High-affinity σ1 protein agonist reduces clinical and pathological signs of experimental autoimmune encephalomyelitis

Abstract

Background and purpose: Selective agonists of the sigma-1 receptor (σ1 protein) are generally reported to protect against neuronal damage and modulate oligodendrocyte differentiation. Human and rodent lymphocytes possess saturable, high-affinity binding sites for compounds binding to the σ1 protein and potential immunomodulatory properties have been described for σ1 protein ligands. Experimental autoimmune encephalomyelitis (EAE) is recognized as a valuable model of the inflammatory aspects of multiple sclerosis (MS). Here, we have assessed the role of a σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, in EAE.

Experimental approach: EAE was induced in SJL/J female mice by active immunization with myelin proteolipid protein (PLP)139-151 peptide. The σ1 protein agonist was injected i.p. at the time of immunization (day 0). Disease severity was assessed clinically and by histopathological evaluation of the CNS. Phenotyping of B-cell subsets and regulatory T-cells were performed by flow cytometry in spleen and cervical lymph nodes.

Key results: Prophylactic treatment of EAE mice with the σ1 protein agonist prevented mononuclear cell accumulation and demyelination in brain and spinal cord and increased T2 B-cells and regulatory T-cells, resulting in an overall reduction in the clinical progression of EAE.

Conclusions and implications: This σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, decreased the magnitude of inflammation in EAE. This effect was associated with increased proportions of B-cell subsets and regulatory T-cells with potential immunoregulatory functions. Targeting of the σ1 protein might thus provide new therapeutic opportunities in MS.

© 2014 The British Pharmacological Society.

Figures

Figure 1

Chemical structure of compound 1(S), the σ1 protein agonist evaluated.

Figure 2

Effect of compound 1(S) on clinical signs of PLP 139–151-induced EAE in SJL/J mice. Mice were immunized on day 0 (D0) and their clinical course was followed for the next 35 days. Data are presented as median ± inter-quartile range IQR. (A) Reduction of EAE clinical score by a single injection of compound 1(S) at D0. Three different groups, EAE-vehicle, EAE-1(S) 1 mg·kg−1 and EAE-1(S) 5 mg·kg−1, were used per experiment with 5–7 animals per treatment group. Data were compiled from three independent experiments (n = 15–19/group). The arrow indicates the time corresponding to the disease peak, when the histological, Tregs and B-cell subsets analyses were carried out. (B) Involvement of the σ1 protein in the beneficial effects of compound 1(S) on the clinical scores. Three different groups, EAE-vehicle, EAE-1(S) 5 mg·kg−1 and EAE-BD1047 and 1(S) 5 mg·kg−1 were used per experiment with nine animals per treatment group. The σ1 protein antagonist BD1047 (10 mg·kg−1) was given i.p., 20 min before compound 1(S) injection. Data are from two separate experiments.

Figure 3

A single injection of compound 1(S) at D0 decreased the infiltration of mononuclear cells and the demyelination in the corpus callosum and the spinal cord in EAE mice. Two different groups, EAE-vehicle and EAE-1(S) 5 mg·kg−1 were used. Mice were immunized to induce EAE and compound 1(S) (5 mg·kg−1) given i.p. Animals were killed at the peak of disease (i.e. on day 14 ± 2), when the clinical grade for the EAE-vehicle group was 4.0 and for the EAE-1(S) 5 mg·kg−1 group was 1.5. This experiment utilized three animals per experimentation group. A total of 24 sections per CNS area per animal were analysed. (A) Quantification of mononuclear cell infiltration in corpus callosum/striatum, cerebellum/brainstem, and cervical, thoracic and lumbar spine. Graph bars indicate the inflammatory score (means ± SD). **P < 0.005, significantly different as indicated. (B) Representative light microscopy of mouse corpus callosum/striatum (panels A–C, G–I) or spinal cord (panels D–F, J–L) sections stained with H&E or LFB. Only EAE-vehicle mice show moderate and extensive inflammatory lesions, in corpus callosum (panels A and B) and spinal cord (panels D and E open arrowhead) respectively. Sections from the same level stained for myelin show loss of myelin in the spinal cord (panels J and K open arrowhead), while white matter was not disrupted in the corpus callosum/striatum area (panels G and H). Minimal infiltration (panels C and F filled arrowhead) and no demyelination (panels I and L filled arrowhead) was observed in sections from EAE-1(S) mice. Scale bars = 100 μm, original magnification: 10×.

Figure 4

Foxp3+ regulatory T-cells are increased in spleen and CLNs by a single injection of the σ1 protein agonist on D0. Foxp3+ Tregs (A and B) counts were analysed in spleen and CLNs in sham and EAE mice. Compound 1(S) (5 mg·kg−1) was given i.p. Animals were killed at the peak of disease (i.e. on day 14 ± 2) after immunization (clinical grades: sham-vehicle and sham-1(S) 5 mg·kg−1 group, 0; EAE-vehicle, 4.0; EAE-1(S) 5 mg·kg−1 group, 1.5). Horizontal bar indicates median values. **P < 0.005, significantly different as indicated. This experiment utilized four animals per experimentation group and is representative of two independent studies.

Figure 5

Transitional 2 (T2) B-cell subset is increased in spleen by a single injection of the σ1 protein agonist on D0. T2 (A), MZ (B), B1a (C) and follicular, FO (D) B-cell sub-population counts were analysed in spleen in sham and EAE mice. Compound 1(S) (5 mg·kg−1) was given i.p. Animals were killed at the peak of disease (i.e. on day 14 ± 2) after immunization (clinical grades: sham-vehicle and sham-1(S) 5 mg·kg−1 group, 0; EAE-vehicle, 4.0; EAE-1(S) 5 mg·kg−1 group, 1.5). Horizontal bar indicates median values. Significant differences are indicated as follows: *P < 0.05, **P < 0.005; significantly different as indicated. This experiment utilized four animals per experimentation group and is representative of two independent studies.

Figure 6

No modulation of B-cell subsets in CLNs by a single injection of σ1 protein agonist on D0. B1a (A) and FO (B) B-cell sub-population counts were analysed in CLNs in sham and EAE mice. Compound 1(S) (5 mg·kg−1) was given i.p. Animals were killed at the peak of disease (i.e. on day 14 ± 2) after immunization (clinical grades: sham-vehicle and sham-1(S) 5 mg·kg−1 group, 0; EAE-vehicle, 4.0; EAE-1(S) 5 mg·kg−1 group, 1.5). Horizontal bar indicates median values. **P < 0.005, significantly different as indicated. This experiment utilized four animals per experimentation group and is representative of two independent studies.