Experimental autoimmune encephalomyelitis in the common marmoset: a translationally relevant model for the cause and course of multiple sclerosis

Abstract

Aging Western societies are facing an increasing prevalence of chronic autoimmune-mediated inflammatory disorders (AIMIDs) for which treatments that are safe and effective are scarce. One of the main reasons for this situation is the lack of animal models, which accurately replicate clinical and pathological aspects of the human diseases. One important AIMID is the neuroinflammatory disease multiple sclerosis (MS), for which the mouse experimental autoimmune encephalomyelitis (EAE) model has been frequently used in preclinical research. Despite some successes, there is a long list of experimental treatments that have failed to reproduce promising effects observed in murine EAE models when they were tested in the clinic. This frustrating situation indicates a wide validity gap between mouse EAE and MS. This monography describes the development of an EAE model in nonhuman primates, which may help to bridge the gap.

Figure 1

Translational research: an iterative process. The main goal of our exploratory preclinical research has been to find new targets in the pathogenic process for safer and more effective therapies. The translation of a new scientific discovery into a safe and effective innovative treatment for patients is indicated as forward translation. In the applied arm of our research, new therapies are tested. Results from such tests can be used to validate scientific concepts. When the process of forward translation fails, the reasons for failure should be investigated and this information should be fed back (i.e., reverse translation) to the animal model in order to make the necessary corrections in the scientific concept and/or the animal model itself.

Figure 2

Clinical and pathological aspects of marmoset EAE induced with MS myelin/CFA.(a) The graphs show the protracted clinical course, which is variable among individual animals. Case EH has relapsing–remitting disease that could be followed for almost 1 year. The other four cases transit to progressive disease, which can worsen quickly (EK, EL) or more slowly (EI, EJ). (b)  Case EI was subjected to in vivo magnetic resonance imaging (MRI) just before sacrifice. The middle and bottom rows show two horizontal brain slices from a T2-weighted image (middle) and a postcontrast (triple dose Gadolinium-DTPA; bottom) with the position of corresponding lesions indicated. After the scans were made, the monkey was humanely killed, the brain was removed and fixated in toto. Then a new postmortem T2-weighted scan was made. This allowed us to determine the exact position of all lesions that were detected in vivo. (c) The top row shows two coronal sections of the same MRI scan with lesions indicated. The middle and bottom rows show a magnification of individual lesions, which allowed us to conclude that brain lesions in this model are presented in different stages. (d) The top image shows an MRP14 staining of macrophages from lesions I and J, illustrating their inflammatory active nature. Notice that lesion I is one of the two gadolinium contrast-enhancing lesions. The three images below show the histological aspect of lesion J, which is characterized by primary demyelination (LFB staining), sparing of axons (Bielschowsky silver impregnation) and inflammation (MRP14). The macrophage staining shows the heterogeneity of this lesion, which is suggestive of confluent lesions of different age.

Figure 3

Characteristic brain pathology of marmoset EAE induced with human MS myelin/CFA. One brain hemisphere was immunostained for PLP to visualize demyelination and for MRP14 (macrophages/microglia) to visualize inflammation. For comparison, a brain hemisphere of a healthy marmoset was stained for PLP. Clearly visible is the different pathological aspect of white and cortical grey matter (cGM).

Figure 4

Clinical aspects of multiple sclerosis. (a) For quantification of the degree of disability, for example in the clinical assessment of new treatments, the expanded disability status scale (EDSS) has been developed (Kurtzke, 1983). (b) A graphical representation of the most common MS phenotype. In the beginning MS is asymptomatic, but with advanced imaging techniques (contrast-enhanced MRI for example) focal abnormalities due to inflammation can be observed inside the brain white matter. This is followed by a period of variable length between patients where the inflammation increases in severity causing discrete episodes of disability (relapse) alternating with complete recovery (remission). In about 50 % of the patients with relapsing–remitting MS the disease becomes progressive, where remissions disappear and neurological functions decrease progressively. Based on data from the marmoset EAE model, we posit that the degeneration of oligodendrocyte/myelin complexes can be differentiated into three types: (1) normal age-appropriate progressive degeneration; (2) progressive degeneration amplified by a newly discovered T cell attack on oligodendrocytes; (3) reversible degeneration induced by a classical autoimmune attack of pro-inflammatory T cells and autoantibodies on myelin sheaths.

