Marmoset Experimental Autoimmune Encephalomyelitis Research Articles

In vivo MRI is sensitive to remyelination in a nonhuman primate model of multiple sclerosis.

Remyelination is crucial to recover from inflammatory demyelination in multiple sclerosis (MS). Investigating remyelination in vivo using magnetic resonance imaging (MRI) is difficult in MS, where collecting serial short-interval scans is challenging. Using experimental autoimmune encephalomyelitis (EAE) in common marmosets, a model of MS that recapitulates focal cerebral inflammatory demyelinating lesions, we investigated whether MRI is sensitive to, and can characterize, remyelination. In six animals followed with multisequence 7 T MRI, 31 focal lesions, predicted to be demyelinated or remyelinated based on signal intensity on proton density-weighted images, were subsequently assessed with histopathology. Remyelination occurred in four of six marmosets and 45% of lesions. Radiological-pathological comparison showed that MRI had high statistical sensitivity (100%) and specificity (90%) for detecting remyelination. This study demonstrates the prevalence of spontaneous remyelination in marmoset EAE and the ability of in vivo MRI to detect it, with implications for preclinical testing of pro-remyelinating agents.

Ultrahigh-resolution MRI Reveals Extensive Cortical Demyelination in a Nonhuman Primate Model of Multiple Sclerosis.

Cortical lesions are a primary driver of disability in multiple sclerosis (MS). However, noninvasive detection of cortical lesions with in vivo magnetic resonance imaging (MRI) remains challenging. Experimental autoimmune encephalomyelitis (EAE) in the common marmoset is a relevant animal model of MS for investigating the pathophysiological mechanisms leading to brain damage. This study aimed to characterize cortical lesions in marmosets with EAE using ultrahigh-field (7T) MRI and histological analysis. Tissue preparation was optimized to enable the acquisition of high-spatial resolution (50-μm isotropic) T2*-weighted images. A total of 14 animals were scanned in this study, and 70% of the diseased animals presented at least one cortical lesion on postmortem imaging. Cortical lesions identified on MRI were verified with myelin proteolipid protein immunostaining. An optimized T2*-weighted sequence was developed for in vivo imaging and shown to capture 65% of cortical lesions detected postmortem. Immunostaining confirmed extensive demyelination with preserved neuronal somata in several cortical areas of EAE animals. Overall, this study demonstrates the relevance and feasibility of the marmoset EAE model to study cortical lesions, among the most important yet least understood features of MS.

Severe oxidative stress in an acute inflammatory demyelinating model in the rhesus monkey.

Oxidative stress is increasingly implicated as a co-factor of tissue injury in inflammatory/demyelinating disorders of the central nervous system (CNS), such as multiple sclerosis (MS). While rodent experimental autoimmune encephalomyelitis (EAE) models diverge from human demyelinating disorders with respect to limited oxidative injury, we observed that in a non-human primate (NHP) model for MS, namely EAE in the common marmoset, key pathological features of the disease were recapitulated, including oxidative tissue injury. Here, we investigated the presence of oxidative injury in another NHP EAE model, i.e. in rhesus macaques, which yields an acute demyelinating disease, which may more closely resemble acute disseminated encephalomyelitis (ADEM) than MS. Rhesus monkey EAE diverges from marmoset EAE by abundant neutrophil recruitment into the CNS and destructive injury to white matter. This difference prompted us to investigate to which extent the oxidative pathway features elicited in MS and marmoset EAE are reflected in the acute rhesus monkey EAE model. The rhesus EAE brain was characterized by widespread demyelination and active lesions containing numerous phagocytic cells and to a lesser extent T cells. We observed induction of the oxidative stress pathway, including injury, with a predilection of p22phox expression in neutrophils and macrophages/microglia. In addition, changes in iron were observed. These results indicate that pathogenic mechanisms in the rhesus EAE model may differ from the marmoset EAE and MS brain due to the neutrophil involvement, but may in the end lead to similar induction of oxidative stress and injury.

Oxidative Injury and Iron Redistribution Are Pathological Hallmarks of Marmoset Experimental Autoimmune Encephalomyelitis.

