The first large scale human clinical trial in patients with relapsing-remitting MS (RRMS) using interferon beta (IFN-β) was published in 1993 and showed that relapse rates were reduced by 34% in high dose IFN-β1b and by 8% in lower dose compared to placebo group, and severity of relapses was also reduced (6).

To study the manifestations of EAE as well as TnP compared to betaseron subcutaneous (s.c.) prophylactic treatment in the CNS, we used the MOG_35–55 peptide-induced EAE model in the susceptible strain (Figures 1A–C). Female C57BL/6J (BL6) mice induced with EAE and treated with vehicle exhibited chronic disease progression with onset around day 11 post-induction (p.i.), exhibiting mean maximal clinical score of 2.3 ± 0.09 at day 17 p.i. that was maintained in chronic phase, grade 2–2.1.

TnP (3 mg/Kg) or betaseron (10,000 UI/mice) treatments started with s.c. daily application by 10 days during induction phase. EAE symptoms appeared on day 12 p.i. for both treated-groups (0.1 ± 0.01 and 0.2 ± 0.01, respectively). TnP as well as betaseron both ameliorated the clinical manifestations of EAE decreasing by 30% the mean maximal clinical score from 2.3 ± 0.09 to 1.6 ± 0.02. TnP delayed the onset of maximal symptoms by 2 days (19) and betaseron delayed the onset of symptoms by 1 day (18). The beneficial effect was stable over time and sustained until day 30 (Figure 1A).

Because immune cell infiltration of the CNS is considered a hallmark of EAE, we investigated the effect of TnP compared to betaseron on leukocyte infiltration in the CNS by the analysis of hematoxylin and eosin (H&E, Figure 1B) or with Luxol fast blue (LFB, Figure 1C) paraffin sections at day 17. While vehicle-treated EAE mice presented extensive infiltration of inflammatory cells per mm² (254 ± 4.3) and areas of demyelination (score 3), tissue from TnP- (34 ± 2.3 and score 1.5) or betaseron-treated mice (45 ± 1.9 and score 1.2) lacked severe signs of inflammation with preserved myelinated areas.

IFNs do not directly exert a neuroprotective effect; however, through their direct effect on encephalitogenic CD4+Th1 cells, altering their activation state trigger a decrease in neuronal demyelination, which prevents further neuronal damage (7). Next, Fluoro-Jade C was used to evaluate the degree of neurodegeneration in damage areas of the spinal cord of vehicle- or treated-EAE mice and the corrected total cell fluorescence (CTCF) was obtained (Figure 2). Vehicle-treated EAE mice exhibited a significantly increased number of degenerating neurons on day 17 (1.12 ± 0.01). Degeneration progressively intensified up to day 30 p.i. in this group, from central to the peripheral areas (2.29 ± 0.01). The TnP-treated group of mice showed a mild degeneration (0.54 ± 0.01) in the central region that remained similar until day 30 (0.65 ± 0.01). In contrast, the intense neuronal degeneration seen on day 17 (3.38 ± 0.01) in betaseron-treated group was observed to increase at day 30 (3.99 ± 0.01). In summary, these data show that subcutaneous treatment with TnP was more effective in the long-term control of neuronal degeneration.

Like IFN-β, glatiramer exerts broad immunomodulatory effects that are incompletely understood. In patients, glatiramer significantly reduced disease symptoms and development of new lesions by up to 30% in RRMS, although it showed no improvement in long-term efficacy on progression of disability (8). Next we examined the role of TnP in the control of EAE symptoms compared with glatiramer in a prophylactic regimen of subcutaneous application.

Mice that received TnP showed substantially fewer neurological deficits by the measurement of the mean maximal clinical score of 0.9 ± 0.03 than mice that received glatiramer (1.5 ± 0.03) or vehicle-treated EAE mice (1.6 ± 0.01). The decrease in mean maximal clinical score induced by glatiramer was 6% in the first 5 days compared to 44% of decrease caused by TnP treatment which lasted for 30 days. In addition, we observed that TnP delayed the peak of symptoms by 4 days, while glatiramer accelerated it by 1 day (Figure 3A).

At the peak of disease (day 17), microglia and macrophage infiltrations were examined in the CNS of vehicle- or treated-groups. The percentage of CD45^highCD11b^high macrophages infiltrating the brain or spinal cord decreased by 74% and 55%, respectively, after TnP treatment compared to a 26% and 24% of decrease caused by glatiramer (Figures 3B, C). In addition, TnP caused a 37% of decrease in CD45^lowCD11b^low microglia in the brain and 79% in the spinal cord compared to percentages of 12% and 60% induced by glatiramer (Figures 3D, E).

