JoAnn P. Palma

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

ABSTRACT Theilers virus infection in the central nervous system (CNS) induces a demyelinating disease very similar to human multiple sclerosis. We have assessed cytokine gene activation upon Theilers murine encephalomyelitis virus (TMEV) infection and potential mechanisms in order to delineate the early events in viral infection that lead to immune-mediated demyelinating disease. Infection of SJL/J primary astrocyte cultures induces selective proinflammatory cytokine genes (interleukin-12p40 [IL-12p40], IL-1, IL-6, tumor necrosis factor alpha, and beta interferon [IFN-β]) important in the innate immune response to infection. We find that TMEV-induced cytokine gene expression is mediated by the NF-κB pathway based on the early nuclear NF-κB translocation and suppression of cytokine activation in the presence of specific inhibitors of the NF-κB pathway. Further studies show this to be partly independent of dsRNA-dependent protein kinase (PKR) and IFN-α/β pathways. Altogether, these results demonstrate that infection of astrocytes and other CNS-resident cells by TMEV provides the early NF-κB-mediated signals that directly activate various proinflammatory cytokine genes involved in the initiation and amplification of inflammatory responses in the CNS known to be critical for the development of immune-mediated demyelination.

Induction of selected chemokines in glial cells infected with Theiler's virus

To elucidate the early events in Theilers virus-induced demyelination, a model for human multiple sclerosis (MS), chemokine gene activation in the central nervous system (CNS) resident cells upon viral infection was investigated. Viral infection selectively upregulated RANTES and IP-10 gene expression in primary astrocyte cultures and broader chemokine genes in oligodendrocyte and microglia cultures. Both RANTES and IP-10 were stimulated by proinflammatory cytokine interferon-gamma (IFNgamma), but only RANTES by tumor necrosis factor alpha (TNFalpha), suggesting that virus infection induces chemokines overlapping with those inducible by proinflammatory cytokines. These results suggest that glial cells, astrocytes in particular, may be critical for early recruitment of inflammatory cells in the initiation of virus-induced, immune-mediated demyelination.

CD8-deficient SJL mice display enhanced susceptibility to Theiler’s virus infection and increased demyelinating pathology

Theiler’s murine encephalomyelitis virus (TMEV) infection of the central nervous system (CNS) induces a chronic, progressive demyelinating disease in susceptible mouse strains characterized by inflammatory mononuclear infiltrates and spastic hind limb paralysis. Our lab has previously demonstrated a critical role for TMEV- and myelin-specific CD4+ T cells in initiating and perpetuating this pathology. It has however, also been shown that the MHC class I loci are associated with susceptibility/resistance to TMEV infection and persistence. For this reason, we investigated the contribution of CD8+ T cells to the TMEV-induced demyelinating pathology in the highly susceptible SJL/J mouse strain. Here we show that β2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however β2M-deficient mice demonstrated earlier onset of clinical disease, elevated in vitro responses to TMEV and myelin proteolipid (PLP) epitopes, and significantly higher levels of CNS demyelination and macrophage infiltration at 50 days post-infection. β2M-deficient mice also displayed a significant elevation in persisting viral titers, as well as an increase in macrophage-derived pro-inflammatory cytokine mRNA expression in the spinal cord at this same time point. Taken together, these results indicate that CD8+ T cells are not required for clinical or histologic disease initiation or progression in TMEV-infected SJL mice. Rather, these data stress the critical role of CD4+ T cells in this capacity and further emphasize the potential for CD8+ T cells to contribute to protection from TMEV-induced demyelination.

Induction of chemokines in human astrocytes by picornavirus infection requires activation of both AP-1 and NF-κB

Infection with different picornaviruses can cause meningitis/encephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astrocytes was investigated following infection with Theilers murine encephalomyelitis virus (TMEV), coxsackievirus B3 (CVB3), or coxsackievirus B4 (CVB4). We report that all these viruses are potent inducers for the expression of interleukin‐8 (IL‐8) and monocyte chemoattractant protein‐1 (MCP‐1) genes in primary human astrocytes, as well as in an established astrocyte cell line (U‐373MG). Further studies indicated that both activator protein‐1 (AP‐1) and NF‐κB transcription factors are required in the activation of chemokine genes in human astrocytes infected with various picornaviruses. Interestingly, the pattern of activated chemokine genes in human astrocytes is quite restricted compared to that in mouse astrocytes infected with the same viruses, suggesting species differences in gene activation. This may result in potential differences in the pathogenic outcome in each species.

Pathogenesis of virus-induced immune-mediated demyelination

Theilers murine encephalomyelitis virus-induced demyelinating disease has been extensively studied as an attractive infectiousmodel for human multiple sclerosis. Virus-specific inflammatory Th1 cell responses followed by autoimmune responses to myelin antigens play a crucial role in the pathogenic processes leading to demyelination. Antibody and cytotoxic T cells (CTL) responses to virus appears to be primarily protective from demyelinating disease. Although the role of Th1 and CTL responses in the induction of demyelinating disease is controversial, assessment of cytokine sproduced locally in the central nervous system (CNS) during the course of disease and the effects of altered inflammatory cytokine levels strongly support the importance of Th1 responses in this virus-induced demyelinating disease. Induction of various chemokines and cytokines in different glial and antigen presenting cells upon viral infection appears to be an important initiation mechanism for inflammatory Th1 responses in the CNS. Coupled with the initial inflammatory responses, viral persistence in the CNS may be acritical factor for sustaining inflammatory responses and consequent immune-mediated demyelinating disease.

