Cutting Edge: TLR3 stimulation suppresses experimental autoimmune encephalomyelitis by inducing endogenous IFN-beta

Experimental autoimmune encephalomyelitis is a well-characterized model of cell-mediated autoimmunity. TLRs expressed on APCs recognize microbial components and induce innate immune responses, leading to the elimination of invading infectious agents. Certain TLR agonists have been reported to have adjuvant properties in CNS autoimmune inflammatory demyelination. We report in this study that TLR3 stimulation by polyinosinic-polycytidylic acid, a double-stranded RNA analog, suppresses relapsing demyelination in a murine experimental autoimmune encephalomyelitis model. Disease suppression is associated with the induction of endogenous IFN-beta and the peripheral induction of the CC chemokine CCL2. These data indicate that a preferential activation of the MyD88-independent, type I IFN-inducing TLR pathway has immunoregulatory potential in this organ-specific autoimmune disease.     

Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis.

Bone marrow (BM)-derived dendritic cells (DC) are potent stimulators of naive CD4+ T cell activation. Because DC are efficient at Ag processing and could potentially present self Ags, we investigated the role of DC in the presentation of an encephalitogenic peptide from myelin basic protein (Ac1-11) in the induction of experimental autoimmune encephalomyelitis (EAE). To determine if DC could prime for EAE, we transferred DC pulsed with Ac1-11 or with medium alone into irradiated mice in combination with CD4+ T cells isolated from a mouse transgenic for a TCR specific for Ac1-11 + I-Au. Mice transferred with Ac1-11-pulsed DC developed EAE 7-10 days later, whereas mice receiving medium-pulsed DC did not. By day 15, all mice given peptide-loaded DC had signs of tail and hind limb paralysis, and by day 20 infiltration of Ac1-11-specific CD4+ T cells was detected in the brain parenchyma. We also demonstrated interactions between Ac1-11-pulsed DC and Ac1-11-specific T cells in the lymph nodes 24 h following adoptive transfer of both cell populations. These data show that DC can efficiently present the self Ag myelin basic protein Ac1-11 to Ag-specific T cells in the periphery of mice to induce EAE.     

Lithium prevents and ameliorates experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) models, in animals, many characteristics of multiple sclerosis, for which there is no adequate therapy. We investigated whether lithium, an inhibitor of glycogen synthase kinase-3 (GSK3), can ameliorate EAE in mice. Pretreatment with lithium markedly suppressed the clinical symptoms of EAE induced in mice by myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization and greatly reduced demyelination, microglia activation, and leukocyte infiltration in the spinal cord. Lithium administered postimmunization, after disease onset, reduced disease severity and facilitated partial recovery. Conversely, in knock-in mice expressing constitutively active GSK3, EAE developed more rapidly and was more severe. In vivo lithium therapy suppressed MOG35-55-reactive effector T cell differentiation, greatly reducing in vitro MOG35-55- stimulated proliferation of mononuclear cells from draining lymph nodes and spleens, and MOG35-55-induced IFN-gamma, IL-6, and IL-17 production by splenocytes isolated from MOG35-55-immunized mice. In relapsing/remitting EAE induced with proteolipid protein peptide139-151, lithium administered after the first clinical episode maintained long-term (90 days after immunization) protection, and after lithium withdrawal the disease rapidly relapsed. These results demonstrate that lithium suppresses EAE and identify GSK3 as a new target for inhibition that may be useful for therapeutic intervention of multiple sclerosis and other autoimmune and inflammatory diseases afflicting the CNS.     

Reduced proteasomal activity contributes to the accumulation of carbonylated proteins in chronic experimental autoimmune encephalomyelitis

We have recently shown that several carbonylated proteins, including glial fibrillary acidic protein, β-actin and β-tubulin, accumulate within cerebellar astrocytes during the chronic phase of myelin-oligodendrocyte glycoprotein (MOG)(35-55) peptide-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. As protein carbonyls cannot be repaired and there is less oxidative stress in chronic than in acute EAE, we hypothesized that the accumulation of carbonylated proteins in these animals may be due to a defect in the degradation of the modified proteins. Alternatively, oxidized proteins in chronic EAE mice may be more resistant to proteolysis. Using lipopolysaccharide-stimulated astrocytes and several protease inhibitors we identified the 20S proteasome as the proteolytic system responsible for the elimination of most oxidized proteins. We also discovered that the chymotrysin-like and caspase-like activities of the 20S proteasome are impaired in chronic EAE, while the amount of proteasome was unchanged. Proteasome failure in these animals was confirmed by the build-up of ubiquitinated proteins, mostly within astrocytes. In a cell-free system, carbonylated proteins from EAE mice with acute and chronic disease seem to be equally sensitive to proteasomal degradation. Altogether, the results support the notion that diminished activity of the 20S proteasome is a major contributor to the accumulation of carbonylated proteins in astrocytes of chronic EAE mice.     

