The effect of FTY720 in the Theiler's virus model of multiple sclerosis

Journal of the Neurological Sciences 308 (2011) 41–48

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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

The effect of FTY720 in the Theiler's virus model of multiple sclerosis Libin Li a,⁎, Makiko Matsumoto a, Timothy J. Seabrook b, 1, Celine Cojean b, Volker Brinkman b, Andrew R. Pachner a a b

Department of Neurology and Neuroscience, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA Novartis Institutes for Biomedical Research, Basel, Switzerland

a r t i c l e

i n f o

Article history: Received 15 March 2011 Received in revised form 9 June 2011 Accepted 14 June 2011 Available online 2 July 2011 Keywords: FTY720 TMEV Multiple sclerosis Animal model Progressive disability Antibody responses

a b s t r a c t FTY720 (fingolimod) has demonstrated efficacy in multiple sclerosis (MS). We evaluated the effects of FTY720 on progressive disability, viral load, and antibody responses in mice infected with Theiler's murine encephalomyocarditis virus (TMEV). FTY720 and phosphorylated FTY720 (FTY720-P) were detected in the brain after intraperitoneal injection of the drug. Bioactivity of FTY720 was confirmed by reduced numbers of mononuclear cells in the spleen and blood after treatment. No significant differences were found in disability progression, viral load, and serum antibody responses between the FTY720-treated versus the PBS-treated mice. There was less production of IgG within the CNS in the FTY-treated group on some measures. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) in humans that causes demyelination and progressive neurological disability. There is no cure for MS, and more effective therapies are needed. FTY720 (also called fingolimod or Gilenya) is an oral therapy recently approved for use in relapsing MS by the FDA, that has demonstrated efficacy in MS superior to placebo as well as to interferon beta, the most commonly used drug for MS treatment [1,2]. FTY720 is effectively phosphorylated by sphingosine kinases in vivo, and the active metabolite FTY720-P targets a new class of G-protein coupled receptors, termed S1P receptors [3]. FTY720-P likely acts as a functional antagonist, down-modulating lymphocytic S1P1 receptors [4], and this is associated with retention of central- but not effector memory T cells in lymphoid organs [5]. FTY720 crosses the blood–brain barrier (BBB) and attains substantial concentrations in the CNS [6]. Thus, the drug may also directly affect neural cells [7,8]. In order to learn more about mechanisms of action of FTY720, we analyzed its effects in the Theiler's murine encephalomyocarditis virus (TMEV)-induced, immune-mediated demyelinating disease (TIDD) model, which is characterized by progressive weakness and robust

⁎ Corresponding author at: Department of Neurology and Neuroscience, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 S.Orange Ave., Newark, NJ 07103, United States. Tel.: + 1 973 972 4509; fax: + 1 973 972 5059. E-mail address: [email protected] (L. Li). 1 Current address: Merck Serono S.A., Geneva, Switzerland. 0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.06.029

systemic and CNS antiviral antibody and cellular responses [9–11]. Our goal was to test the effect of FTY720 on the progressive neurological disability, CNS histology, the viral load, and the antiviral immune response in TIDD. 2. Materials and methods 2.1. FTY720 FTY720 was obtained from Novartis Institutes for Biomedical Research (Basel, Switzerland) and was dissolved in distilled water. A dose of 0.3 mg/kg/day, called “low dose” (FTY-L) or 3.0 mg/kg/day, called “standard dose” (FTY-S), was given daily by intraperitoneal (IP) injection in a volume of 0.1 ml until necropsy. The selection of doses of FTY720 was based on earlier studies in murine viral infection [12] or experimental autoimmune encephalomyelitis (EAE) in which 3 mg/kg/day was effective in decreasing disease [13,14]. There was no peritoneal serositis induced by the IP injections, based on examination at necropsy. 2.2. Mice and virus All animal work utilized protocols reviewed and approved by the University Animal Care and Use Committee at UMDNJ-New Jersey Medical School. SJL mice purchased from Harlan Laboratories (Indianapolis, IN) were 4–8-week old females, and were housed in isolator cages in the Research Resource Facility at New Jersey Medical School. The virus used was the BeAn strain of TMEV, obtained originally

