Dynamic changes and molecular analysis of cell death in the spinal cord of SJL mice infected with the BeAn strain of Theiler’s murine encephalomyelitis virus

Theiler’s murine encephalomyelitis (TME) is caused by the TME virus (TMEV) and represents an important animal model for multiple sclerosis (MS). Oligodendroglial apoptosis and reduced apoptotic elimination of encephalitogenic leukocytes seem to participate in autoimmune demyelination in MS. The present study quantified apoptotic cells in BeAn–TMEV-induced spinal cord white matter lesions at 14, 42, 98, and 196 days post infection (dpi) using immunostaining. Apoptotic cells were identified by transmission electron microscopy and double-immunofluorescence. The mRNA expression of apoptosis-related genes was investigated using microarray analysis. Oligodendroglial apoptosis was already detected in the predemyelinating phase at 14 dpi. Apoptotic cell numbers peaked at 42 dpi and decreased until 196 dpi partly due to reduced T cell apoptosis. In addition to genes involved in the classical pathways of apoptosis induction, microarray analysis detected the expression of genes related to alternative mechanisms of cell death such as pyroptosis, necroptosis, and endoplasmic reticulum stress. Consequently, oligodendroglial apoptosis is involved in the initiation of the TME demyelination process, whereas the development of apoptosis resistance of T cells potentially favors the maintenance of inflammation and myelin loss.     

Monoclonal anti-I-A antibody reverses chronic paralysis and demyelination in Theiler's virus-infected mice: critical importance of timing of treatment

Susceptibility to demyelination caused by the WW isolate of Theiler's murine encephalomyelitis viruses is linked to class II genes of the major histocompatibility complex. SJL/J (H-2s) mice, expressing only I-As class II gene products of the major histocompatibility complex, are highly susceptible to Theiler's murine encephalomyelitis virus infection with the WW virus isolate, with chronic paralysis and severe inflammation and demyelination in the central nervous system. The effect of in vivo administration of anti-I-As monoclonal antibodies on Theiler's murine encephalomyelitis virus infection was observed. SJL/J mice were treated in various protocols pre- or postinfection. Anti-I-As monoclonal antibody reversed chronic paralysis and reduced inflammation and demyelination when given after the establishment of persistent infection. The effect was long lasting, but clinical signs, inflammation, and demyelination recurred 2 months after treatment ceased. Anti-I-As antibodies had no effect on viral titers within the central nervous system. The timing of the administration of monoclonal antibodies was critical. Administration of anti-I-As before the establishment of the persistent infection resulted in fatal encephalitis.     

Demyelination induced by Theiler's virus: influence of the H-2 haplotype

Demyelination induced by Theiler's virus was examined in nonrecombinant H-2 congeneic strains of mice on common backgrounds expressing independent haplotypes. Light and electron microscopy of spinal cord sections from mice with s, f, p, r, v, or q haplotypes on a C57BL/10 background showed perivascular inflammation and multifocal areas of demyelination in the white matter. The demyelination in these mice was usually associated with clinical neurologic deficits. In contrast, mice with identical genetic backgrounds but b, k, or d haplotypes showed no pathologic or clinical abnormalities. Qa or Tla haplotypes did not appear to influence demyelination. The observations support the hypothesis that susceptibility to Theiler's murine encephalomyelitis virus-induced demyelination is influenced by genes that are linked to the H-2 complex. The findings suggest that the demyelination may be mediated by immune cells rather than being a direct cytolytic effect of virus on oligodendrocytes.     

Characterization of the inflammatory response in the central nervous system of mice susceptible or resistant to demyelination by Theiler's virus