Attenuation of experimental autoimmune encephalomyelitis and nonimmune demyelination by IFN-beta plus vitamin B12: treatment to modify notch-1/sonic hedgehog balance

Interferon-beta is a mainstay therapy of demyelinating diseases, but its effects are incomplete in human multiple sclerosis and several of its animal models. In this study, we demonstrate dramatic improvements of clinical, histological, and laboratory parameters in in vivo mouse models of demyelinating disease through combination therapy with IFN-beta plus vitamin B(12) cyanocobalamin (B(12)CN) in nonautoimmune primary demyelinating ND4 (DM20) transgenics, and in acute and chronic experimental autoimmune encephalomyelitis in SJL mice. Clinical improvement (p values <0.0001) was paralleled by near normal motor function, reduced astrocytosis, and reduced demyelination. IFN-beta plus B(12)CN enhanced in vivo and in vitro oligodendrocyte maturation. In vivo and in vitro altered expression patterns of reduced Notch-1 and enhanced expression of sonic hedgehog and its receptor were consistent with oligodendrocyte maturation and remyelination. IFN-beta-B(12)CN combination therapy may be promising for the treatment of multiple sclerosis.     

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis.

4-1BB, a member of the TNFR superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB Abs enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of an agonistic anti-4-1BB mAb (2A) dramatically reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). Adoptive transfer of T cells from such treated mice failed to induce EAE, whereas anti-4-1BB treatment following adoptive transfer of encephalitogenic T cells did not prevent EAE pathogenesis. These results suggest that anti-4-1BB treatment during the induction phase inhibits autoreactive T cell immune responses rather than preventing T cell trafficking into the CNS. This was substantiated by the observations that draining lymph node cells from anti-4-1BB-treated mice failed to respond to Ag stimulation in vitro. In addition, we found that such treatment initially promotes the activation and proliferation of Ag-specific CD4+ T cells but subsequently increases their probability of undergoing activation-induced cell death, thereby inhibiting effector T cell responses. More importantly, 2A treatment also inhibits the relapse of EAE in a clinically relevant murine model of multiple sclerosis. This study indicates that the agonistic Ab against 4-1BB can potentially be used as a novel immunotherapeutic agent for treating autoimmune diseases.     

The origin and application of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a model of the neuroimmune system responding to priming with central nervous system (CNS)-restricted antigens. It is an excellent model of post-vaccinal encephalitis and a useful model of many aspects of multiple sclerosis. EAE has been established in numerous species and is induced by priming with a large number of CNS-derived antigens. As a consequence, the pathogenesis, pathology and clinical signs vary significantly between experimental protocols. As I describe in this Timeline article, the reductionist approach taken in some lines of investigation of EAE resulted in a reliance on results obtained under a narrow range of conditions. Although such studies made important contributions to our molecular understanding of inflammation, T-cell activation, and MHC restriction, they did not advance as effectively our knowledge of the polyantigenic responses that usually occur in CNS immunopathology and autoimmunity.     

Characteristics of initial and reinduced experimental autoimmune encephalomyelitis

 The factors which influence expression of autoimmune disease in the central nervous system are still poorly understood. We determined the characteristics of experimental autoimmune encephalomyelitis (EAE) in twelve different inbred strains of mice using either mouse spinal cord homogenate or synthetic peptides as the encephalitogen. We also determined whether these strains were susceptible to reinduction of EAE at six weeks after the initial injection. The incidence, time of onset, severity, duration, and number of spontaneous relapses varied widely among the different strains. Duration of initial EAE correlated significantly with incidence of spontaneous relapses, and was greatest in C57L mice and in mice with a C57BL/10 background. Most strains of mice recovered from initial EAE, but recovery was unusual in A.SW and PERA mice. Incidence of reinduced EAE differed from incidence of initial EAE in some strains and did not correlate with incidence of spontaneous relapse. We conclude that the same factors control disease duration and incidence of spontaneous relapse, and that these factors are independent of the factors which control initial incidence. The factors controlling incidence of reinduced EAE are distinct from those controlling spontaneous relapse, and may also differ from those controlling initial incidence. Further investigation of the mechanisms effecting recovery from EAE and the genetic background underlying those mechanisms may help us understand human diseases such as multiple sclerosis. Received: 24 April 1996 / Revised: 26 May 1996     

Antigen-induced suppression of mitogen responses and resistance to experimental autoimmune encephalomyelitis