Oxidative damage and iron redistribution are associated with the pathogenesis and progression of multiple sclerosis (MS), but these aspects are not entirely replicated in rodent experimental autoimmune encephalomyelitis (EAE) models. Here, we report that oxidative burst and injury as well as redistribution of iron are hallmarks of the MS-like pathology in the EAE model in the common marmoset. Active lesions in the marmoset EAE brain display increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p22phox, p47phox, and gp91phox) and inducible nitric oxide synthase immunoreactivity within lesions with active inflammation and demyelination, coinciding with enhanced expression of mitochondrial heat-shock protein 70 and superoxide dismutase 1 and 2. The EAE lesion-associated liberation of iron (due to loss of iron-containing myelin) was associated with altered expression of the iron metabolic markers FtH1, lactoferrin, hephaestin, and ceruloplasmin. The enhanced expression of oxidative damage markers in inflammatory lesions indicates that the enhanced antioxidant enzyme expression could not counteract reactive oxygen and nitrogen species-induced cellular damage, as is also observed in MS brains. This study demonstrates that oxidative injury and aberrant iron distribution are prominent pathological hallmarks of marmoset EAE thus making this model suitable for therapeutic intervention studies aimed at reducing oxidative stress and associated iron dysmetabolism.

An essential role of virus-infected B cells in the marmoset experimental autoimmune encephalomyelitis model.

Infection with Epstein–Barr virus (EBV) has been associated with an enhanced risk of genetically susceptible individuals to develop multiple sclerosis (MS). However, an explanation for the contrast between the high EBV infection prevalence (60–90%) and the low MS prevalence (0.1%) eludes us. Here we propose a new concept for the EBV–MS association developed in the experimental autoimmune encephalomyelitis model in marmoset monkeys, which are naturally infected with the EBV-related γ1-herpesvirus CalHV3. The data indicate that the infection of B cells with a γ1-herpesvirus endows them with the capacity to activate auto-aggressive CD8+ T cells specific for myelin oligodendrocyte glycoprotein.

Magnetic resonance imaging of experimental autoimmune encephalomyelitis in the common marmoset

Magnetic resonance imaging (MRI) is an invaluable tool for the diagnosis and monitoring of patients with multiple sclerosis (MS) as well as for the study of the disease pathophysiology. Because of its strong clinical, radiological and histopathological similarities with the human disease, experimental autoimmune encephalomyelitis (EAE) in the common marmoset has been studied more intensively over the past several years. Here, we review the current knowledge on MRI in the marmoset EAE, and we outline the physiopathological significance and translational values of these studies with respect to MS. Accumulating evidences suggest that the application of conventional, as well as non-conventional, MRI techniques in the marmoset EAE is a promising approach to elucidate the pathological processes underlying the development of inflammatory demyelinated lesions in the central nervous system, potentially improving the identification and development of new therapeutics.

The common marmoset as an indispensable animal model for immunotherapy development in multiple sclerosis

New drugs often fail in the translation from the rodent experimental autoimmune encephalomyelitis (EAE) model to human multiple sclerosis (MS). Here, we present the marmoset EAE model as an indispensable model for translational research into MS. The genetic heterogeneity of this species and lifelong exposure to chronic latent infections and environmental pathogens create a human-like immune system. Unique to this model is the presence of the pathological hallmark of progressive MS, in particular cortical grey matter lesions. Another great possibility of this model is systemic and longitudinal immune profiling, whereas in humans and mice immune profiling is usually performed in a single compartment (i.e. blood or spleen, respectively). Overall, the marmoset model provides unique opportunities for systemic drug-effect profiling.

Immune modulation by a tolerogenic myelin oligodendrocyte glycoprotein (MOG)10-60 containing fusion protein in the marmoset experimental autoimmune encephalomyelitis model.

