IL-27 has been shown to play a suppressive role in experimental autoimmune encephalomyelitis (EAE) as demonstrated by more severe disease in IL-27R-deficient (WSX-1−/−) mice. However, whether IL-27 influences the induction or effector phase of EAE is unknown. This is an important question as therapies for autoimmune diseases are generally started after autoreactive T cells have been primed. In this study, we demonstrate maximal gene expression of IL-27 subunits and its receptor in the CNS at the effector phases of relapsing-remitting EAE including disease peak and onset of relapse. We also show that activated astrocyte cultures secrete IL-27p28 protein which is augmented by the endogenous factor, IFN-γ. To investigate functional significance of a correlation between gene expression and disease activity, we examined the effect of IL-27 at the effector phase of disease using adoptive transfer EAE. Exogenous IL-27 potently suppressed the ability of encephalitogenic lymph node and spleen cells to transfer EAE. IL-27 significantly inhibited both nonpolarized and IL-23-driven IL-17 production by myelin-reactive T cells thereby suppressing their encephalitogenicity in adoptive transfer EAE. Furthermore, we demonstrate a strong suppressive effect of IL-27 on active EAE in vivo when delivered by s.c. osmotic pump. IL-27-treated mice had reduced CNS inflammatory infiltration and, notably, a lower proportion of Th17 cells. Together, these data demonstrate the suppressive effect of IL-27 on primed, autoreactive T cells, particularly, cells of the Th17 lineage. IL-27 can potently suppress the effector phase of EAE in vivo and, thus, may have therapeutic potential in autoimmune diseases such as multiple sclerosis.

Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS.Recent studies have demonstrated that significant axonal injury also occurs in MS patients and correlates with neurological dysfunction, but it is not known whether this neuronal damage is a primary disease process, or occurs only secondary to demyelination. In the current studies, neurotropic strains of mouse hepatitis virus (MHV) that induce meningitis, encephalitis, and demyelination in the CNS, an animal model of MS, were used to evaluate mechanisms of axonal injury. The pathogenic properties of genetically engineered isogenic spike protein recombinant demyelinating and nondemyelinating strains of MHV were compared. Studies demonstrate that a demyelinating strain of MHV causes concomitant axonal loss and macrophage-mediated demyelination. The mechanism of axonal loss and demyelination in MHV infection is dependent on successful transport of virus from gray matter to white matter using the MHV host attachment spike glycoprotein. Our data show that axonal loss and demyelination can be independent direct viral cytopathic events, and suggest that similar direct axonal damage may occur in MS. These results have important implications for the design of neuroprotective strategies for CNS demyelinating disease, and our model identifies the spike protein as a therapeutic target to prevent axonal transport of neurotropic viruses.

Demyelination is the pathologic hallmark of the human immune-mediated neurologic disease multiple sclerosis, which may be triggered or exacerbated by viral infections. Several experimental animal models have been developed to study the mechanism of virus-induced demyelination, including coronavirus mouse hepatitis virus (MHV) infection in mice. The envelope spike (S) glycoprotein of MHV contains determinants of properties essential for virus-host interactions. However, the molecular determinants of MHV-induced demyelination are still unknown. To investigate the mechanism of MHV-induced demyelination, we examined whether the S gene of MHV contains determinants of demyelination and whether demyelination is linked to viral persistence. Using targeted RNA recombination, we replaced the S gene of a demyelinating virus (MHV-A59) with the S gene of a closely related, nondemyelinating virus (MHV-2). Recombinant viruses containing an S gene derived from MHV-2 in an MHV-A59 background (Penn98-1 and Penn98-2) exhibited a persistence-positive, demyelination-negative phenotype. Thus, determinants of demyelination map to the S gene of MHV. Furthermore, viral persistence is insufficient to induce demyelination, although it may be a prerequisite for the development of demyelination.Primary demyelination is a pathologic process in which myelin is destroyed, while neuronal axons remain relatively preserved. The demyelinating process can occur by either a direct attack on oligodendrocytes, the cells that produce and maintain myelin, or an autoimmune attack against myelin components, resulting in secondary destruction of oligodendrocytes. The most prevalent primary demyelinating disease in humans is multiple sclerosis (MS), which affects over a quarter of a million individuals in the United States (2). The etiology and pathogenesis of MS have long been investigated; however, causation has not been proven. There is a consensus that the process involves a T-cell-mediated autoimmune phenomenon that may be triggered by one or more viral infections (1). Several experimental animal models have been developed in order to better understand the mechanism of demyelination. Experimental autoimmune encephalomyelitis and several virus-induced experimental models, including coronavirus infection in mice (5), have been instrumental in providing insight into the pathogenesis of demyelination.Coronaviruses, members of the order Nidovirales, form a group of enveloped single-stranded RNA viruses (22,33,36,46). Many members of the nidoviruses, including coronaviruses and arteriviruses, infect the central nervous system and provide experimental animal model systems for neurologic diseases (30). The A59 and JHM strains of the murine member of the coronaviruses (mouse hepatitis virus [MHV]) produce demyelination in mice that mimics many of the pathologic features of MS (12,15,26,39,42,46,47). Chronic MHV-induced demyelination is immune mediated (11, 45), may be partially T-cell dependent (8), and is associated with viral persistence (25, 39) and concomit...