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L. Li et al. / Journal of the Neurological Sciences 308 (2011) 41–48

from Steven Miller (Northwestern University), and passaged in a hamster fibroblast line, BHK, as previously described [15]. PFU were determined by a cytopathic effect (CPE) assay. 25–30 μl of a PBS solution containing 3 × 10 8 PFU/ml BeAn (i.e., 7–9 million PFU per mouse) was injected into the right frontal lobe of mouse brain. Injections of FTY720 were begun at 3 different time points prior to or after the development of weakness, at 30(n= 81), 60(n= 30), or 85(n = 30) days postinfection (DPI); the results did not substantively vary according to time post-infection (p.i.). I.e., for all 141 infected mice, comparisons between FTY720- and PBS-treated mice showed no differences, and there were no significant differences in outcome of any measures according to when treatment with FTY720 or PBS was initiated. Injections of therapy, FTY720 or PBS, were continued in each animal until the animal was necropsied. Mice were necropsied at any average of 150 days p.i., or earlier if their severe neurological disability endangered their life, but never prior to 100 days p.i. Techniques used in the necropsies were performed as previously described [16–18], including anesthesia, intracerebral (IC) injection, perfusion with PBS, CSF collection by cisternal tap, and the collection of blood and other tissues. 2.3. Isolation of mononuclear cells (MNCs) MNCs were obtained from spleen and spinal cord (SC) as previously described [16,19]. In brief, spleen was minced on frosted glass slides to obtain single cell suspensions; a Percoll gradient was used to isolate cerebral MNCs. Ammonium chloride was then used to lyse red blood cells. 2.4. Quantification of FTY720 and FTY720-P in the blood and brain Whole blood was collected by cardiac puncture. Brains were collected after cardiac perfusion with 5 ml PBS. Blood and brains were snap-frozen and kept in −80 °C until analysis. Concentrations of FTY720 and FTY720-P were determined by high-pressure liquid chromatography (Agilent 1100; Agilent, Waldbronn, Germany) with mass spectrometric detection as described previously [6]. 2.5. ELISA for albumin, total IgG, anti-TMEV binding antibody (Bab) CSF and serum albumin, total IgG, as well as anti-TMEV IgG (TMEV-IgG) were assayed by ELISA as previously described [16]. The indices were calculated according to the following formulas: Quotient Albumin (Qalb) = albuminCSF /albuminserum × 1000; Quotient IgG (Q IgG) = IgG CSF/IgGserum × 1000 IgG Index = Q IgG/Qalb. TMEV-specific binding antibody (TMEV-Bab) was determined as previously described. In brief, total IgG concentrations in the serum and CSF were diluted to the same level. Each sample was then assigned an