Glatiramer treatment results in the skewing of auto-reactive lymphocytes away from the pathogenic effector cell responses towards regulatory functions (9). The next step was to compare the ability of prophylactic treatment with TnP to induce regulatory T and B cells in the presence of EAE. As showed in Figure 4A, an increase in the percentage of activated CD19⁺CD1d⁺CD5⁺B regulatory (Breg) during the priming phase was also observed in the spleen of TnP- (2.9-fold) as well as glatiramer- (2.3-fold) compared to vehicle-treated EAE mice, consistent with a systemic effect of both treatments.

We also examined the percentage of conventional CD4⁺CD25⁺FOXP3⁺T regulatory (Treg) cells in the CNS (Figures 4B, C). A higher percentage of CD4+ Treg cells was detected in the CNS mainly in the spinal cord of TnP-treated mice during the peak phase of the disease (2.3-fold and 8-fold increase), compared to a percentage (2-fold and 6-fold increase) of these cells in the CNS of glatiramer-treated mice.

Fingolimod was granted Food and Drug Administration approval in 2010 for oral therapy (10). Fingolimod is effective in reducing clinical exacerbations and delays the disability progression in RRMS patients based on reduction of encephalitogenic lymphocytes egress from secondary lymphoid organs (11–14).

After onset at day 7, EAE progressed, and vehicle-treated mice reached the peak of the disease (mean = 2.6 ± 0.19) at day 18. After prophylactically administered oral TnP, clinical signs became evident at 11 ± 1 p.i. and developed a disease that peaked at day 16 ± 1 p.i. with clinical score = 1.3 ± 0.09, indicating a decrease of 50%. In contrast, in prophylactically fingolimod-treated EAE mice, clinical symptoms were observed starting from 14 ± 1 p.i. with the maximal gravity (clinical score = 2.1 ± 0.03) being evident at day 19 p.i. ± 1, representing 19% of decrease. These data show that both treatments delayed onset of disease from 7 to 11 or 14, but only TnP significantly reduced peak symptoms by 50% (Figure 5A).

In order to further analyze the impact of prophylactic treatment by TnP or fingolimod on inflammation and myelin distribution, spinal cords from vehicle- and treated-mice were assessed by H&E (Figure 5B) and LFB (Figure 5C) staining. The cell infiltration was moderately prevented in the spinal cord white matter of TnP (67 ± 3) or fingolimod-treated EAE mice (70 ± 4) when compared to the control or vehicle-treated EAE groups (159 ± 6). Seventeen days after EAE induction, demyelination was clearly evident in the white matter of spinal cord of vehicle-treated EAE mice. Levels of intense remyelination were observed after TnP and fingolimod-treated mice.

In addition, analysis of the spinal cord by Fluoro-Jade C revealed that the tissues of mice treated prophylactically with oral TnP showed a large extent of degenerated neurons on day 17 (2.35 ± 0.01), compared to vehicle-treated EAE mice (0.88 ± 0.01) that were reduced dramatically until day 30 (TnP: 0.58 ± 0.01 versus vehicle: 2.67 ± 0.01). Fingolimod-treated mice showed a smaller area of neuronal degeneration at day 17 (0.28 ± 0.01) compared to TnP, but analysis of progression shows that the injury got worse after 30 days in fingolimod-treated mice (1.16 ± 0.01) (Figure 6). Although the number of degenerated neurons in the fingolimod-treated mice is lower than the vehicle-treated mice at day 30 by 56%, they are 2-fold higher than the TnP-treated mice. Together, these data confirmed that TnP promoted an improvement in long-term neuronal regeneration.

Finally, all vehicle-treated EAE mice displayed a prominent body weight loss from day 10, onset of symptoms peaking on day 14. The low weight observed in EAE mice remained until day 21 when it slightly increased and stabilized. The improvement of clinical score induced by TnP corresponded to an increase in body weight immediately after disease induction. The s.c. treatment of mice with TnP was more effective than oral administration in improving weight loss as it prevented weight loss between days 10 to 21 and, unlike untreated mice, it induced weight gain from day 22 to 30. Oral TnP-treatment of EAE mice induced partial protection between days 10 to 17, but from day 18, weight gain was similar to s.c. administration (Figure 7).

