty-four hours later, cells were mock infected or infected with JEV (NT113) for another 36 h. The cells were then lysed, and the lysates were harvested. Firefly and Renilla luciferase activities were detected by the dual-luciferase reporter assay system. Firefly luciferase activity was normalized to Renilla luciferase activity. The mock-infected pGL-A group was the control (n = 3).

FIG. 9.

Induction of transcription factors in JEV-infected cells. Mixed glia were mock infected or infected with JEV (NT113) for 8 h. Nuclear extracts were prepared and subjected to the electrophoretic mobility shift assay with the A/B, E, NF-κB, and NF-IL-6 oligonucleotides. Lane 1, free probe only; lane 2, mock infection; lane 3, JEV infection. Similar results were obtained from two independent experiments.

FIG. 10.

Specificity of NF-κB and NF-IL-6 binding. Mixed glia were infected with JEV (NT113) for 8 h. The isolated nuclear extracts were subjected to the electrophoretic mobility shift assay with the biotin-labeled A/B (A) or E (B) oligonucleotide. The characteristics of A/B-protein and E-protein complex were assessed by the competition assay (lanes 2 to 5) with unlabeled oligonucleotides (50-fold). Lane 1, labeled A/B or E control; lane 2, unlabeled A/B; lane 3, unlabeled E; lane 4, unlabeled NF-κB; lane 5, unlabeled NF-IL-6. Similar results were obtained from two independent experiments.

FIG. 11.

Induction of transcription factors depends on activation of ERK. After mock or JEV (NT113) infection, mixed glia were cultivated for a further 8 h in medium alone or in the presence of U0126 (10 μM) or NAC (5 mM). The isolated nuclear extracts were subjected to the electrophoretic mobility shift assay with the A/B, E, NF-κB, and NF-IL-6 oligonucleotides. Similar results were obtained from three independent experiments.

FIG. 12.

Chemotaxis of RANTES. Mixed glia were mock infected or infected with JEV (NT113) for 24 h. Supernatants isolated from JEV-infected cells were modified by treatments before chemotactic assay, including inactivation by boiling for 15 min and neutralization with RANTES and MCP-1 neutralizing antibodies (50 μg/ml) for 30 min. Chemotactic activity against RAW264.7 monocytes/macrophages was measured by Transwell assay as described in Materials and Methods. Mock infection was the control. *, P < 0.05, and **, P < 0.01 versus JEV; and ##, P < 0.01 versus the mock-infected control, n = 4 (A). Mixed glia were infected with the mock-infected control or JEV (NT113) for 24 h. Total RNAs were isolated and subjected to RT-PCR for the detection of MCP-1, Mip-1α, cytokine-induced neutrophil chemoattractant (CINC), and GAPDH mRNAs. One of three independent experiments is shown (B).