Current therapies for multiple sclerosis (MS), a chronic autoimmune neuroinflammatory disease, mostly target general cell populations or immune molecules, which may lead to a compromised immune system. A more directed strategy would be to re-enforce tolerance of the autoaggressive T cells that drive tissue inflammation and injury. In this study, we have investigated whether the course of experimental autoimmune encephalomyelitis (EAE) in mice and marmosets can be altered by a potent tolerizing fusion protein. In addition, a multi-parameter immunological analysis was performed in marmosets to assess whether the treatment induces modulation of EAE-associated cellular and humoral immune reactions. The fusion protein, CTA1R9K-hMOG10-60-DD, contains a mutated cholera toxin A1 subunit (CTA1R9K), a dimer of the Ig binding D region of Staphylococcus aureus protein A (DD), and the human myelin oligodendrocyte glycoprotein (hMOG) sequence 10-60. We observed that intranasal application of CTA1R9K-hMOG10-60-DD seems to skew the immune response against myelin oligodendrocyte glycoprotein (MOG) towards a regulatory function. We show a reduced number of circulating macrophages, reduced MOG-induced expansion of mononuclear cells in peripheral blood, reduced MOG-induced production of interleukin (IL)-17A in spleen, increased MOG-induced production of IL-4 and IL-10 and an increased percentage of cells expressing programmed cell death-1 (PD-1) and CC chemokine receptor 4 (CCR4). Nevertheless, the treatment did not detectably change the EAE course and pathology. Thus, despite a detectable effect on relevant immune parameters, the fusion protein failed to influence the clinical and pathological outcome of disease. This result warrants further development and improvement of this specifically targeted tolerance inducing therapy.

The formation of inflammatory demyelinated lesions in cerebral white matter.

Vascular permeability and inflammatory demyelination are intimately linked in the brain, but what is their temporal relationship? We aimed to determine the radiological correlates of the earliest tissue changes accompanying demyelination in a primate model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) in the common marmoset. By 7T magnetic resonance imaging (MRI), T1 maps, proton density, and T2-weighted images were acquired before and after EAE induction in 5 marmosets (every other week before lesions appeared, weekly thereafter). From scans before and after intravenous injection of contrast material, we measured the evolution of lesional blood-brain barrier (BBB) permeability, comparing in vivo MRI to postmortem tissue examination. On average, BBB permeability increased 3.5-fold (p < 0.0001) over the 4 weeks prior to lesion appearance. Permeability gradually decreased after lesion appearance, with attendant changes in the distribution of inflammatory cells (predominantly macrophages and microglia) and demyelination. On tissue analysis, we also identified small perivascular foci of microglia and T cells without blood-derived macrophages or demyelination. These foci had no visible MRI correlates, although permeability within the foci, but not outside, increased in the weeks before the animals died (p < 0.0001). This study provides compelling evidence that in marmoset EAE, which forms lesions strongly resembling those of MS, early changes in vascular permeability are associated with perivascular inflammatory cuffing and parenchymal microglial activation but precede the arrival of blood-derived monocytes that accompany demyelination. Prospective detection of transient permeability changes could afford an opportunity for early intervention to forestall tissue damage in newly forming lesions.

Experimental Autoimmune Encephalomyelitis in Marmosets.

Experimental autoimmune encephalomyelitis (EAE) in the common marmoset, a small-bodied Neotropical primate, is a well-known and validated animal model for multiple sclerosis (MS). This model can be used for exploratory research, i.e., investigating the pathogenic mechanisms involved in MS, and applied research, testing the efficacy of new potential drugs.In this chapter, we will describe a method to induce EAE in the marmoset. In addition, we will explain the most common immunological techniques involved in the marmoset EAE research, namely isolation of mononuclear cells (MNC) from peripheral blood and lymphoid tissue, assaying T cell proliferation by thymidine incorporation, MNC phenotyping by flow cytometry, antibody measurement by ELISA, generation of B cell lines and antigen-specific T cell lines, and assaying cytotoxic T cells.

The Different Clinical Effects of Anti-BLyS, Anti-APRIL and Anti-CD20 Antibodies Point at a Critical Pathogenic Role of γ-Herpesvirus Infected B Cells in the Marmoset EAE Model