Figure 5

Two opposing paradigms explaining the cause of autoimmunity in MS. The prevailing concept is the outside-in paradigm, namely that infection of individuals who are genetically predisposed to MS with an as yet unidentified microorganism activates autoreactive T and B cells present in the normal immune repertoire. The autoimmune attack on the CNS induces cytodegeneration. Less commonly accepted is the inside-out paradigm, which states that a pathogenic event inside the CNS elicits the release of myelin antigens that activate autoreactive T and B cells present in the normal immune repertoire. The principle difference between both paradigms is that in the outside-in paradigm infection is the direct trigger of autoimmunity, whereas in the inside-out paradigm infections create a higher responsive state of the immune system.

Box 1

Epstein–Barr virus (EBV).

Figure 6

What is a marmoset? Depicted is an artist's impression of two marmosets (Callithrix jacchus) showing their small body size (oil on canvas painting by Sir Edwin Landseer, 1803–1873, the Royal Collection, United Kingdom). The lower part of the figure depicts the phylogenetic tree of primates with the evolutionary distance to humans.

Box 2

Dual role of MOG, as a tolerogen or immunogen.

Figure 7

Clinical and pathological aspects of the marmoset EAE model induced with rhMOG/CFA. (a) Depicted is the EAE course in a representative selection of $\mathrm{30}$ unrelated marmosets receiving a single immunization with rhMOG/CFA on day 0. All monkeys developed clinically evident EAE, but the time of onset varied from 2 to 16 weeks. (b) Serial imaging of a case with late EAE onset, showing early onset of brain lesion formation. The white arrow points to the first detectable lesion. Clearly visible is that formation of new lesions in the depicted brain slice (0.5 mm) is disseminated in time and space. Around the time that clinical signs were diagnosed, lesion colonization of cortical grey matter is detectable (inserted magnifications). (c) PLP staining of a brain hemisphere shows the dramatic demyelination in white and grey matter (a). Different grey matter lesion types identified in the MS brain can be distinguished (b). Lesions are paucicellular with regard to T (CD3) and B (CD20) cells, but contain abundant MRP14$+$ myeloid cells, representing microglia and macrophages.

Box 3

Why are the MOG24-36 and MOG40-48 immunodominant?

Figure 8

Two autoimmune pathways. EAE development in the rhMOG/CFA model involves two distinct pathogenic mechanisms. The 100 % EAE incidence (see Fig. 7a) maps to the MHC class II/Caja-DRB*W1201-restricted activation of Th1 cells specific for MOG epitope 24–36. The signature cytokine of this pathway, which essentially replicates mouse EAE models, is IFN$\mathit{\gamma }$. Early blockade of IL-12/IL-23 with a mAb against IL-12p40 abrogates the activation of this pathway. The observation that the EAE initiation pathway is activated in rhMOG/IFA model indicates that the responsive Th1 cells may be antigen-experienced cells. The variable EAE onset maps to another pathogenic mechanism, namely the MHC class I/Caja-E-restricted activation of CTL specific for MOG epitope 40–48, which shares almost complete sequence similarity with an epitope from the major capsid protein of CMV. The signature cytokine of this pathway is IL-17A. The characteristics and specificity of the CTLs suggest that they originate from the effector memory T cells that keep CMV under control. We hypothesize that the variable activation of the latter pathway is induced by antigens released from white matter lesions induced by the former pathway.

Figure 9

Clinical and pathological aspects of an atypical EAE model induced with MOG34-56/IFA. Immunization with MOG34-56/IFA at a 28 d interval (arrows) induces 100 % clinical EAE with a variable time of onset. Notice that the only information relayed to the marmoset's immune system is the sequence of 23 letters (CTL core epitope in red). Immunostaining for PLP shows lesions in the white and grey matter (A). Rectangle 1 indicates a demyelinated region in the cingulate cortex, which is characterized by complete demyelination (B), presence at the lesion edge of macrophages containing a PLP$+$ inclusion particle (C), activated MRP14$+$ microglia (E) and depletion of oligodendrocytes (F). Immune cells (CD3) were only detected in meninges. Rectangle 2 (A) indicates a leukocortical and an intracortical lesion (G), which are also depleted from oligodendrocytes (H) and contain abundant MRP14$+$ microglia. Mf is macrophage, TPPP is tubulin polymerization-promoting protein, ODC is oligodendrocyte.