Four to six-week-old female C57BL/6J (BL6) wild type mice were obtained from a colony at the Butantan Institute, São Paulo, Brazil. Mice were kept in the same SPF animal unit maintained in sterile micro-isolators with sterile rodent feed and acidified water and housed in positive-pressure air-conditioned units (25°C, 50% relative humidity) on a 12 h light/dark cycle. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Brazilian College of Animal Experimentation. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Butantan Institute (#747/10; #1203/14; #1205/14).

TnP trifluoroacetate compound (P13821401, C₆₃H₁₁₄N₂₂O₁₃S₄, purity 97.3%) was manufactured under the patent holder’s proprietary method (Laboratório Cristália Produtos Químicos Farmacêuticos LTDA). Myelin oligodendrocyte glycoprotein trifluoroacetate lyophilized powder (MOG)_35-55 peptide (316131-3 part P1822131, MEVGWYRSPFSRVVHLYRNGK, 2581,4 Da e 95.2%) was purchased from GenScript (order 20615, Piscataway NJ USA). Copaxone (glatiramer acetate, P53888, P63924, or P63033) was purchased by Teva Pharmaceutical Industries Ltd (Petah Tikva, Israel). rhIFNβ-1b (betaseron) was purchased by Bayer Health Care Pharmaceuticals. Fingolimod (FTY720 hydrochloride, Gilenya; Novartis) was purchased by Cayman Chemical.

EAE was induced according to Mendel et al. (26). Briefly, four to six-week-old female BL6 (n = 5 per group) received a subcutaneous injection (s.c.) in the tail base of 300 µg of MOG_35–55 per animal emulsified in 100 µL incomplete Freund’s adjuvant (IFC, 263910, Difco) containing 500 µg of Mycobacterium tuberculosis H37RA (231141, part 0239413, Difco) on day 0. Immediately thereafter and again 48 h later, mice received an intraperitoneal (i.p.) injection of 500 ng of Pertussis toxin (P7208, part SLBK1874V, Sigma-Aldrich, St Louis, MO, USA) diluted in 200 µL of sterile 0.9% saline. EAE progression was monitored for 30 d after immunization with MOG. Clinical sign scores of EAE were daily assigned as follows: 1, tail limpness; 2, impaired righting reflex; 3, hind limb paralysis; 4, hind- and forelimb paralysis; 5, death. The mean of monthly scores was calculated. Mice were weight every day. All behavioral measurements were done in awake, unrestrained, age matched female mice. All tests were performed in an appropriate quiet room between 10 am and 4 pm. If necessary, food was provided on the cage floor. Prior injection of Pertussis toxin mice were anaesthetized with isoflurane. A humane endpoint was fixed using specific parameters as follow: EAE mice consistently scored higher (≥4, complete hind limb paralysis or quadriparesis, and weight loss greater than 30%) were removed from the study and killed.

For subcutaneous treatment, mice (n = 5/group) were injected with 100 µL of TnP at dose of 3 mg/Kg mice according to Komegae et al. (4); glatiramer at 2 mg/mice according to Aharoni et al. (27) or betaseron at 10,000 UI/mice according to Inoue et al. (28) at, all diluted in 0.9% saline on the back. For oral treatment, mice previously submitted to a 4-hour fast received 30 µL of TnP at 3 mg/Kg or fingolimod at 0.3 mg/Kg mice according to Bonfiglio et al. (29), all diluted in 0.9% saline administered by oral gavage. Mice were treated every day from day 0 to 9 (Prophylactic treatment - during induction phase). The EAE controls were injected with 0.9% saline alone (Vehicle).

Healthy, vehicle-treated EAE, and EAE treated-mice (n = 5/group) were killed and spleens were removed at day 7 p.i. for analysis during the induction phase. The brain and the spinal cord were excised from mice perfused transcardially with ice-cold phosphate buffered saline (PBS) at peak of disease (17). Single-cell suspensions of splenic tissue were prepared by digestion with 1 mg/mL of type II collagenase (Roche) and 500 U DNase I (Sigma-Aldrich) following by mechanical disruption in GentleMacs dissociator (Miltenyi). Erythrocytes in spleens were lysed with 0.14 M NH4Cl and 17 mM Tris-Cl (pH 7.4). Additionally, the brain and spinal cord cell suspensions were prepared and centrifuged at 200 g for 10 min and resuspended in 4 mL of 30% isotonic Percoll (P1644, Sigma) diluted in HBSS and overlaid by equal volumes of 37% and 70% isotonic Percoll. The gradient was centrifuged at 800 g for 20 min and leukocytes were harvested from the 37% - 70% interface, washed, and counted.

