TGF-beta1 suppresses T cell infiltration and VP2 puff B mutation enhances apoptosis in acute polioencephalitis induced by Theiler's virus

GDVII and DA strains of Theiler's murine encephalomyelitis virus (TMEV) differ in VP2 puff B. One week after GDVII virus infection, SJL/J mice had large numbers of TUNEL+ apoptotic cells with a relative lack of T cell infiltration in the brain. DA viruses with mutation in puff B induced higher levels of apoptosis than wild-type DA virus, but levels of inflammation in brains were similar between DA and DA virus mutants. The difference in inflammation among TMEVs could be due to TGF-beta1 expression that was seen only in GDVII virus infection and negatively correlated with CD3+ T cell infiltration.     

Theiler’s virus strain-dependent induction of innate immune responses in RAW264.7 macrophages and its influence on viral clearance versus viral persistence

Infection of susceptible mice with the DA strain of Theiler's murine encephalomyelitis virus (TMEV) induces a persistent central nervous system infection accompanied by demyelination that resembles multiple sclerosis. In contrast, Theiler's GDVII strain does not persist, because infected animals either clear the virus or die. Previously, the authors have shown that in vitro infection of RAW264.7 macrophages displays a similar strain-dependent outcome, resulting in the establishment of a persistent infection with the DA strain and clearance of the GDVII strain. Here, the authors show that when RAW264.7 cells were infected with both strains, the antiviral response triggered by the GDVII virus interfered with the DA virus' ability to induce a persistent infection. Treatment of cells with 2-aminopurine, a protein kinase R inhibitor, increased GDVII virus yields in contrast to DA virus yields. By comparing the antiviral activity of RAW264.7 macrophages against TMEV, it was found that GDVII-infected macrophages mounted a five times more potent antiviral response than the DA-infected ones, indicating that there are strain-dependent differences in the induction of host innate immune responses. Measurements of interferon (IFN) production confirmed this finding. In addition, it was found that the macrophages' antiviral response is dependent on the multiplicity of infection. The antiviral activity resulting from GDVII-infected macrophages could be partially neutralized with antibodies against IFN-alpha or IFN-gamma, but not with an anti-IFN-beta antibody. Because only a partial neutralization was reached, the authors speculate that apart from the investigated IFNs, other cellular factors contribute to the observed antiviral activity. Taken together, these results demonstrate the importance of host innate immune responses in determining the balance between viral clearance and viral persistence.     

Peripheral nerve protein, P0, as a potential receptor for Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) belongs the family Picornaviridae. TMEV not only replicates in the gastrointestinal tract but also spreads to the central nervous system (CNS) either by a hematogenous or a neural pathway during natural infection. The DA strain of TMEV infects neurons during the acute phase, and glial cells and macrophages during the chronic phase, leading to a demyelinating disease similar to multiple sclerosis. Different virus-host receptor interactions in the peripheral and the neuronal cells could explain the pathways of viral spread from the peripheral to the CNS and neurons to glial cells. However, the receptor for TMEV remains unknown. P0 protein, a 28-31 kD glycoprotein, belongs to the immunoglobulin superfamily and constitutes 50% of the total myelin protein in the peripheral nerve. Other picornaviruses use members of the immunoglobulin superfamily as receptors. Thus we hypothesized P0 protein could act as a receptor for TMEV. In a virus overlay assay, radiolabeled TMEV bound to a 28-30 kD protein from the peripheral nerve of wild-type C57BL/6, but no binding was found in the peripheral nerve from P0-knockout mice. TMEV replicated fourfold higher in P0-transfected BW5147.G.1.4 cells than in mock-transfected cells. The increase in virus replication in the P0-transfected cell line was blocked by preincubation of the cells with anti-P0 antibody. A virus binding study showed that TMEV bound to P0-transfected cells but not to mock-transfected cells. The use of the P0 protein in Schwann cells as a receptor may be one mechanism by which TMEV spreads from the gastrointestinal tract to the CNS.     