Box 4

Why do humans develop MS and chimpanzees not? A hypothesis.

Box 5

Destructive and productive processing of MOG34-56.

Box 6

Autophagy.

Figure 10

T and B cell epitopes plotted on the 3-D structure of MOG monomer. (a) The conformational antibody epitope is formed by the three loops that connect the B–C, C'–C” and F–G $\mathit{\beta }$ sheets. Notice that the B–C connecting loop (residues 27–36) overlaps with the CD4 T cell epitope and contains the Asn31 residue to which in the native molecule the N-linked glycan is attached. (b) The positions of the three dominant T cell epitopes and a linear antibody epitope are indicated. Notice that these epitopes all overlap with $\mathit{\beta }$-sheet connecting loops. (c) An excision of the critical MOG34-56 peptide, which appears to be composed of two large antiparallel $\mathit{\beta }$ sheets and a small one. Notice that the three critical Arg residues in the MOG34-56 peptide where the peptide can be cleaved by cathepsin G (positions 41, 46 and 52) are located at contact points of loop and $\mathit{\beta }$ sheet. (d) Depicts a space-filling model of monomeric MOG (pdb accession number 1PKO) in molecular surface representation, colored according to B factor (blue low rms/rigid; red high rms/flexible. The surface-exposed MOG40-48 epitope (YRSPFSRVV) is indicated in white/purple. The P43 and F44 residues, which stick out of the plane towards the reader, are not resolved in the structure, probably due to the high flexibility of this part of the sequence resulting in a diffuse diffraction pattern (Breithaupt et al., 2003), the V48 residue is buried in the interior of the protein, and therefore not visible. The putative LIR motif (xSxF${}_{\mathrm{43}}$SRV${}_{\mathrm{47}}$), which is part of the 40–48 epitope is shown in purple. The surface exposure of this motif enables interaction with the LC3 docking molecule of autophagosomes. (e) In a ribbon representation of monomeric MOG the three Arg residues are highlighted. It is clear from this figure that the Arg${}^{\mathrm{46}}$ and Arg${}^{\mathrm{52}}$ residues stick out while the Arg${}^{\mathrm{41}}$ residue is somewhat buried.

Figure 11

Concept for T cell activation within the CLNs. (a) MOG draining via interstitial fluid (ISF) or cerebrospinal fluid (CSF) to the CLNs is captured by DC-like phagocytic cells (de Vos et al., 2002). (b, c) T cells against the two dominant epitopes (24–36 and 40–48) are probably not deleted during thymic negative selection as both epitopes are vulnerable to destruction by TSSP, which cleaves at SP/PP residues present in both epitopes (Serre et al., 2017). Processing of MOG in myeloid APCs and B cells is led by catG, which cleaves at arginine ($R$) residues (Jagessar et al., 2016). This mechanism prevents peripheral activation of autoaggressive T cells that have escaped thymic selection. (b) The peptide sequence left after cleavage of the MOG40-48 epitope at Arg${}^{\mathrm{41}}$ and Arg${}^{\mathrm{46}}$ (the 4-mer 42–45) is too short for filling the peptide-binding cleft of the MHC class I molecule. In the BLC, however, the MOG40-48 epitope is protected against destructive processing. (c) Cleavage of the MOG24-36 epitope at Arg${}^{\mathrm{25}}$ may have little effect as a peptide of sufficient length (the 9-mer 26–34) is left intact for filling the peptide-binding cleft of the MHC class II molecule.