Random-bred Hartley and inbred Strain 2 and Strain 13 guinea pigs were inoculated for acute experimental autoimmune encephalomyelitis (EAE). Sixty-six percent (69/103) of the Hartleys developed signs of EAE while the remaining 34% (34/103) were resistant. No Strain 2 and all Strain 13 guinea pigs developed EAE. Histologic examination of nervous tissue revealed that susceptible Hartleys and Strain 13 and Strain 2 animals had lesions characteristic of EAE. Tissue from resistant Hartleys showed fewer and less severe changes. Lymphocyte-transformation assays with EAE-inducing and noninducing antigens and T-cell mitogens revealed three different sets of responses in vitro: (i) lymphocytes from all animals responded to mitogens; (ii) lymphocytes from susceptible animals responded to EAE-inducing antigens; and (iii) lymphocytes from resistant Hartleys were suppressed from responding to the mitogens solely by EAE-inducing antigens. Plasmas from all EAE-sensitized animals had equivalent anti-myelin basic proteins (MBP) antibody titers and skin tests of EAE-inoculated Hartleys were all positive for MBP sensitization. Therefore, resistance and reduced histologic changes characteristic of EAE correlated with a disease-specific antigen-induced suppression of lymphocyte responses to T-cell mitogens. This suggests that clinical resistance to EAE in Hartley guinea pigs is mediated by an immunologic suppressor mechanism.     

Taenia crassiceps infection abrogates experimental autoimmune encephalomyelitis

Helminth infections induce strong immunoregulation that can modulate subsequent pathogenic challenges. Taenia crassiceps causes a chronic infection that induces a Th2-biased response and modulates the host cellular immune response, including reduced lymphoproliferation in response to mitogens, impaired antigen presentation and the recruitment of suppressive alternatively activated macrophages (AAMФ). In this study, we aimed to evaluate the ability of T. crassiceps to reduce the severity of experimental autoimmune encephalomyelitis (EAE). Only 50% of T. crassiceps-infected mice displayed EAE symptoms, which were significantly less severe than uninfected mice. This effect was associated with both decreased MOG-specific splenocyte proliferation and IL-17 production and limited leukocyte infiltration into the spinal cord. Infection with T. crassiceps induced an anti-inflammatory cytokine microenvironment, including decreased TNF-α production and high MOG-specific production of IL-4 and IL-10. While the mRNA expression of TNF-α and iNOS was lower in the brain of T. crassiceps-infected mice with EAE, markers for AAMФ were highly expressed. Furthermore, in these mice, there was reduced entry of CD3⁺Foxp3⁻ cells into the brain. The T. crassiceps-induced immune regulation decreased EAE severity by dampening T cell activation, proliferation and migration to the CNS.     

Immunopathology of the lesion in chronic relapsing experimental autoimmune encephalomyelitis in the mouse

To analyze immunopathologic events within the central nervous system (CNS) during various stages of actively induced chronic relapsing EAE in SJL/J mice, animals were sampled at various timepoints post inoculation (PI) and T cells, T-cell subsets, Ia+ cells and Ig+ cells, albumin, and Ig deposits were localized in frozen sections by immunocytochemical techniques. Furthermore, sections were stained for the demonstration of Ia antigen, myelin basic protein (MBP), and galactocerebroside (GC) on endothelial cells and astrocytes. During the acute phase of the disease, large numbers of all types of inflammatory cells studied (Lyt-1.2+, L3T4+, Lyt-2+, Ia+, Ig+) were randomly distributed throughout lesions, a finding similar to that described previously for acute EAE. A more distinct distribution pattern of infiltrating T cells was found during active chronic disease in that L3T4+ cells predominated within the CNS parenchyma, while Lyt-2+ cells were more numerous in meningeal and perivascular areas. During all chronic stages, a low-grade diffuse infiltration of the neuraxis by hematogenous cells was present. Ia and myelin antigens were detectable on some endothelial cells and astrocytes. Damage to the blood-brain barrier, as indicated by albumin and Ig deposits, was more extensive during the acute than during chronic stages of the disease. Taken in concert, the results further support the possibility of local antigen presentation on endothelial and astroglial cells and an essential involvement of helper (L3T4+) T cells in CNS lesion formation. These findings correlate well with events reported previously in acute and chronic multiple sclerosis lesions.     

Oral zinc aspartate treats experimental autoimmune encephalomyelitis

The essential trace element zinc plays a critical role in the regulation of immune homeostasis. Zinc deficiency or excess can cause severe impairment of the immune response, which points to the importance of the physiological and dietary control of zinc levels for a functioning immune system. We previously reported that injection of zinc aspartate suppresses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), as well as effector T cell functions in vitro. Among the preferred characteristics of novel therapeutics for the treatment of autoimmune diseases such as MS are oral availability and a tolerable effective dose to minimize side effects. In this study, we investigated whether oral administration of zinc aspartate, an approved drug to treat zinc deficiency in humans, is effective in controlling EAE at clinically approved doses. We show that oral administration of 6 µg/day [0.3 mg/kg body weight (BW)] or 12 µg/day [0.6 mg/kg BW] of zinc aspartate reduces clinical and histopathological signs during the relapsing remitting phase of the disease in SJL mice. The clinical effect in mice was accompanied by suppression of IFN-γ, TNF-α, GM-CSF and IL-5 production in stimulated human T cells and mouse splenocytes in a dose-dependent manner. Furthermore, a large array of proinflammatory cytokines was modulated by zinc aspartate exposure in vitro. These data suggest that administration of oral zinc aspartate may have beneficial effects on autoimmune diseases like MS.     