Cytokine/chemokine profile in J774 macrophage cells persistently infected with DA strain of Theiler’s murine encephalomyelitis virus (TMEV)

Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus and persists in the spinal cords of mice, followed by inflammatory demyelinating disease. Viral persistence is a key determinant for the TMEV-induced demyelination. Macrophages are thought to serve as the site of TMEV persistence during the chronic demyelinating phase. We previously demonstrated that two nonstructural proteins of TMEV, L and L*, were important for virus growth in J774.1 macrophage cells. However, the key factors of macrophage cells related to virus persistence and demyelination remain poorly understood. The inflammatory response is heavily dependent on cytokine and chemokine production by cell of both the immune system and the central nervous system (CNS). In this study, we established the macrophage cells persistently infected with DA strain, and then analyzed the cytokine expression pattern in those cells. The present results are the first to demonstrate the up-regulation of B-lymphocyte chemoattractant (BLC) and granulocyte colony-stimulating factor (G-CSF) in the macrophage cells persistently infected with DA strain. Furthermore, up-regulation of interleukin (IL)-10 and down-regulation of interferon (IFN)-α4, IFN-β, and IFN-γ were shown in those cells. The data suggest that these cytokines/chemokines may contribute to the virus persistence and the acceleration of TMEV-induced demyelination.     

Brain CD8+ and cytotoxic T lymphocytes are associated with, and may be specific for, human immunodeficiency virus type 1 encephalitis in patients with acquired immunodeficiency syndrome

CD8+ T cells infiltrate brains with human immunodeficiency virus type-1 (HIV-1) encephalitis (HIVE) and related animal models; their perineuronal localization suggests cytotoxic T cell (CTL)-mediated neuronal killing. Because CTLs have not been identified in acquired immunodeficiency syndrome (AIDS) brains, the authors identified their cytotoxic granules in autopsy AIDS brains with HIVE and without HIVE (HIVnE) plus controls (7 to 13 cases/group) and determined gene expression profiles of CTL-associated genes in a separate series of cases. CD3+ and CD8+ T cells were significantly increased (P < .01) in perivascular spaces and inflammatory nodules in HIVE but were rare or absent in brain parenchyma in HIVnE and control brains. Eight HIVE brains contained granzyme B+ T cells and five contained perforin+ T cells. Their T-cell origin was confirmed by colocalization of CD8 and granzyme B in the same cell and the absence of CD56+ natural killer cells. The CTLs directly contacted with neurons, as the authors showed previously for CD3+ and CD8+ T cells. CTLs were rare or absent in HIV nonencephalitis (HIVnE) and controls. Granzyme B and H precursor gene expression was up-regulated and interleukin (IL)-12A precursor, a maturation factor for natural killer cells and CTLs, was down-regulated in HIVE versus HIVnE brain. This study demonstrates, for the first time, CTLs in HIVE and shows that parenchymal T cells and CTLs are sensitive biomarkers for HIVE. Consequently, CD8+ T cells and CTLs could mediate brain injury in HIVE and may represent an important biomarker for productive brain infection by HIV-1.     

CD8(+) T cells from Theiler's virus-resistant BALB/cByJ mice downregulate pathogenic virus-specific CD4(+) T cells

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus which induces an immune-mediated demyelinating disease in susceptible strains of mice and serves as a relevant animal model for multiple sclerosis. Treatment with low dose irradiation prior to infection with the BeAn strain of TMEV renders the genetically resistant BALB/cByJ (C/cByJ) mice susceptible to disease. Previous studies have shown that disease resistance in the C/cByJ is mediated by a 'regulatory' CD8(+) T cell population, which does not appear to function via a cytolytic mechanism. We show here that TMEV-specific CD4(+) T cell blasts transferred into susceptible, irradiated C/cByJ accelerate clinical disease and enhance TMEV-specific DTH and proliferation in these animals. Significantly, CD8(+) cells from infected, resistant C/cByJ mice specifically downregulate the in vivo disease potentiation and diminish virus specific DTH, and proliferative and pro-inflammatory cytokine responses (IFNgamma and IL-2) in recipients of TMEV-specific CD4(+) T cell blasts. These results indicate that TMEV infection of resistant C/cByJ mice induces a radiosensitive population of regulatory CD8(+) T cells which actively downregulate inherent Th1 responses which have disease initiating potential.     

Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis

Nonfatal paralysis, induced by the attenuated Pasteur strain of rabies virus, is characterised by local and irreversible flaccid paralysis of the inoculated limbs. We characterised the spread and localisation of virus in the CNS of infected mice, determined the nature of cell injury and examined the role of the immune response. Data indicate that infection of BALB/c mice induced paralysis in 60% of infected mice, the others recovering without sequelae. In both groups of mice, virus was detected in restricted sub-populations of neurons from the brain and spinal cord, and intensity of the neuropathology correlated with levels of rabies RNA and apoptotic infected neurons. However, apoptosis of neurons and paralysis were not due to a direct deleterious effect of the virus, but induced by a T-dependent immune response, as evidenced by their absence in nude mice. Paralysed and asymptomatic mice developed a similar rabies virus-specific IgG2a antibody response, thus excluding the role of any modification of the humoral immune response. In contrast, three events were critically associated with the development of neurological symptoms: the amount of virus in the CNS, the level of apoptosis in both infected neurons and uninfected surrounding cells and the progressive parenchymal infiltration of CD4+ and CD8+ T cells at the site of infection. These data suggest that during nonfatal rabies infection, the levels of viral replication and primary degeneration of infected neurons by apoptosis could be responsible for the infiltration of T lymphocytes capable of inducing secondary degeneration of neural cells.     

A comparative study of acute and chronic diseases induced by two subgroups of Theiler’s murine encephalomyelitis virus

Theiler’s murine encephalomyelitis viruses (TMEV) are divided into two subgroups on the basis of their different biological activities. The GDVII strain produces acute polioencephalomyelitis in mice, whereas the DA strain produces demyelination with virus persistence in the spinal cord. A comparative study of GDVII and DA strains suggested that low host immune responses are responsible for the development of acute GDVII infection and that the persistence of infected macrophages plays a crucial role in the development of chronic white matter lesions in DA infection. All 78 mice infected with GDVII died or became moribund by day 13, while none of 54 mice infected with DA died. In the acute stage, the distribution of viral antigens in the central nervous system (CNS) tissue was similar in both GDVII and DA infections, although the virus titer was higher in GDVII infection. In DA infection, a substantial number of T cells were recruited to the CNS on day 6 when they were virtually absent in GDVII infection. The titer of neutralizing antibody was already high on day 6 in DA infection but was negligible in GDVII infection. Development of chronic paralytic disease from day 35 of the DA infection was accompanied by focal accumulation of viral antigen-positive macrophages in the spinal white matter. In addition, white matter lesions comparable to those in chronic DA infection were induced in the spinal cord within 7 days after intracerebral injection of DA-infected murine macrophages. Received: 26 June 1995 / Revised, accepted: 27 December 1995     

The role of cellular immune response in Theiler's virus-induced central nervous system demyelination

Theiler's murine encephalomyelitis virus (TMEV) persists in spinal cord white matter of susceptible mice (e.g., SJL/J), resulting in chronic inflammation and demyelination. Reconstitution of severe combined immunodeficient (SCID) mice with CD4(+) T- or CD8(+) T-lymphocytes results in extensive TMEV-induced demyelination, and depletion of CD8(+) T-lymphocytes in the early or late phase of the disease decreases the extent of demyelination, indicating that the cellular immune response against the virus plays a key role in myelin destruction. In susceptible mice, the demyelinated lesions are characterized by infiltration of a large numbers of B- and T-lymphocytes; whereas in mice resistant to TMEV-induced demyelination (e.g., C57BL/6), virus clearance requires infiltration of between 2.9 x 10(5) and 5.7 x 10(5) CD8(+) T-lymphocytes and between 3.4 x 10(5) and 6.1 x 10(5) CD4(+) T-lymphocytes per mouse in the brain 5-9 days post infection. Transgenic expression of capsid proteins of TMEV abrogates resistance in C56BL/6 mice, rendering the mice susceptible to TMEV persistence and demyelination. Comparison of the kinetics of virus replication and B- and T-lymphocyte infiltration in mice lacking key adhesion molecules (L-selectin (L-sel(-/-)), P-selectin (P-sel(-/-)), intracellular adhesion molecule-1 (ICAM-1(-/-)), or leukocyte function-associated antigen-1 (LFA-1(-/-))) demonstrates a role for individual adhesion molecules in recruitment of immune cells into central nervous system (CNS), but the role is not significant to prevent eventual virus clearance.     

CD8+ T cells reduce in vitro interferon-gamma production in Theiler's murine encephalomyelitis virus-induced demyelinating disease model

In the Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease model for multiple sclerosis, regulatory CD8+ T cells prevent demyelinating disease and reduce in vivo interferon (IFN)-gamma production by anti-TMEV CD4+ blast cells in BALB/c mice. We describe here that regulatory CD8+ T cells reduce in vitro IFN-gamma production by lymph node cells from both TMEV and fowl gamma globulin immunized mice without affecting interleukin (IL)-4, IL-10, tumour growth factor-beta or tumour necrosis factor-alpha production.     