The robust and rapid clinical effect of depleting anti-CD20 monoclonal antibodies (mAb) in multiple sclerosis (MS) demonstrates a critical pathogenic contribution of B cells. The clinical effect of anti-CD20 mAb has been replicated in a relevant preclinical MS model, experimental autoimmune encephalomyelitis (EAE) in marmoset monkeys (Callithrix jacchus). By contrast, treatment with mAbs against two essential cytokines in B cell activation growth and survival, i.e. BlyS/BAFF and APRIL, was only partially effective. All three mAbs induced depletion of CD20+ B cells from the circulation, albeit with different kinetics and based on distinct mechanisms of action. In the current study we analyzed whether the different clinical effect of anti-CD20 mAb or the anti-BLyS and anti-APRIL mAbs is due to different depletion of B cells infected with the EBV of marmosets, CalHV3. Employing a novel PCR-based assay, half of the colony of group-housed marmosets was tested positive for CalHV3 DNA in secondary lymphoid organs. The same prevalence was observed in placebo-treated monkeys. In marmosets treated with anti-CD20 mAb the load of CalHV3 DNA in lymphoid organs was substantially reduced, while this was not observed in the monkeys treated with anti-BLyS or anti-APRIL mAbs. To examine the pathogenic role of virus-transformed B cells, we infused EBV-transformed B lymphoblastic cell (BLC) lines presenting the immunodominant MOG34-56 peptide. We observed in the recipients of MOG34-56 pulsed BLC, but not in their fraternal siblings infused with non-pulsed BLC, activation of anti-MOG34-56T cells and meningeal inflammation. Collectively, the data show that among CD20+ B cells, the herpesvirus-transformed subset has a particularly important pathogenic role in the marmoset EAE model.

Unravelling the T‐cell‐mediated autoimmune attack on CNS myelin in a new primate EAE model induced with MOG34–56 peptide in incomplete adjuvant

Induction of experimental autoimmune encephalomyelitis (EAE) has been documented in common marmosets using peptide 34-56 from human myelin/oligodendrocyte glycoprotein (MOG(34-56) ) in incomplete Freund's adjuvant (IFA). Here, we report that this EAE model is associated with widespread demyelination of grey and white matter. We performed an in-depth analysis of the specificity, MHC restriction and functions of the activated T cells in the model, which likely cause EAE in an autoantibody-independent manner. T-cell lines isolated from blood and lymphoid organs of animals immunized with MOG(34-56) displayed high production of IL-17A and specific lysis of MOG(34-56) -pulsed EBV B-lymphoblastoid cells as typical hallmarks. Cytotoxicity was directed at the epitope MOG(40-48) presented by the non-classical MHC class Ib allele Caja-E, which is orthologue to HLA-E and is expressed in non-inflamed brain. In vivo activated T cells identified by flow cytometry in cultures with MOG(34-56,) comprised CD4(+) CD56(+) and CD4(+) CD8(+) CD56(+) T cells. Furthermore, phenotypical analysis showed that CD4(+) CD8(+) CD56(+) T cells also expressed CD27, but CD16, CD45RO, CD28 and CCR7 were absent. These results show that, in the MOG34-56/IFA marmoset EAE model, a Caja-E-restricted population of autoreactive cytotoxic T cells plays a key role in the process of demyelination in the grey and white matter.

Effects of Early IL-17A Neutralization on Disease Induction in a Primate Model of Experimental Autoimmune Encephalomyelitis

We report on the effect of antibody-mediated neutralization of interleukin (IL)-17A in a non-human primate experimental autoimmune encephalomyelitis (EAE) model induced with recombinant human myelin oligodendrocyte glycoprotein (rhMOG). We tested a human-anti-human IL-17A-antibody in two doses (3 and 30 mg/kg) against placebo (PBS). The treatment was started 1 day before EAE induction and continued throughout the experiment. Although all monkeys developed clinically evident EAE, the onset of neurological signs was delayed in some monkeys from both treatment groups. Total CNS lesion volumes, demyelination, or inflammation did not differ between the different groups. Immune profiling revealed an altered distribution of IL-17A producing cells in the lymphoid organs of antibody-treated monkeys. Comparable numbers of IL-17A producing cells were observed in the brain. RhMOG-induced T cell proliferation in the lymph nodes was slightly reduced after anti-IL-17A antibody treatment. To summarize, we found that anti-IL-17A antibody as a single treatment from disease induction effects a trend towards delayed neurological disease progression in the marmoset EAE model, although the effect did not reach statistical significance. This suggests a role of IL-17A in late stage disease in the marmoset EAE model, but IL-17A may not be the dominant pathogenic cytokine.