arbitrary unit (AU) of anti-TMEV binding activity according to the standard curve in the anti-TMEV ELISA. The Bab index was calculated as the following ratio: TMEV-Bab Index = AUCSF/AUserum. 2.6. Neutralizing antibody (Nab) assay TMEV-specific neutralizing antibody (TMEV-Nab) was measured using the modified cytopathic effect assay (CPE) [20,21]. In brief, 1500 PFU of TMEV was incubated with serial three-fold dilutions of mouse serum and CSF for 2 h, followed by incubation with BHK cells for 48 h in a 96-well microplate format. Live BHK cells were then exposed to naphthol blue-black dye, and absorbance read on an ELISA reader at 620 nm. Neutralizing titers were expressed as the inverse of that dilution of serum able to block 90% of the cytopathic effect of the virus; i.e. to lower the cytopathic effect of 1500 PFU of virus to 150 PFU of virus under conditions in which the change from 1500 PFU to 150 PFU is in the linear part of the curve of the cytopathic effect dilution curve. The Nab index was calculated as the ratio: Quotient TMEV-Nab (QTMEV-Nab)=TMEVNabCSF/TMEV-Nabserum ×1000; then the TMEV-NAb index was calculated as: TMEV-Nab Index=QTMEV-Nab/QIgG. 2.7. Real-time RT-PCR for in situ IgG production and TMEV load Expression of IgG and TMEV RNA was performed essentially as previously described with specific primers and probes for murine IgG1 and TMEV [18,22]. Total RNA was isolated from fresh, homogenized tissue samples using the Trizol One-Step Isolation method; reverse transcription (RT) was performed in a Gene Amp PCR system 9700 (Perkin-Elmer Applied Biosystems, Foster City, CA). Taqman RT-PCR was performed in an ABI 7000 Sequence Detection System (PE Applied Biosystems). To determine the viral load in tissue, a standard curve was obtained when the threshold cycle (CT) values of the samples were plotted against known TMEV concentration. Viral load in tissue was determined by referring the CT values of experimental samples to the standard curve. Results were expressed as estimated TMEV numbers in each 0.5 μg RNA from tissues. Mouse GAPDH mRNA levels were used as a measure of RNA quality. The IgG1 mRNA level was expressed as the relative expression index of 2 to the power of −ΔCT, where −ΔCT = − (CT-IgG − CT-GAPDH). 2.8. Rotarod testing for progressive disability Progressive disability in mice was assessed as previously described [17]. Rotarod data were expressed as a neurological function index (NFI). The NFI value at any time point was the mean of the last 3 time indices divided by the mean time indices from day 15 to day 45 after infection. Time indices were the time on the Rotarod for that day divided by the mean of the 2 maximum times for that mouse. Data shown are pooled data for the 3 different times post-infection in which FTY720 was administered. There were no significant differences between the FTY720- and PBS-treated groups at any of these time periods. 2.9. Histology

Fig. 1. Viral load in mouse tissues. Brain and spinal cord (SC) were collected at necropsy. Viral load was expressed as mean copy number of virus in each 0.5 ug RNA from tissues as calculated from a standard of known virus copy number. Error bars represented standard error. n = 15 in FTY-S, n = 30 in FTY-L, n = 36 in PBS.

Sections of lumbar spinal cord were harvested and postfixed in 4% formaldehyde overnight. The tissues were then transferred to PBS until processing into paraffin blocks using standard techniques. Two separate lumbar areas were examined at least 2 mm apart. Sections of 10 μm were cut, placed on glass slides and histochemical staining performed using standard methods for overall morphology (Haematoxylin and Eosin, H&E), myelin (solochrome) and axonal density (Bielshowsky's silver stain). T cells were examined using anti-CD3 (AbD Serotec, Oxford, UK), microglia/macrophages by anti-ionized calcium binding adaptor molecule 1 (Iba-1, Wako Pure Chemical Industries, Osaka, JP) and astrocytes were examined using anti-glial fibrillary acidic protein

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Fig. 2. Anti-TMEV IgG antibody responses in sera of FTY720 and PBS-treated TIDD mice. TMEV-Bab levels were expressed as binding units referring to a positive standard as described in Materials and methods. Error bars represented standard error. (A) Time course of TMEV-Bab response. Sera samples were collected at different time points during the course of infection. Data showed average units of at least 5 animals in each group. (B) TMEV-Bab levels in sera collected at necropsy, which were 140–160 DPI. Open circles (○) represented TMEV-Bab units of sera from each mouse. Mean unit values were represented as horizontal bars. Total serum samples tested: n = 15 in FTY-S group, n = 51 in FTY-L group, n = 41 in PBS group. Sera from uninfected mice had readings of less than 100 anti-TMEV units. (C) TMEV-Nab levels in sera collected at necropsy. TMEV-Nab titers were expressed as tenfold reduction units (TRU). Data showed average TRUs of serum specimens. n = 10 in FTY-S group, n = 12 in FTY-L group, n = 12 in PBS group. No TMEV-Nab could be detected in uninfected animals.

Fig. 3. Antibody level in the CSF. CSF samples were collected at necropsy about 140–160 DPI. n = 10 in FTY-S group, n = 12 in FTY-L group, n = 12 in PBS group, n = 6 in uninfected group. Error bars represented standard error. (A) Total IgG concentration in the CSF of TIDD mice with different treatments. *, p b 0.05 for FTY groups compared with PBS group; (B) TMEV-Bab level in the CSF; (C) TMEV-Nab level in the CSF. Comparison between the groups showed no statistical significance (p N 0.05).