Figure 12

Antigenicity and cytotoxicity of citrullination MOG34-56. EBV-BLC cocultures with lymph node cells from EAE marmosets. (a) Marmoset EBV-induced BLCs were lethally irradiated and incubated for 1 h with titrating concentrations of unmodified MOG34-56 (peptide 10), or peptide citrullinated at positions 41$+$46 (peptide 11) or positions 46$+$52 (peptide 12). Subsequently, lymph node or spleen cells from marmosets immunized with MOG${}_{\text{34–56}}$ were added. T-cell responses to the peptides were assayed by proliferation and are expressed as stimulation index per culture condition. The experiment was conducted with $N=\mathrm{6}$ (marmosets) and with three biological replicates. Data are presented as mean $\pm$ standard error of the mean (SEM). (b) To test which cell type is targeted by the peptides, EBV BLCs (from two marmosets, M1 and M2, respectively) were incubated with Celltrace dye before incubation with peptide (white circles) and mixture with the spleen/lymph node cells (red triangles). Lymphocytes that are not subjected to coculturing were used as controls (black circles). Cultured cells were harvested and stained for Annexin V as a marker of late apoptotic/dead cells. Final analysis was done utilizing flow cytometry.

Figure 13

LC3 staining of EBV-infected human B lymphoblastoid cells (BLCs). Cytospin preparations of stably growing cells were prepared, fixed and stained with anti-LC3 $+$ GAM-FITC conjugate. Arrows point to small LC3$+$ bodies, which seem to be shed from the BLCs.

Figure 14

Monitoring the uptake and aggregation of bio-orthogonal, site-specific citrullinated MOG peptides via confocal microscopy. Human EBV-infected BLCs were incubated for 48 h with either none (peptide conc. 0 $\mathrm{\mu }$M), 6.2 or 25 $\mathrm{\mu }$M of unmodified MOG34-56 (peptide 10), or peptide citrullinated at positions $\mathrm{41}+\mathrm{46}$ (peptide 11) or positions $\mathrm{46}+\mathrm{52}$ (peptide 12) (highlighted in yellow). Cells were fixed with 4 % paraformaldehyde and processed for immunofluorescence with the following primary antibodies; the nucleus was stained with DAPI (blue) and LC-3 was used as an autophagosome marker (green). The bio-orthogonal peptides were stained using CuAAC chemistry with azide Alexa-647 (Thermo Fisher, the Netherlands).

Figure 15

Effect of the dietary modification (2014) on incidence of EAE and CalHV3 infection in the BPRC marmoset colony. Since the discovery in 2012 that full-blown clinical EAE could be induced with the rhMOG/IFA model, 19 marmosets have been tested. (a) Time to clinical score 2 (disease-free survival) or the clinical endpoint (overall survival) of monkeys tested before ($n=\mathrm{13}$ marmosets, water-based supplement is WBS) and after ($n=\mathrm{6}$ marmosets) the introduction of the yogurt-based supplement (YBS). The graphs show a significant effect of the dietary modification on the incidence and course of EAE (log-rank test). (b) Periodic blood samples were collected from the colony at the dates on the $x$ axis and tested for the presence of CalHV3 DNA using qPCR. The percentages of monkeys tested positive and negative for the viral DNA are given on the $y$ axis. The figure shows that after the dietary change the percentage of monkeys in which CalHV3 could be detected was reduced from $\pm \mathrm{70}$ % to $\pm \mathrm{30}$ %.

Figure 16

Schematic overview of immune processes in the EAE model. (a) Autoreactive CD4$+$ T cells that have been activated in peripheral lymphoid organs by the injection of antigen-adjuvant emulsion infiltrate the CNS via passage through the blood–brain barrier. This passage is mediated by interaction adhesion molecules, such as the $\mathit{\alpha }$4$\mathit{\beta }$1-integrin VLA-4 with ICAM-1 on blood–brain barrier endothelial cells (see insert). By local cognate interactions with local antigen-presenting cells, including dendritic cells, macrophages and microglia, the T cells elicit a cascade of pathophysiological reactions leading to inflammation and demyelination. (b) A second infiltrating lymphocyte type is the B cell, which secrete antibodies that, via binding to myelin sheaths and oligodendrocytes, elicit macrophage-mediated and complement-dependent cytotoxicity (ADCC and CDC, respectively). However, this classical role of B cells requires adjustment, because B cells have a much more elaborate pathogenic role. (c) The target of the autoimmune process is the axon–myelin unit, which comprises axons, the enwrapping myelin sheaths and the myelin-forming oligodendrocytes. In the healthy CNS, damaged oligodendrocytes can be replaced by infiltrating oligodendrocyte precursor cells (OPCs), but this repair capacity seems impaired in MS.