Theiler's murine encephalomyelitis virus and mechanisms of its persistence

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups: GDVII and DA (or TO). The GDVII subgroup strains are highly virulent and produce acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination occurs. In contrast, the DA (or TO) subgroup strains are less virulent and establish a persistent infection in the spinal cords of mice, despite a vigorous humoral immune response, followed by chronic demyelination. Theiler's murine encephalomyelitis virus is an excellent animal model for the human demyelinating disease multiple sclerosis. Several interesting features regarding TMEV persistence have been reported. A majority of TMEV-infected cells during the chronic stage of the disease contain only 100–500 copies of the genome, which is less than the number necessary for detectable expression of viral proteins. The limited viral protein synthesis probably induces relatively little cytopathic effect and also allows the virus to bypass immunological clearance. Sequence analysis of escape mutant viruses resistant to DA neutralizing monoclonal antibodies identified four separate amino acid mutations of capsid proteins, all of which are located on or near the rim of the ‘pit’, a putative receptor binding region of the virus. In addition, mutant viruses demonstrated a decrease of demyelinating activity. These findings suggest that antibody mediates neutralization by preventing the binding of virus to the cell receptor and that demyelination with virus persistence can be interfered with by preventing viral attachment to receptors of particular cell types. Finally, a 17 kDa protein, designated as L*, is synthesized in DA subgroup strains from an alternative, out-of-frame initiation site. Studies of a DA mutant virus without L* protein synthesis demonstrated that this protein is important for virus growth in particular cell types and is critical for DA-induced demyelinating disease and virus persistence.     

Persistent infection of RAW264.7 macrophages with the DA strain of Theiler’s murine encephalomyelitis virus: An in vitro model to study viral persistence

Theiler's murine encephalomyelitis virus (TMEV) is a member of the Picornaviridae family and causes a virus strain dependent pathology in the central nervous system of mice. The GDVII strain induces an acute and mostly fatal encephalomyelitis. In the few mice that survive, the virus is cleared by the immune system. In contrast, infection with the DA strain leads to a persistent infection, marked by inflammation and demyelination that resembles multiple sclerosis. In the DA-induced disease, macrophages play a crucial role because they contribute to demyelination by the secretion of toxic mediators. Moreover, they represent the main viral reservoir, hereby also underlining their essential role in TMEV persistence. The mechanism of this persistence is not yet understood and tools to investigate it directly, without the complexity imposed by experimental animals, are largely missing. By studying TMEV infection of RAW264.7 macrophages, we found that the DA strain establishes a persistent infection in these cells, in contrast to the neurovirulent GDVII strain. Whereas the GDVII strain was cleared within 4 to 5 days post infection, DA virions were still present after 1 year of cell cultivation. This persistently DA-infected macrophage cell line, which we have called DRAW, provides a model to investigate the interactions between the cellular and viral factors influencing persistence and to screen for anti-TMEV agents.     

Flow cytometry evaluation of the T-cell receptor Vbeta repertoire among human T-cell lymphotropic virus type-1 (HTLV-1) infected individuals: effect of interferon alpha therapy in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

Human T-cell lymphotropic virus type-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is chronic inflammatory disease of the spinal cord characterized by perivascular lymphocytic cuffing and parenchymal lymphocytic infiltration. In this study using flow cytometry, we have investigated the T-cell receptor (TCR) Vbeta repertoire of peripheral blood T lymphocytes in 8 HAM/TSP patients, 10 HTLV-1 infected healthy carriers, and 11 uninfected healthy controls to determine if there is a biased usage of TCR Vbeta. We found that TCR Vbeta7.2 was under-utilized and Vbeta12 was over-utilized in CD4+ T cells of HTLV-1 infected individuals compared with healthy uninfected controls, whereas there were no such differences in CD8+ T cells. Comparison of Vbeta repertoire changes before and after interferon-alpha (IFN-alpha) treatment for HAM/TSP revealed that one out of five patients showed dramatic decrease of specific Vbeta in CD8+ T cells. Our results suggest that dominant Vbeta subpopulations in CD4+ T cells evolved associated with chronic HTLV-1 infection, and IFN-alpha treatment for HAM/TSP does not induce a specific pattern of TCR Vbeta changes.     

