Immunopathology of Japanese macaque encephalomyelitis is similar to multiple sclerosis

Abstract

Japanese macaque encephalomyelitis (JME) is an inflammatory demyelinating disease that occurs spontaneously in a colony of Japanese macaques (JM) at the Oregon National Primate Research Center. Animals with JME display clinical signs resembling multiple sclerosis (MS), and magnetic resonance imaging reveals multiple T2-weighted hyperintensities and gadolinium-enhancing lesions in the central nervous system (CNS). Here we undertook studies to determine if JME possesses features of an immune-mediated disease in the CNS. Comparable to MS, the CNS of animals with JME contain active lesions positive for IL-17, CD4+ T cells with Th1 and Th17 phenotypes, CD8+ T cells, and positive CSF findings.

Keywords: Demyelination; Inflammatory; Interleukin 17 (IL-17); Intrathecal IgG; Magnetic resonance imaging (MRI); Th1; Th17.

Figure 1. MRI from animals with JME

Axial 3T MRI from JM 22019 (A and B) and JM 30760 (C and D) 1 day after presentation with clinical signs of JME. (A) Axial T₂-weighted image of cerebellum from JM 22019 shows hyperintense signal identified by arrow. (B) Axial T₁-weighted MPRAGE image acquired 40 minutes post-0.2 mmol/kg gadoteridol administration shows an enhancing lesion in cerebellum; more readily visualized in the inset which represents a difference image T₁-weighted (pre-gadoteridol) subtracted from T₁-weighted (post-gadoteridol). (C) Axial T₂-weighted image of JM 30760 shows hyperintense signal in the internal capsule region of the cerebral cortex (arrow). (D) Axial post-gadoteridol T₁-weighted image shows enhancing lesion in the internal capsule; expanded in the inset showing difference image as in panel (B). The lesion enhances on a T₁- weighted MPRAGE image acquired 6 minutes after the administration of gadoteridol. MPRAGE = magnetization prepared rapid acquisition gradient echo.

Figure 2. Histopathology and immunostained images of gadolinium-enhancing CNS lesions acquired from JME animals

(A) Low magnification (2.5x) image of luxol fast blue (LFB) and hematoxylin-eosin (H&E) stain of cerebellar lesion from JM 22019 shows demyelinated region adjacent to perivascular cuff surrounding the blood vessel. Arrowhead points to the demyelinated region. Scale bar = 100 μM. (B) High magnification (40x) of immunostained cerebellar lesion showing numerous CD163+ macrophage/activated microglia (brown) immunoreactive for myelin basic protein (MBP, gray). Arrowheads point to a representative macrophage/activated microglia immunoreactive for MBP. Scale bar = 10 μM. (C) Low magnification (2.5x) image of LFB/H&E stain of internal capsule lesion shows extent of demyelination. Arrowhead points to the demyelinated region. Scale bar = 100 μM. (D) High magnification (40x) image of immunostained internal capsule lesion showing fewer CD163+ macrophage/activated microglia (brown) immunoreactive for MBP (gray). Arrowhead points to a representative macrophage/activated microglia immunoreactive for MBP. Scale bar = 10 μM. (E) Low magnification (2.5x) image of LFB/H&E stain of internal capsule lesion of JM 30773. Arrowhead points to large demyelinated region of the lateral geniculate nucleus. (F) High magnification (40x) of immunostained periventricular surface of inflammatory cell aggregates with arrowhead showing accumulation of CD20+ B cells. Scale bar = 10uM.

Figure 3. IL-17 production in the active cerebellar lesion of animal JM 26174 with JME

High magnification (40x) images of double immunofluorescence staining for IL-17 (A, D, and G; green), CD3⁺ T cells (B, red), olig2+ oligodendrocytes (E, red), and GFAP+ activated astrocytes (H, red) taken from the demyelinating cerebellar lesion in JM 26174. The overlay demonstrates expression of IL-17 in CD3+ T cells (C, arrowheads), oligodendrocytes (F, arrowheads), and in astrocytes (I, arrowheads). Scale bars = 10μM.

Figure 4. Analysis of CNS mononuclear cell infiltrates in active CNS lesions of JME animals

A. Flow cytometry was used to analyze T cell populations infiltrating CNS lesions isolated from five JME animals (JMs 22019, 31509, 27624, 31852 and 31522). Mononuclear cells (MNCs) were stained with anti-CD4 and anti-CD8. Bars represent the percentage of lymphocytes expressing CD4⁺ (white) or CD8⁺ (grey). B. Infiltrating MNCs were stimulated for 6 hours with PMA and ionomycin in the presence of Brefeldin A followed by intracellular cytokine staining for IL-17 and IFN-γ. Plots show the percentage of IL-17 and IFN-γ expressing cells within CD4⁺ or CD8⁺ T cell populations gated on CD3⁺.

Figure 5