Intracerebral inoculation of mice with Theiler's murine encephalomyelitis virus results in an intense inflammatory response of mononuclear leukocytes which infiltrate into the central nervous system. Resistant strains of mice have the ability to clear virus whereas susceptible strains become infected persistently and are associated with chronic demyelination which is proposed to be immune-mediated. In an attempt to better understand the role of the immune response during demyelination, mononuclear leukocytes were isolated from the central nervous system of infected mice and stained by an immunoperoxidase technique with anti-Thy-1.2, anti-L3T4, anti-Lyt-2 and anti-MAC-1 mAb. Infection of susceptible SJL/J mice resulted in a biphasic immune response which peaked on days 7 and 27 post-infection. In contrast, a single peak (day 7) was observed in resistant C57BL/10SNJ mice. The presence of Thy-1.2, L3T4, and MAC-1+ cells was similar between the two strains. However, although the number of Lyt-2+ cells peaked on day 7 in C57BL/10SNJ mice, they were not detected in SJL/J mice until 14 days post-infection and gradually increased in number over the course of infection. To further study the role of T cells in demyelination, serial frozen sections of brain and spinal cord were stained for the presence of Lyt-2 and L3T4+ cells in the lesions of chronically infected SJL/J mice. L3T4+ cells were observed predominantly in perivascular regions while Lyt-2+ cells were observed infiltrating the parenchyma. These results provide further evidence that Lyt-2+ lymphocytes are important in the mechanism of susceptibility/resistance to Theiler's murine encephalomyelitis virus-induced demyelination.     

Expression of human HLA-B27 transgene alters susceptibility to murine Theiler's virus-induced demyelination

Infection of certain strains of mice with Theiler's murine encephalomyelitis virus results in persistence of virus and an immune-mediated primary demyelination in the central nervous system that resembles multiple sclerosis. Because susceptibility/resistance to demyelination in B10 congeneic mice maps strongly to class I MHC genes (D region) we tested whether expression of a human class I MHC gene (HLA-B27) would alter susceptibility to Theiler's murine encephalomyelitis virus-induced demyelination. Transgenic HLA-B27 mice were found to co-express human and endogenous mouse class I MHC genes by flow microfluorimetry analysis of PBL. In the absence of the human transgene, H-2stf, or v mice but not H-2b mice had chronic demyelination and persistence of virus at 45 days after infection. No difference in degree of demyelination, meningeal inflammation, or virus persistence was seen between transgenic HLA-B27 and nontransgenic littermate mice of H-2f or H-2v haplotype. In contrast, H-2s (HLA-B27+) mice showed a dramatic decrease in extent of demyelination and number of virus-Ag+ cells in the spinal cord compared with H-2s (HLA-B27-) littermate mice. In addition, none of the eight H-2s mice homozygous for HLA-B27 gene had spinal cord lesions even though infectious virus was isolated chronically from their central nervous system. Expression of HLA-B27 transgene did not interfere with the resistance to demyelination normally observed in B10 (H-2b) mice. These experiments demonstrate that expression of a human class I MHC gene can modulate a virus-induced demyelinating disease process in the mouse.     

Clonal expansion of infiltrating T cells in the spinal cords of SJL/J mice infected with Theiler's virus

Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus results in immune-mediated inflammatory demyelination in the white matter and consequent clinical symptoms. This system has been utilized as an important virus model for human multiple sclerosis. Although the potential involvement of virus-specific Th cells has been studied extensively, very little is known about the nature of T cells infiltrating the CNS during viral infection and their role in the development of demyelinating disease. In this study, the clonal nature of T cells in the spinal cord during the disease course was analyzed using size spectratyping and sequencing of the TCR beta-chain CDR3 region. These studies clearly indicate that T cells are clonally expanded in the CNS after viral infection, although the overall TCR repertoire appears to be diverse. The clonal expansion appears to be Ag-driven in that it includes Th cells specific for known viral epitopes. Interestingly, such restricted accumulation of T cells was not detectable in the infiltrates of mice with proteolipid protein peptide-induced experimental autoimmune encephalomyelitis. The initial T cell repertoire (7-9 days postinfection) seems to be more diverse than that observed in the later stage (65 days) of virally induced demyelination, despite the more restricted utilization of Vbeta subfamilies. These results strongly suggest continuous stimulation and clonal expansion of virus-specific T cells in the CNS of Theiler's murine encephalomyelitis virus-infected mice during the entire course of demyelinating disease.     