Spread of HSV-1 to the suprachiasmatic nuclei and retina in T cell depleted BALB/c mice

Following uniocular anterior chamber inoculation of the KOS strain of HSV-1 in euthymic BALB/c mice, virus spreads from the injected eye to the brain, and from the brain to the optic nerve and retina of the uninjected eye by day 7 post inoculation (P.I.), but the optic nerve and retina of the injected eye are not infected with virus. Infection of the optic nerve and retina of the injected eye is observed only in athymic mice or in mice depleted of both CD4⁺ and CD8⁺ T cells. To determine the role of T cells in virus spread, adult female BALB/c mice were thymectomized and T cell depleted. Mice were co-injected with the KOS strain of HSV-1 and RH116, a thymidine kinase-negative mutant of KOS containing the Escherichia coli lac Z gene. Animals were sacrificed on days 3–7 P.I., and the eyes and brains were examined for blue-stained, virus-infected cells. A difference in the timing of virus infection was observed in the area of the suprachiasmatic nuclei only in mice depleted of both CD4⁺ and CD8⁺ T cells, and in this group, the contralateral suprachiasmatic nucleus was infected two days earlier. Since one route by which virus could infect the retina of the injected eye is via connections of the contralateral suprachiasmatic nucleus to the ipsilateral optic nerve, these findings suggest that (a) retinitis observed in the injected eyes of mice depleted of both CD4⁺ and CD8⁺ T cells results from virus infection of the contralateral suprachiasmatic nucleus followed by spread of virus to the ipsilateral optic nerve and retina and (b) early HSV-1 infection of the contralateral suprachiasmatic nucleus is prevented by a T cell dependent mechanism.     

Immunization with structural and non-structural proteins of Theiler’s murine encephalomyelitis virus alters demyelinating disease

Theiler’s murine encephalomyelitis virus (TMEV) causes a demyelinating disease similar to multiple sclerosis in the central nervous system (CNS) of susceptible SJL/J mice. Immune responses to TMEV contribute to viral clearance as well as to demyelination. We constructed recombinant vaccinia viruses (VV) that encode each or all of the capsid proteins (VV_VP1, VV_VP2, VV_VP3, VV_VP4, and VV_all) or non-structural proteins (VV_P2, VV_P2P3, and VV_3′P3) of the Daniels strain of TMEV. To determine the role of each of the coding regions of TMEV in vivo, we immunized SJL/J mice with each recombinant VV, with or without subsequent TMEV infection. The groups of mice were compared clinically, immunologically, and histologically. No mice immunized with any recombinant VV without subsequent TMEV infection developed demyelination. However, antibody responses to TMEV were detected in mice immunized with VV_all. In addition, in some mice, VV_P2 immunization induced mild meningitis. VV_VP3 or VV_VP4 immunization of mice prior to TMEV infection ameliorated TMEV-induced pathology or clinical signs of disease. The beneficial effect of VP4 immunization was also seen through DNA immunization with a plasmid encoding VP4 and leader prior to TMEV infection. Therefore, vaccination against not only surface capsid proteins (VV_VP3 and VV_all) but also non-surface capsid protein (VV_VP4), and non-structural proteins (VV_P2) can elicit immune responses to virus or modulate subsequent viral-induced CNS disease.     

Chronic social stress impairs virus specific adaptive immunity during acute Theiler's virus infection

Prior exposure to social disruption (SDR) stress exacerbates Theiler's murine encephalomyelitis virus (TMEV) infection, a model of multiple sclerosis. Here we examined the impact of SDR on T cell responses to TMEV infection in SJL mice. SDR impaired viral clearance and exacerbated acute disease. Moreover, TMEV infection alone increased CD4 and CD8 mRNA expression in brain and spleen while SDR impaired this response. SDR decreased both CD4⁺ and CD8⁺ virus-specific T cells in CNS, but not spleen. These findings suggest that SDR-induced suppression of virus-specific T cell responses contributes to impairments in viral clearance and exacerbation of acute disease.     

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 beta2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however beta2M-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. beta2M-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.     

Persistence of measles virus in rat brain neurons is promoted by depletion of CD8+ T cells

Fourteen-day-old Lewis rats were injected intracerebrally with the hamster neurotropic (HNT) strain of measles virus. At the same time, CD8+ T cytotoxic cells were eliminated by a single injection of a mouse monoclonal antibody (Ox8) directed against this lymphocyte phenotype. The lymphocyte depletion, which endured for more than 7 weeks, markedly reduced the elimination of measles virus antigen from the brain, but did not affect the induction of major histocompatibility complex (MHC) molecules in the early phase of infection. These results demonstrate a role for MHC class I-restricted CD8+ T cells in controlling persistence of measles virus infection in neurons.