Preferential Induction of IL-10 in APC Correlates with a Switch from Th1 to Th2 Response Following Infection with a Low Pathogenic Variant of Theiler’s Virus

Theiler’s murine encephalomyelitis virus induces immune-mediated demyelination in susceptible mice after intracerebral inoculation. A naturally occurring, low pathogenic Theiler’s murine encephalomyelitis virus variant showed a single amino acid change within a predominant Th epitope from lysine to arginine at position 244 of VP1. This substitution is the only one present in the entire viral capsid proteins. In this paper, we demonstrate that the majority of T cells specific for VP1233–250 and VP274–86 from wild-type virus-infected mice are Th1 type and these VP1-specific cells poorly recognize the variant VP1 epitope (VP1K244R) containing the substituted arginine. In contrast, the Th2-type T cell population specific for these epitopes predominates in variant virus-infected mice. Immunization with UV-inactivated virus or VP1 epitope peptides could not duplicate the preferential Th1/Th2 responses following viral infection. Interestingly, the major APC populations, such as dendritic cells and macrophages, produce IL-12 on exposure to the pathogenic wild-type virus, whereas they preferentially produce IL-10 in response to the low pathogenic variant virus. Thus, such a spontaneous mutant virus may have a profoundly different capability to induce Th-type responses via selective production of cytokines involved in T cell differentiation and the consequent pathogenicity of virally induced immune-mediated inflammatory diseases.

Enhanced susceptibility to Theiler's virus-induced demyelinating disease in perforin-deficient mice

Theilers virus induces immune-mediated demyelinating disease similar to human MS in susceptible mice. Though the MHC class II-restricted T cell response is critical, susceptibility/resistance is also associated with a MHC class I haplotype. Here we report that perforin-deficient C57BL/6 mice (pKO) are susceptible to demyelination and develop clinical disease. The levels of primary demyelination, proliferation, Th1 responses, and viral load were also markedly enhanced. In addition, immunization of pKO mice with UV-inactivated virus further enhanced clinical incidence and accelerated the disease course. Thus, perforin is most likely involved in viral clearance, hence protection from the disease.

Innate immune response induced by Theiler's murine encephalomyelitis virus infection.

Although the causative agents of human multiple sclerosis (MS) are not known, it is suspected that a viral infection may be associated with the initiation of the disease. Several viral disease models in mice have been studied to understand the pathogenesis of demeylination. In particular, Theilers murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) has been extensively studied as a relevant model. Various cytokines and chemokines are produced upon viral infection by different cell types, including antigen-presenting cells (APCs) such as macrophages; dendritic cells (DCs); and glial cells, such as astrocytes, microglia, and oligoden-drocytes. The upregulation of the corresponding molecules are also found in MS and are likely to play an important role in the protection and/or pathogenesis of chronic inflammatory demyelinating disease. In this review, the type of cells and molecules, gene-activation mechanisms as well as their potential roles in protection and pathogenesis will be discussed.

Treatment with lipopolysaccharide enhances the pathogenicity of a low-pathogenic variant of Theiler's murine encephalomyelitis virus.

Intracerebral infection of susceptible mouse strains with Theilers murine encephalomyelitis virus (TMEV) results in an immune‐mediated demyelinating disease (TMEV‐IDD) similar to human multiple sclerosis (MS). Although the etiology of MS remains unknown, a role of an infectious agent has been implicated in its onset. Previously we have shown the ability of bacterial lipopolysaccharide (LPS) to alter susceptibility to TMEV‐IDD in genetically resistant C57BL/6 mice. In this study, the potential of LPS to alter pathogenicity of a low/non‐pathogenic variant of TMEV was investigated. After intraperitoneal treatment of genetically susceptible SJL/J mice with LPS before and during viral infection, 80–100% of the mice developed clinical symptoms, while without LPS treatment none of the mice were affected. However, clinical severity in these LPS‐treated mice was much milder than the level induced by the wild type pathogenic virus. Increased susceptibility to the disease after LPS treatment did not correlate with splenic T cell proliferative responses against viral antigens. However, by reverse transcriptase polymerase chain reaction (RT‐PCR) analyses, an early increase in the production of Th1‐type proinflammatory cytokine messages (e.g., interferon‐γ [IFN‐γ] and enhancement of viral persistence was observed in the CNS of LPS‐treated, virus‐infected animals as compared to mice infected with the variant virus alone. These results indicate that environmental factors such as a bacterial infection (e.g., LPS) promoting proinflammatory cytokine production can significantly enhance the pathogenicity of demyelination induced by a normally non‐pathogenic virus.

Pathogenic Immunity in Theiler’s Virus-Induced Demyelinating Disease: A Viral Model for Multiple Sclerosis

Multiple sclerosis involves inflammatory immune responses in the central nervous system (CNS) and is considered as an autoimmune disease potentially associated with viral infection. The majority of experimental models rely heavily on the autoimmune components since similar diseases can be induced following immunization with various myelin antigens. A very attractive alternative model is the Theilers murine encephalomyelitis virus-induced demyelinating disease. This disease is primarily a CD4+ T cell-mediated, inflammatory demyelinating disease induced following viral infection. Virus-specific inflammatory Th1 cell responses, rather than cytotoxic T lymphocyte response, play a critical role in the pathogenic immune responses. The major pathogenic epitopes have been identified and these are correlated with a Th1 type response to the epitopes following viral infection. In addition, the initial virus-specific immune response is followed by the autoimmune responses to myelin antigens. Assessment of cytokines produced locally in the CNS during the course of disease suggests involvement of inflammatory cytokines in the disease. Furthermore, the manipulation of inflammatory cytokine levels by administration of either recombinant cytokines or antibodies to the cytokines strongly influences the induction and/or progression of disease, supporting the importance of these inflammatory cytokines in this virus-induced demyelinating disease.