Time-course expression of metallothioneins and tissue metals in chronic relapsing form of experimental autoimmune encephalomyelitis

To elucidate the role of metallothioneins (MTs) in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MTs I+II and the tissue concentrations of Zn2+ and Cu2+ in the brain, spinal cord (SC) and in the liver during the periods of attacks and remissions in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE). Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control rats were treated with CFA. The data, obtained by clinical assessment, immunohistochemistry and inductivity coupled plasma spectrometry, have shown that during the first attack (on the 12th day) MTs I+II were markedly upregulated in subarachnoid regions and perivascular space on astrocytes, microglia and on spinal neurons. Simultaneously, the concentrations of zinc in the SC and zinc and copper in the liver have found to be increased. During the second attack (on the 22nd day) a new overexpression of MTs was found in the cerebellum, in sulcus hippocampi, in spinal neurons and particularly in hepatocytes around the central vein. Concomitantly, in the brain and SC the concentration of copper increased. The data point to a neuroprotective role of MTs and to an important regulatory role of essential metals and hepatic MTs in the pathogenesis of CR-EAE.     

Cytoskeletal protein carbonylation and degradation in experimental autoimmune encephalomyelitis

Protein carbonylation, the non-enzymatic addition of aldehydes or ketones to specific amino acid residues, has been implicated in the pathophysiology of multiple sclerosis. In this study, we investigated whether protein carbonyls also accumulate in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE). Western blots analysis after derivatization with dinitrophenyl hydrazine (oxyblot) showed elevated protein carbonylation at the time of maximal clinical disability. During the same period glutathione levels were substantially reduced, suggesting a causal relationship between these two markers. In contrast, lipid peroxidation products accumulated in EAE spinal cord well before the appearance of neurological symptoms. Carbonyl staining was not restricted to inflammatory lesions but present throughout the spinal cord particularly in neuronal cell bodies and axons. By 2-dimensional-oxyblot, we identified several cytoskeletal proteins, including β-actin, glial acidic fibrillary protein, and the neurofilament proteins as the major targets of carbonylation. These findings were confirmed by pull-down experiments, which also showed an increase in the number of carbonylated β-actin molecules and a decrease in that of oxidized neurofilament proteins in EAE. These data suggest the possibility that oxidation targets neurofilament proteins for degradation, which may contribute to axonal pathology observed in multiple sclerosis and EAE.     

Hyperphosphorylation and aggregation of tau in experimental autoimmune encephalomyelitis

Axonal damage is a major morphological correlate and cause of permanent neurological deficits in patients with multiple sclerosis (MS), a multifocal, inflammatory and demyelinating disease of the central nervous system. Hyperphosphorylation and pathological aggregation of microtubule-associated protein tau is a common feature of many neurodegenerative diseases with axonal degeneration including Alzheimer's disease. We have therefore analyzed tau phosphorylation, solubility and distribution in the brainstem of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Tau was hyperphosphorylated at several sites also phosphorylated in Alzheimer's disease and became partially detergent-insoluble in EAE brains. Morphological examination demonstrated accumulation of amorphous deposits of abnormally phosphorylated tau in the cell body and axons of neurons within demyelinating plaques. Hyperphosphorylation of tau was accompanied by up-regulation of p25, an activator of cyclin-dependent kinase 5. Phosphorylation of tau, activation of cdk5, and axonal pathology were significantly reduced when diseased rats were treated with prednisolone, a standard therapy of acute relapses in MS. Hyperphosphorylation of tau was not observed in a genetic or nutritional model of axonal degeneration or demyelination, suggesting that inflammation as detected in the brains of rats with EAE is the specific trigger of tau pathology. In summary, our data provide evidence that axonal damage in EAE and possibly MS is linked to tau pathology.     

Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence). Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs. The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.     

Systemic infections in multiple sclerosis and experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). It has been suggested that viral and bacterial infections contribute to the pathogenesis of MS. This review will give an overview about the influence of viral and bacterial infections on MS and experimental autoimmune encephalomyelitis (EAE). It will focus on bacterial infections and will also emphasise therapeutic consequences such as the impact of antibiotic treatment on the course of EAE. In summary, a growing body of evidence suggests that systemic infections are a risk factor for the initiation of autoimmune processes including the induction of acute events in MS. Experimental and clinical data strongly suggest early treatment of bacterial infections in MS patients to avoid aggravation and relapse.