Depletion of Blood-Borne Macrophages Does Not Reduce Demyelination in Mice Infected with a Neurotropic Coronavirus

Mice infected with the neurotropic coronavirus mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating disease with symptoms of hindlimb paralysis. Histological examination of the brains and spinal cords of these animals reveals the presence of large numbers of activated macrophages/microglia. In two other experimental models of demyelination, experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus-induced demyelination, depletion of hematogenous macrophages abrogates the demyelinating process. In both of these diseases, early events in the demyelinating process are inhibited by macrophage depletion. From these studies, it was not possible to determine whether infiltrating macrophages were required for late steps in the process, such as myelin removal. In this study, we show that when macrophages are depleted with either unmodified or mannosylated liposomes encapsulating dichloromethylene diphosphate, the amount of demyelination detected in MHV-infected mice is not affected. At a time when these cells were completely depleted from the liver, approximately equivalent numbers of macrophages were present in the spinal cords of control and drug-treated animals. These results suggest that blood-borne macrophages are not required for MHV-induced demyelination and also suggest that other cells, such as perivascular macrophages or microglia, perform the function of these cells in the presence of drug.     

Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus.

The DA strain of Theiler's virus, a murine picornavirus, causes a persistent infection of glial cells of the white matter of the spinal cord, associated with chronic inflammation and primary demyelination. The GDVII strain causes an acute fatal grey matter encephalomyelitis. We characterized the target cells of GDVII and DA viruses 4 days following intracerebral inoculation, and we compared the levels of viral RNA within these cells. GDVII virus infected approximately 10 times more cells than DA virus. Whereas GDVII virus infected neurons exclusively, DA virus infected also astrocytes and possible macrophage-microglial cells. The levels of viral RNA in neurons infected with GDVII and DA viruses were of the same order. These results show that DA virus infects glial cells already at the beginning of the disease and that the more efficient spread of GDVII virus is probably not due to a higher level of RNA replication per cell.     

The interaction of two groups of murine genes determines the persistence of Theiler''s virus in the central nervous system.

Theiler's murine encephalomyelitis virus is responsible for a chronic inflammatory demyelinating disease of the central nervous system of the mouse. The disease is associated with persistent viral infection of the spinal cord. Some strains of mice are susceptible to viral infection, and other strains are resistant. The effect of the genetic background of the host on viral persistence has not been thoroughly investigated. We studied the amount of viral RNA in the spinal cords of 17 inbred strains of mice and their F1 crosses with the SJL/J strain and observed a large degree of variability among strains. The pattern of viral persistence among mouse strains could be explained by the interaction of two loci. One locus is localized in the H-2D region of the major histocompatibility complex, whereas the other locus is outside this complex and is not linked to the Tcrb locus on chromosome 6.     

Astrocytic YAP prevents the demyelination through promoting expression of cholesterol synthesis genes in experimental autoimmune encephalomyelitis

Cholesterols are the main components of myelin, and are mainly synthesized in astrocytes and transported to oligodendrocytes and neurons in the adult brain. It has been reported that Hippo/yes-associated protein (YAP) pathways are involved in cholesterol synthesis in the liver, however, it remains unknown whether YAP signaling can prevent the demyelination through promoting cholesterol synthesis in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis characterized by neuroinflammation and demyelination. Here, we found that YAP was upregulated and activated in astrocytes of spinal cords of EAE mice through suppression of the Hippo pathway. YAP deletion in astrocytes aggravated EAE with earlier onset, severer inflammatory infiltration, demyelination, and more loss of neurons. Furthermore, we found that the neuroinflammation was aggravated and the proliferation of astrocytes was decreased in YAP^GFAP-CKO EAE mice. Mechanically, RNA-seq revealed that the expression of cholesterol-synthesis pathway genes such as HMGCS1 were decreased in YAP^−/− astrocytes. qPCR, western blot, and immunostaining further confirmed the more significant reduction of HMGCS1 in spinal cord astrocytes of YAP^GFAP-CKO EAE mice. Interestingly, upregulation of cholesterol-synthesis pathways by diarylpropionitrile (DPN) (an ERβ-ligand, to upregulate the expression of HMGCS1) treatment partially rescued the demyelination deficits in YAP^GFAP-CKO EAE mice. Finally, activation of YAP by XMU-MP-1 treatment promoted the expression of HMGCS1 in astrocytes and partially rescued the demyelination and inflammatory infiltration deficits in EAE mice. These findings identify unrecognized functions of astrocytic YAP in the prevention of demyelination through promoting cholesterol synthesis in EAE, and reveal a novel pathway of YAP/HMGCS1 for cholesterol synthesis in EAE pathology.Subject terms: Multiple sclerosis, Astrocyte, Multiple sclerosis     