Spleen, brain, and the spinal cord from healthy, vehicle-treated EAE, and EAE treated-mice were harvested and cell suspensions were prepared. Immune cell infiltration and resident microglia activation were assessed per cytofluorometric (FACS) by cytometer analysis. For surface staining, single-cell suspensions (1 x 106 cells in 100 µL) were treated with 3% mouse serum of naive mice and then incubated for 30 min in ice with specific anti-mouse antibodies (Abs) fluorochromes-conjugated or purified Abs followed by secondary Abs fluorochromes-conjugated purchased from BD Biosciences, R&D Systems or eBioscience: PerCP-Cy5.5-CD11b (550993, BD Biosciences), PE-CD11b (557397, BD), FITC-CD4 (553729, BD Biosciences), PE-CD4 (557308, BD Biosciences), PE-CD25 (553075, BD Biosciences), FITC-CD19 (557398 or 553785, BD Biosciences), APC-CD19 (17-0193-82, eBioscience), PE-CD5 (553022, BD Biosciences), FITC-CD5 (11-0051-82, eBioscience), allophycocyanin-CD1d (17-0011-82, eBioscience), PE-CD1d (12-0011-82, eBioscience), unlabeled rat anti-mouse CD45 (MAB114, R&D Systems) and anti-rat Ig PerCP5.5 (F0115, R&D Systems) and Goat PerCP-Rat F(ab)2 IgG (H+L) (F-0115), for 30 min on ice. For intracellular staining, cells were washed, fixed and permeabilized with Cytofix/Cytoperm solution (554722, BD Biosciences) for 20 min in ice, washed (554723, BD Biosciences) and stained with PerCPCy5.5-FOXP3 (45-5773-82, eBioscience). Cells were washed three times in RPMI medium and re-suspended in 1% formaldehyde for the cytofluorometric analysis. Negative-controls were used to set the flow cytometer photomultiplier tube voltages, and single-color positive controls were used to adjust instrument compensation settings. Cells were examined for viability by flow cytometry using side/forward scatter characteristics or 7-aminoactinomycin D (7-AAD, SML1633, Merck) exclusion. Data (100,000 events acquired per sample) were acquired using a four-color FACSCalibur flow cytometer equipped with CellQuest software (Becton-Dickinson, San Jose, CA). Data were recorded as percent of fluorescent positive cells per organ..

To evaluate the histological manifestations of EAE, healthy, vehicle-treated EAE, and EAE treated-mice (n = 5/group) were killed on days 17 or 30. The spinal cords were removed and fixed in buffered formalin 4%. Paraffin-embedded sections of spinal cord were stained with hematoxylin and eosin (H&E) or with Luxol fast blue (LFB) for analysis of inflammation or demyelination, respectively. Histopathological examination was performed in a blinded fashion.

The 10 micron-thick slices of spinal cord at 17 or 30 day were first deparaffinized in a slice warmer, and then rehydrated through a graduated alcohol series followed by a wash in 70% ethanol. The tissue was briefly rinsed with distilled water and incubated in 0.06% KMnO₄ solution for 10 min, afterwards, the slices were washed and transferred to a 0.0001% solution of Fluoro-Jade C (AG325, Millipore) dissolved in 0.1% acetic acid for 10 min in dark. The slices were rinsed through three changes of distilled water for 1 min per change. Hundred microliters of 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, sc-3000415) solution at 1.5 µg/mL for 10 min were used to label nuclear DNA. The slices were rinsed through three changes of distilled water for 1 min per change. Excess water was drained onto a paper towel, and the slides were air dried on a slide warmer at 37°C for 35 min. The air-dried slices were cover-slipped with 20 µL of Fluoromount G non-fluorescent (00-4958, eBioscience) mounting media and blocked with nail polish. All images were acquired using the OLYMPUS BX51 microscope at a 10x magnification and scale indicates 50 micrometers. The blue nuclear label conferred by DAPI was visualized via ultraviolet light excitation, while the Green Fluoro-Jade C staining was quantified by the use of Image J Software v.1.8.0_172. The corrected total cell fluorescence (CTCF) was obtained utilizing the formula CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings).

All values were expressed as mean ± SEM (standard error of the mean). Experiments using 5 mice per group were performed independently two times. Parametric data were evaluated using analysis of variance, followed by the Bonferroni test for multiple comparisons. Non-parametric data were assessed using the Mann-Whitney test. Differences were considered statistically significant at p < 0.05 using GraphPad Prism (Graph Pad Software, v6.02, 2013, La Jolla, CA, USA).