E-WE thrombin, a protein C activator, reduces disease severity and spinal cord inflammation in relapsing-remitting murine experimental autoimmune encephalomyelitis

Abstract

Objective: Relapses in patients with relapsing-remitting multiple sclerosis (RRMS) are typically treated with high-dose corticosteroids including methylprednisolone. However, high-dose corticosteroids are associated with significant adverse effects, can increase the risk for other morbidities, and often do not impact disease course. Multiple mechanisms are proposed to contribute to acute relapses in RRMS patients, including neuroinflammation, fibrin formation and compromised blood vessel barrier function. The protein C activator, E-WE thrombin is a recombinant therapeutic in clinical development for its antithrombotic and cytoprotective properties, including protection of endothelial cell barrier function. In mice, treatment with E-WE thrombin reduced neuroinflammation and extracellular fibrin formation in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). We therefore tested the hypothesis that E-WE thrombin could reduce disease severity in a relapsing-remitting model of EAE.

Methods: Female SJL mice were inoculated with proteolipid protein (PLP) peptide and treated with E-WE thrombin (25 μg/kg; iv) or vehicle at onset of detectable disease. In other experiments, E-WE thrombin was compared to methylprednisolone (100 mg/kg; iv) or the combination of both.

Results: Compared to vehicle, administration of E-WE thrombin significantly improved disease severity of the initial attack and relapse and delayed onset of relapse as effectively as methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment, and the combination of both treatments had an additive effect.

Conclusion: The data presented herein demonstrate that E-WE thrombin is protective in mice with relapsing-remitting EAE, a widely used model of MS. Our data indicate that E-WE thrombin is as effective as high-dose methylprednisolone in improving disease score and may exert additional benefit when administered in combination. Taken together, these data suggest that E-WE thrombin may be an effective alternative to high-dose methylprednisolone for managing acute MS attacks.

Keywords: Animal model; Demyelination; Inflammation; Multiple sclerosis; Relapsing/remitting; Steroids; Thrombin.

Figure 1. Administration of E-WE thrombin during attack and relapse reduces disease severity in relapsing remitting EAE.

Female SJL mice (n = 7–8/group) were inoculated with a 0.2 mL emulsion (sc) containing 150 μg PLP 139–151 in saline and equal volume of CFA containing 200 μg of Mycobacterium tuberculosis H37RA. Once a clinical score of 2 was observed, mice were administered E-WE thrombin (25 μg/kg; iv) or vehicle every other day for four days at the first attack and again during relapse. Shown are average disease scores (± SEM) and cumulative disease score (± SEM) for two separate experiments (A, B) and (C, D). Statistical differences between treatment group for average disease score were evaluated by 2-way ANOVA, p < 0.05, *compared to vehicle. Cumulative disease index was analyzed by Mann-Whitney, p < 0.05, *compared to vehicle.

Figure 2. E-WE thrombin delays relapse onset and attenuates disease severity.

Female SJL mice (n = 6–8/group) were inoculated with a 0.2 mL emulsion (sc) containing 150 μg PLP 139–151 in saline and equal volume of CFA containing 200 μg of Mycobacterium tuberculosis H37RA. Once a clinical score of 2 was observed, mice were administered E-WE thrombin (25 μg/kg; iv) or vehicle every other day for four days at the first attack and assessed without additional treatment during relapse. Shown are the average disease scores (± SEM) and cumulative disease score (± SEM) for two separate experiments (A, B) and (C, D). Statistical differences between treatment group for average disease score were evaluated by 2-way ANOVA, p < 0.05, *compared to vehicle. Cumulative disease index was analyzed by Mann-Whitney, p < 0.05, *compared to vehicle.

Figure 3. Effect of E-WE thrombin in methylprednisolone-treated SJL mice.

Female SJL mice were inoculated as described above and treated once a clinical score of 2 was observed. Four treatment groups were evaluated: vehicle, E-WE thrombin (E-WE)(25 μg/kg; iv), methylprednisolone (MP)(100 mg/kg; iv) or the combination of E-WE thrombin and methylprednisolone (E-WE + MP). For methylprednisolone, a single dose was administered (black arrow) and for E-WE thrombin or vehicle, mice were dosed every other day for four days (gray arrows). Data are the means ± SEM of two separate experiments (A, B) and (C, D), n=3–7 mice/group. Statistical differences in average disease score were analyzed by repeated measures ANOVA, p < 0.05,* compared to vehicle. Cumulative disease index was analyzed by 1-way ANOVA, using Dunnett’s Multiple Comparison test, p < 0.05, *compared to vehicle.

Figure 4. E-WE thrombin reduces CD45+ immune cell infiltration in the lumbar spinal cord sections.

Lumbar spinal cord sections collected from mice on Day 29 were fixed, embedded in paraffin and immunostained for CD45+ cells (red). Cell nuclei were visualized by staining with DAPI (blue). Representative sections are portrayed for each treatment group (A-D). Magnification bar is 200 microns. The number of CD45 positive cells were quantified in the (E) meninges and (F) parenchyma using Image J. Data are the means ± SD of two experiments, n = 6–10 mice/group. Statistical differences in treatment group were analyzed by 1-way ANOVA, using Bonferroni’s Multiple Comparison test, p < 0.05, *compared to vehicle.

Figure 5. E-WE thrombin reduces CD3+ immune cell infiltration and improves MBP+ staining in lumbar spinal cord sections.

Lumbar spinal cord sections collected from mice on Day 29 were fixed, embedded in paraffin and immunostained for MBP and CD3. Cell nuclei were visualized by staining with DAPI (blue). Representative photos for each treatment group are shown for MBP+ staining (A-D; red), CD3+ staining (E-H; green), the overlay of MBP+ and CD3+ at 5X magnification (I-L) and at 20X (M-P) magnification. In the third row, white arrows designate regions depicted at 20X magnification in the fourth row. Magnification bars are 200 microns (A, E, I) and 50 microns (M). The number of CD3 positive cells were quantified in the (Q) meninges and (R) parenchyma using Image J. The percentage of MBP positive area (S) was quantified using Image J. Data are the means ± SD of one experiment, n=3–4 mice/group. Statistical differences in treatment groups were analyzed by 1-way ANOVA, using Bonferroni’s Multiple Comparison test, p < 0.05, *compared to vehicle, **compared to methylprednisolone.