Immunoglobulins and complement in demyelination induced in mice by Theiler's virus

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) produces chronic demyelination and persistent infection in the central nervous system (CNS) of susceptible SJL mice. This series of experiments examined the contribution of humoral immunity and C to myelin destruction. As in multiple sclerosis, mice persistently infected with TMEV had elevated levels of IgG and oligoclonal bands in the cerebrospinal fluid (CSF). Immunoblot studies revealed that even in animals exhibiting profound demyelination, IgG in the serum and CSF was directed primarily at virus antigen rather than at normal myelin components. Inflammatory cells positive for Ig were distributed mainly around blood vessels, but occasionally they infiltrated the spinal cord parenchyma. Rare examples of myelin sheaths positive for IgG were found by immunoelectron microscopy in spinal cord sections from infected mice; the third component of complement (C3) was commonly found in the walls of CNS blood vessels but not on myelin. Neither serum nor CSF IgG from infected mice bound to myelin sheaths or other CNS components in sections of normal syngeneic spinal cord. There were significantly more demyelinating lesions in infected mice depleted of C components with cobra venom factor. These data do not support a humoral autoimmune basis for the CNS demyelination that occurs in association with persistent TMEV infection. However, the humoral immune response directed at TMEV antigens may either limit virus spread or promote virus persistence.     

Strains from both Theiler's virus subgroups encode a determinant for demyelination.

The GDVII strain and other members of the GDVII subgroup of Theiler's murine encephalomyelitis viruses (TMEV) cause an acute lethal neuronal infection in mice, whereas the DA strain and other members of the TO subgroup of TMEV cause a chronic demyelinating disease associated with a persistent virus infection. We used GDVII/DA chimeric infectious cDNAs to produce intratypic recombinant viruses in order to clarify reasons for the TMEV subgroup-specific difference in demyelinating activity. We found that both the GDVII and DA strains contain a genetic determinant(s) for demyelinating activity. No demyelination occurs following GDVII strain inoculation because this strain produces an early neuronal disease that kills mice before white matter disease and persistent infection can occur.     

Infection with Theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via NF-kappaB activation: potential role for the initiation of demyelinating disease

Theiler's 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 Theiler's 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-beta]) important in the innate immune response to infection. We find that TMEV-induced cytokine gene expression is mediated by the NF-kappaB pathway based on the early nuclear NF-kappaB translocation and suppression of cytokine activation in the presence of specific inhibitors of the NF-kappaB pathway. Further studies show this to be partly independent of dsRNA-dependent protein kinase (PKR) and IFN-alpha/beta pathways. Altogether, these results demonstrate that infection of astrocytes and other CNS-resident cells by TMEV provides the early NF-kappaB-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.     

Expression of lymphotoxin gene inserted into Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) belongs to the Picornaviridae genus. DA subgroup strains of this virus induce early, non-fatal polioencephalomyelitis followed by demyelination in the spinal cord, with virus persistence. We investigated the use of DA strain as a vector for the introduction of a foreign gene into the central nervous system. Human lymphotoxin (LT) gene was inserted in the L region, the most upstream part of the polyprotein coding region of DA genome. Expression of LT was demonstrated by an immunoblot and an enzyme-linked immunosorbent assay on BHK-21 cells that were infected with the recombinant virus. In addition, the expressed LT showed cytotoxicity against L-929 cells.     