Induction of Progressive Demyelinating Autoimmune Encephalomyelitis in Common Marmoset Monkeys Using MOG34-56Peptide in Incomplete Freund Adjuvant

Experimental autoimmune encephalomyelitis in the neotropical primate common marmoset (Callithrix jacchus) is a relevant autoimmune animal model of multiple sclerosis. T cells specific for peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) have a central pathogenic role in this model. The aim of this study was to assess the requirement for innate immune stimulation for activation of this core pathogenic autoimmune mechanism. Marmoset monkeys were sensitized against synthetic MOG34-56 peptide alone or in combination with the nonencephalitogenic peptide MOG74-96 formulated in incomplete Freund adjuvant, which lacks microbial components. Experimental autoimmune encephalomyelitis development was recorded by monitoring neurological signs, brain magnetic resonance imaging, and longitudinal profiling of cellular and humoral immune parameters. All monkeys developed autoimmune inflammatory/demyelinating central nervous system disease characterized by massive brain and spinal cord demyelinating white matter lesions with activated macrophages and CD3+ T cells. Immune profiling ex vivo demonstrated the activation of mainly CD3+CD4+/8+CD56+ T cells against MOG34-56. Upon ex vivo stimulation, these T cells produced more interleukin 17A compared with TH1 cytokines (e.g. interferon-gamma) and displayed peptide-specific cytolytic activity. These results indicate that the full spectrum of marmoset experimental autoimmune encephalomyelitis can be induced by sensitization against a single MOG peptide in incomplete Freund adjuvant lacking microbial compounds for innate immune activation and by eliciting antigen-specific T-cell cytolytic activity.

Autoimmune Optic Neuritis in the Common Marmoset Monkey: Comparison of Visual Evoked Potentials with MRI and Histopathology

To assess the use of visual evoked potentials (VEPs) for the in vivo detection of impaired visual function in a marmoset model of multiple sclerosis. The sensitivity of the VEP recordings was determined by comparison with magnetic resonance imaging (MRI) and histopathology. Baseline VEPs were recorded in six healthy marmoset monkeys in response to light-flash stimulation. Experimental autoimmune encephalomyelitis (EAE) was induced in four of the six monkeys. Clinical scores were assessed daily, and VEPs were recorded every second week. In vivo MRI and subsequent histopathology of the brains and optic nerves were performed at the end of the study. After induction of EAE, all four marmosets exhibited clinical signs between day 26 and 38 after immunization. VEPs were normal during the induction phase of the disease, but deteriorated in amplitude with the occurrence of clinical symptoms in all animals. MRI revealed bilateral optic neuritis and signal alterations in the optic tracts and occipital subcortical white matter in two of the animals. In the remaining two animals, MRI detected signal alterations in the occipital subcortical white matter. Histopathologic results were concordant with the MRI findings. VEPs are an easily accessible noninvasive tool for measuring visual function and diagnosing impairment of the visual pathway in a marmoset EAE model.

Diffuse cortical atrophy in a marmoset model of multiple sclerosis

Marmoset experimental autoimmune encephalomyelitis (EAE) has previously been shown to replicate the essential features of both white matter and grey matter lesions of MS. This study set out to investigate whether cortical atrophy occurs in marmoset EAE and whether cortical thinning is related to the presence of focal, demyelinated cortical lesions. Seventeen leucocortical lesions and 13 subpial lesions were identified in 6 EAE cases. Cortical thickness surrounding these lesions was recorded and compared with matched cortical areas from five control animals. We found a diffuse 13-21% loss of cortical thickness in all areas of EAE cortex compared with control animals but there was no additional loss seen in demyelinated versus myelinated EAE cortex. These findings could not be accounted for by effects of age, sex and disease duration. We conclude that localised cortical demyelination is not responsible for the major part of the atrophy observed and that cortical thinning is largely due to more diffuse or more remote factors. Marmoset EAE is an invaluable tool which can be used to further investigate the cause and the substrate of cortical loss in demyelinating diseases.

Protection of marmoset monkeys against EAE by treatment with a murine antibody blocking CD40 (mu5D12).