Targeted recombination was used to select mouse hepatitis virus isolates with stable and efficient expression of the gene encoding the enhanced green fluorescent protein (EGFP). The EGFP gene was inserted into the murine coronavirus genome in place of the nonessential gene 4. These viruses expressed the EGFP gene from an mRNA of slightly slower electrophoretic mobility than mRNA 4. EGFP protein was detected on a Western blot of infected cell lysates and EGFP activity (fluorescence) was visualized by microscopy in infected cells and in viral plaques. Expression of EGFP remained stable through at least six passages in tissue culture and during acute infection in the mouse central nervous system. These viruses replicated with similar kinetics and to similar final extents as wild-type virus both in tissue culture and in the mouse central nervous system (CNS). They caused encephalitis and demyelination in animals as wild-type virus; however, they were somewhat attenuated in virulence. Isogenic EGFP-expressing viruses that differ only in the spike gene and express either the spike gene of the highly neurovirulent MHV-4 strain or the more weakly neurovirulent MHV-A59 strain were compared; the difference in virulence and patterns of spread of viral antigen reflected the differences between parental viruses expressing each of these spike genes. Thus, EGFP-expressing viruses will be useful in the studies of murine coronavirus pathogenesis in mice.

BackgroundInterleukin-17A (IL-17A) is the founding member of a novel family of inflammatory cytokines that plays a critical role in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-17A signals through its receptor, IL-17RA, which is expressed in many peripheral tissues; however, expression of IL-17RA in the central nervous system (CNS) and its role in CNS inflammation are not well understood.MethodsEAE was induced in C57Bl/6 mice by immunization with myelin oligodendroglial glycoprotein. IL-17RA expression in the CNS was compared between control and EAE mice using RT-PCR, in situ hybridization, and immunohistochemistry. Cell-type specific expression was examined in isolated astrocytic and microglial cell cultures. Cytokine and chemokine production was measured in IL-17A treated cultures to evaluate the functional status of IL-17RA.ResultsHere we report increased IL-17RA expression in the CNS of mice with EAE, and constitutive expression of functional IL-17RA in mouse CNS tissue. Specifically, astrocytes and microglia express IL-17RA in vitro, and IL-17A treatment induces biological responses in these cells, including significant upregulation of MCP-1, MCP-5, MIP-2 and KC chemokine secretion. Exogenous IL-17A does not significantly alter the expression of IL-17RA in glial cells, suggesting that upregulation of chemokines by glial cells is due to IL-17A signaling through constitutively expressed IL-17RA.ConclusionIL-17RA expression is significantly increased in the CNS of mice with EAE compared to healthy mice, suggesting that IL-17RA signaling in glial cells can play an important role in autoimmune inflammation of the CNS and may be a potential pathway to target for therapeutic interventions.

