Infection with Theiler's Murine Encephalomyelitis Virus Directly Induces Proinflammatory Cytokines in Primary Astrocytes via NF-κB Activation: Potential Role for the Initiation of Demyelinating Disease

Theiler's virus induces an immune-mediated demyelinating disease similar to human multiple sclerosis. Studies have shown the importance of genetic factors like major histocompatibility complex (MHC), T-cell receptor, and gender in the susceptibility to the disease (21). Intracerebral infection of susceptible strains such as SJL/J mice results in an acute, polio-like phase of the gray matter that later develops into a chronic demyelination in the white matter manifested by severe hind limb paralysis and incontinence (25). Demyelination is characterized by mononuclear cell infiltration with the primary involvement of activated macrophages that correlates with virus-specific delayed-type hypersensitivity responses (7, 51). The Theiler's murine encephalomyelitis virus (TMEV)-specific T-cell responses to viral determinants are well characterized in this Th1-mediated disease (21). However, the early innate immune response of central nervous system (CNS)-resident cells that lead to the proinflammatory milieu critical in the recruitment and development of virus-specific Th1 responses, are largely unknown. An innate immune response to viral and bacterial infection often results in the production of immune molecules, including cytokines, chemokines, MHC, and enzymes, etc., that act in concert to control the infectious agents (13). These very same molecules can however induce dysregulated inflammation that leads to target tissue destruction (9, 26). Recent studies suggest that toll-like receptors are involved in the induction of innate immune responses following exposure to bacterial and/or viral components (18, 34). In particular, TLR-3 (as well as double-stranded RNA [dsRNA]-dependent protein kinase [PKR]) is involved in activation of a variety of proinflammatory cellular genes through the NF-κB pathway upon recognition of dsRNA, a replication intermediate of TMEV (2, 55). NF-κB represents a family of dimeric transcription factors that play a central role in these inflammatory responses by regulation of gene expression and inhibition of apoptosis (11, 20). The sequestration of NF-κB by IκB in the cytoplasm and IκB phosphorylation leading to proteasomal degradation resulting in activation and translocation of NF-κB to the nucleus is essential in the transcription of many proinflammatory chemokines and cytokines such as IP-10, RANTES, monocyte chemotactic protein 1 (MCP-1), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), and IL-6, (3, 4). Many investigators have extensively investigated the potential roles of cytokines in TMEV-induced, immune-mediated inflammatory demyelination (5, 6, 21, 44, 49). The accumulation of proinflammatory cytokines has been demonstrated in the CNS of TMEV-infected mice and precedes that of anti-inflammatory cytokines (IL-4 and IL-10) during the course of disease (37). Recent studies have also shown sustained upregulation of proinflammatory cytokines (gamma interferon [IFN-γ], IL-6, IL-12, and TNF-α) and transforming growth factor β, downregulating cytolytic responses in the CNS of SJL mice compared to resistant C57BL/6 mice. These results correlate well with susceptibility to demyelinating disease (6). However, the mechanisms that initiate and expand cytokine gene expression in the CNS inflammatory disease induced following TMEV infection are less clear. We and others have previously reported that TMEV is able to directly activate selective chemokine and cytokine genes in the CNS-resident glial cell populations (32, 36, 38, 46). However, the range of cytokines induced and the signal transduction mechanisms involved in this virus-mediated cytokine gene activation has not been previously explored. In order to delineate the potential pathogenic role of the innate immune response in virus-induced CNS demyelination, we have examined the molecular mechanisms underlying early cytokine gene activation after TMEV infection of primary astrocyte cultures. Here we demonstrate the ability of TMEV to directly induce selective proinflammatory cytokines (IL-12, IL-1, IL-6, TNF-α, and IFN-β) in glial cells. The expression of cytokine genes was evident for some, but not for all, as early as 30 min postinfection, and the activation of NF-κB was demonstrated within 5 min of TMEV infection. Additionally, various NF-κB inhibitors, as well as an IκB super-repressor, were shown to inhibit TMEV-induced cytokine gene expression. These results clearly indicate that NF-κB activation is necessary in TMEV-induced cytokine gene activation. The PKR as well as IFN-α/β pathways do not appear to play a major role in the cytokine gene activation. Altogether, these results strongly support the crucial role of CNS resident cells in the initiation of inflammatory demyelination by production of various proinflammatory cytokines via an NF-κB-dependent innate immune response to TMEV infection.