CD40-CD40 ligand interactions are crucial to cognate interactions between T cells, B cells and antigen-presenting cells (APC), and contribute to non-antigen-specific effector functions of APC in inflammatory disorders. Here we demonstrate that functional blockade of CD40 with an antagonist mouse anti-human CD40 monoclonal antibody (mAb mu5D12) effectively prevents clinical expression of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in outbred marmoset monkeys, a preclinical model of multiple sclerosis. Anti-CD40 mAb interfered with development of clinical symptoms of marmoset EAE during the treatment period, even when treatment was started several weeks after T cell priming. Magnetic resonance imaging demonstrated inflammatory activity in the brain at initiation of antibody treatment, confirming that treatment interfered with the disease process. Access of therapeutic anti-CD40 to potential sites of action, the secondary lymphoid organs and the brain white matter lesions, was visualized in situ. The present data are the first to demonstrate the clinical potential of blocking APC and effector cell functions using murine antagonist anti-CD40 mAb in the treatment of chronic inflammatory diseases.

Preclinical assessment of anti-CD40 Mab 5D12 in cynomolgus monkeys

Monoclonal antibody (Mab) 5D12 is a potent antagonist of the CD40–CD40L pathway. This cellular interaction has been validated in a large number of experimental animal models where dys-regulation of the immune system plays a role. Chimeric 5D12 (ch5D12) was constructed to reduce the potential immunogenicity and enhance the in vivo half-life when used in humans. ch5D12 is a molecularly engineered human IgG 4 antibody containing the variable domains of the heavy and light chains of the murine version of 5D12 (mu5D12). This new chimeric Mab was tested in a marmoset experimental autoimmune encephalomyelitis model and was shown to effectively prevent disease symptoms. The results of this in vivo evaluation supported clinical use of ch5D12 for immune targeted diseases. Therefore GMP material was prepared and a GLP-compliant tissue cross-reactivity study on human tissues (3 donors/37 tissues) and cynomolgus tissues (2 donors/37 tissues) was performed. ch5D12 stained on the surface of B cells and selected dendritic cells and no unexpected cross-reactivity was observed. The identical staining patterns in human and cynomolgus tissues justified the use of cynomolgus monkeys as a relevant model for humans. A GLP-compliant safety and tolerability evaluation for ch5D12 in cynomolgus monkeys was performed using the GMP produced material. Weekly administration of ch5D12 at two dose levels for 4 weeks was shown to be safe and without any side effects in all monkeys.

Novel monoclonal antibodies against proteolipid protein peptide 139–151 demonstrate demyelination and myelin uptake by macrophages in MS and marmoset EAE lesions

Experimental autoimmune encephalomyelitis (EAE) induced by immunization of mice with epitopes of the proteolipid protein (PLP), a major myelin constituent, forms a useful model for the study of multiple sclerosis (MS). In addition, MS patients display PLP-specific T- and B-cell responses, suggesting that PLP reactivity is relevant to pathogenesis. Here, the generation and characterization of a panel of mouse monoclonal antibodies (Mab) against PLP139–151, the prominent encephalitogenic sequence in SJL/J mice is described. Five Mab were generated by conventional immunization of an SJL/J mouse and hybridoma generation. These Mab reacted well with the PLP139–151 peptide in ELISA and belonged to the IgG2a and IgG2b subclasses, consistent with CD4+ T helper 1-cell-supported antibody formation. The Mab also efficiently detected PLP peptide–BSA conjugates in Western blot, confirming their multi-assay applicability. The Mab were subsequently used to determine the occurrence of demyelination in brains of MS patients and marmoset monkeys with EAE. Immunohistochemistry on both paraffin and frozen sections demonstrated a homogeneous expression of PLP139–151 in normal myelin, and a complete absence in lesions containing demyelinated areas, confirming that the Mab can be used as a general myelin marker. In active demyelinating MS lesions, the Mab visualized the peptide in the cytoplasm of macrophages containing phagocytosed myelin. In conclusion, this panel of Mab against the encephalitogenic PLP139–151 epitope forms a useful tool for further study of autoantigen expression, demyelination/remyelination and the staging of lesional activity in MS patients, as well as in EAE models in distinct animal species.