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.Multiple sclerosis is a common disabling neurological disease, pathologically characterized by demyelination, loss of oligodendroglial cells, and axonal degeneration (25,26). The mechanisms that culminate in the destruction of oligodendrocytes and loss of central nervous system (CNS) myelin are not well understood. The process is believed to involve a T-cellmediated autoimmune phenomenon that may be triggered by one or more viral infections (1). Several experimental viral model systems have been instrumental in providing these insights (2). One of the animal models is based on mouse hepatitis virus (MHV)-induced demyelination in mice that mimics the pathology of multiple sclerosis (12,19,21,39,42). Infection with neurotropic MHV strains produces a biphasic neurological disease, with acute meningoencephalitis preceding the onset of chronic demyelination (21).Following intracranial inoculation, MHV is first observed in the brain and subsequently spreads into the spinal cord (30). The viral titer reaches its peak at approximately day 5 postinfection. Infectious viral particles are cleared within the first 7 to 10 days after infection, although viral RNA persists in the white matter of the spinal cord for several months (20,29). Viral RNA persistence has previously been demonstrated in spinal cords of mice infected with demyelinating strains of MHV, including MHV-A59 and JHM (17,20,29), and has previo...

Optic neuritis (ON), an inflammatory demyelinating optic nerve disease, occurs in multiple sclerosis (MS).Pathological mechanisms and potential treatments for ON have been studied via experimental autoimmune MS models. However, evidence suggests that virus-induced inflammation is a likely etiology triggering MS and ON; experimental virus-induced ON models are therefore required. We demonstrate that MHV-A59, a mouse hepatitis virus (MHV) strain that causes brain and spinal cord inflammation and demyelination, induces ON by promoting mixed inflammatory cell infiltration. In contrast, MHV-2, a nondemyelinating MHV strain, does not induce ON. Results reveal a reproducible virus-induced ON model important for the evaluation of novel therapies.Significant neuronal damage, with loss of retinal ganglion cell (RGC) axons that comprise the optic nerve, occurs following inflammatory optic neuritis (ON) and correlates with permanent vision loss (2,6,28). Experimental ON is a useful model to examine mechanisms of neuronal damage in multiple sclerosis (MS) because RGCs can readily be labeled and quantified (23). Rodents immunized with myelin proteins develop experimental autoimmune encephalomyelitis (EAE), a model of MS with inflammation in the brain, spinal cord, and optic nerves (12). Mechanisms of neuronal damage during ON in EAE have been examined but vary between chronic (16, 19) and relapsing (23) EAE models, suggesting that different causes of ON may mediate vision-threatening neuronal damage by distinct mechanisms.MS may be caused by a viral infection triggering an immune response against myelin (1, 26). Studies of virus-mediated models of MS are therefore important for our understanding of disease mechanisms and the development of novel therapies. While virus-induced models of MS exist (15, 26), the incidence of ON has not been characterized.Mouse hepatitis virus (MHV) infection in mice has been used as a model for virus-induced demyelination that mimics many pathological features of MS (9,13,15,27,29). Some neurotropic strains of MHV induce a biphasic neurological disease with acute meningoencephalitis, followed by chronic demyelination (15). Similarly to results for autoimmune mod- els of MS, Dandekar et al. recently demonstrated that axonal damage occurs in mice with MHV-induced demyelination (3).It is not known whether the inflammatory demyelination and axonal loss observed to occur in the MHV-infected mouse brain and spinal cord also affect the optic nerve.In the current study, we inoculated mice with plaque-purified demyelinating strain MHV-A59 (14, 15) and nondemyelinating strain MHV-2 (10). MHV-A59 infects a variety of cell types, including neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells (11,13,15,30), in the central nervous system (CNS) and causes acute encephalitis, meningitis, hepatitis, and chronic demyelination. In contrast, MHV-2, a strain closely related to MHV-A59, has a limited ability to invade the brain and spinal cord, causing meningitis without encephalitis or demyelination ...