Analysis of Cellular Mutants Resistant to Theiler’s Virus Infection: Differential Infection of L929 Cells by Persistent and Neurovirulent Strains

Theiler's murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse and belongs to the Picornaviridae family. TMEV strains are divided into two subgroups on the basis of their pathogenicity. The first group contains two neurovirulent strains, FA and GDVII, which cause a rapid fatal encephalitis. The second group includes persistent strains, like DA and BeAn, which produce a biphasic neurological disease in susceptible mice. Persistence of these viruses in the white matter of the spinal cord leads to chronic inflammatory demyelination. L929 cells, which are susceptible to TMEV infection, were subjected to physicochemical mutagenesis. Cellular clones that became resistant to TMEV infection were selected by viral infection. Three such mutants resistant to strain GDVII were characterized to determine the step of the virus cycle that was inhibited. The mutation present in one of these mutant cell lines inhibited, by more than 1,000-fold, the entry of strain GDVII but hardly decreased infection by strain DA. In the two other cellular mutants, replication of the viral genome was slowed down. Interestingly, one of these mutant cell lines resisted infection by both the persistent and neurovirulent strains while the second cell line resisted infection by strain GDVII but remained susceptible to the persistent virus. These results show that although they have 95% identity at the amino acid sequence level, neurovirulent and persistent viruses use partly distinct pathways for both entry into cells and genome replication.     

Transgenic expression of Theiler's murine encephalomyelitis virus genes in H-2(b) mice inhibits resistance to virus-induced demyelination

We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2(b)) congenic mice expressing three independent contiguous coding regions of the Theiler's murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8(+) T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8(+) T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.     

Remyelination induced by a DNA aptamer in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler's encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination in vivo. Here we show that a 40-nucleotide single-stranded DNA aptamer selected for affinity to murine myelin shows this property. This aptamer binds multiple myelin components in vitro. Peritoneal injection of this aptamer results in distribution to CNS tissues and promotes remyelination of CNS lesions in mice infected by Theiler's virus. Interestingly, the selected DNA aptamer contains guanosine-rich sequences predicted to induce folding involving guanosine quartet structures. Relative to monoclonal antibodies, DNA aptamers are small, stable, and non-immunogenic, suggesting new possibilities for MS treatment.     

TGF-beta 2 reduces demyelination, virus antigen expression, and macrophage recruitment in a viral model of multiple sclerosis

TGF-beta 2 is a potent immunoregulatory mediator that influences B cell, T cell, and macrophage function. To test whether this cytokine alters pathology in a model of virus-induced demyelinating disease, we treated SJL/J mice with TGF-beta 2 either before or after infection with Theiler's murine encephalomyelitis virus. Treatment continued three times weekly through day 35 postinfection. TGF-beta 2 administration resulted in significantly smaller lesions and fewer virus Ag-positive cells in the spinal cords of infected SJL/J mice. Mice treated with TGF-beta 2 had similar levels of virus-specific IgG as infected, control-treated mice. TGF-beta 2 administration significantly increased the level of non-virus-specific activated CTLs, but had no effect on virus-specific CTLs. TUNEL revealed a decrease in the number of apoptotic nuclei in the spinal cord white matter of mice treated in vivo with TGF-beta 2. Immunostaining with an Ab to F4/80 revealed that TGF-beta 2-treated mice had significantly fewer F4/80-positive cells in the white matter of the spinal cord as compared with infected control-treated mice. These data suggest that TGF-beta 2 may control virus-induced demyelination via an immunomodulatory mechanism that reduces macrophage infiltration.     

Virus persistence in an animal model of multiple sclerosis requires virion attachment to sialic acid coreceptors

Persistent Theiler's virus infection in the central nervous system (CNS) of mice provides a highly relevant animal model for multiple sclerosis. The low-neurovirulence DA strain uses sialic acid as a coreceptor for cell binding before establishing infection. During adaptation of DA virus to growth in sialic acid-deficient cells, three amino acid substitutions (G1100D, T1081I, and T3182A) in the capsid arose, and the virus no longer used sialic acid as a coreceptor. The adapted virus retained acute CNS virulence, but its persistence in the CNS, white matter inflammation, and demyelination were largely abrogated. Infection of murine macrophage but not oligodendrocyte cultures with the adapted virus was also significantly reduced. Substitution of G1100D in an infectious DA virus cDNA clone demonstrated a major role for this mutation in loss of sialic acid binding and CNS persistence. These data indicate a direct role for sialic acid binding in Theiler's murine encephalomyelitis virus persistence and chronic demyelinating disease.