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Table 1 CSF data analysis. CSF data from FTY720, PBS mice was analyzed. Calculation of Qalb, QIgG, QTMEV-Bab, IgG Index, TMEV-Bab Index and TMEV-Nab Index was described in Materials and methods. Data was shown as mean± S.E. n = 10 in FTY-S group, n = 12 in FTY-L group, n = 12 in PBS group. Indices

QAlb QIgG QTMEV-Bab QTMEV-Nab IgG Index TMEV-Bab Index TMEV-Nab Index

Groups PBS

FTY-S

FTY-L

4.93 ± 1.19 47.25 ± 10.10 55.40 ± 17.03 602.84 ± 257.80 11.22 ± 2.76 1.62 ± 0.54 25.42 ± 8.46

4.97 ± 0.75 23.73 ± 3.99 30.83 ± 12.39 216.93 ± 87.06 5.69 ± 1.25 1.26 ± 0.32 7.65 ± 2.08

3.73 ± 1.59 32.07 ± 6.24 48.06 ± 10.06 345.18 ± 123.40 11.12 ± 4.42 1.93 ± 0.88 12.56 ± 4.8

(GFAP, Dako, Glostrup, DK). Immunohistochemistry stainings were performed using a Benchmark XT system (Ventana, Tucson, USA). Quantification was performed using a slide scanner and software (ScanScope XT, Aperio, Oxford, UK) to delineate the total area of the spinal cord section and the area demyelinated or immunopositive for Iba-1 staining. T cells were counted in the entire spinal cord area and determined to be either perivenular or parenchymal. To quantify H&E stained sections the section was visually divided into quadrants and the number of quadrants containing either perivenular cuffs or infiltrating inflammation was determined and expressed as a percentage. 2.10. Statistical analysis Data were compared by Student's t test and Excel software. For H&E histological analysis a Mann–Whitney non-parametric ttest was used. A P value of equal or less than 0.05 was considered significant. 3. Results 3.1. FTY720 treatment did not change the viral load level in the spinal cord We wished to know if FTY720 treatment would affect viral load in the CNS. TMEV load was measured by real-time RT-PCR of TMEV RNA in tissues collected at necropsy an average of 150 days post-infection. Although a slightly higher load was observed in the spinal cord in

Fig. 4. IgG1 mRNA levels in different tissues of FTY720 and PBS mice. IgG1 expression was expressed as relative IgG mRNA level in each 0.5 μg total RNA from tissue, expressed as 2 to the power of inversed delta Ct (IgG1-GAPDH). IgG1 expression was indexed to the expression of the housekeeping gene GAPDH, the usual Ct reading of which was 18.0–22.0. n = 10 in FTY-S group, n = 26 in FTY-L group, n = 30 in PBS group. Data from FTY-S mice was presented as FTY group. Data showed means of IgG mRNA level, and error bars represented standard error.

Fig. 5. Comparison of progressive disability between FTY720-treated and PBS-treated animals in time course of TIDD. The average values of the neurological function index from FTY720- and PBS-treated animals are shown on y-axis and time post-infection is shown on the x-axis. Data shown are pooled data for the 3 different time points when FTY720 was administered.

FTY720-treated mice (Fig. 1), the differences between PBS- and FTY720-treated mice were not significant. Higher levels of TMEV were found in the spinal cord than in the brain, and no viral RNA was detected in the spleen. Thus, in this study, as in previous studies, TMEV infection was limited to the CNS, mostly within the spinal cord, and FTY720 administration did not result in dissemination of the infection to sites outside of the CNS. 3.2. FTY720 treatment did not significantly affect the systemic anti-TMEV humoral immune responses in TIDD After infection with TMEV, serum TMEV-BAb levels increased until the end point of necropsy (Fig. 2A), consistent with previous studies [17,18]. Comparison of sera obtained at necropsy, an average of 150 days post-infection, between the groups showed comparable Bab levels (Fig. 2B). We also measured Nab levels using a CPE assay. FTY720 treatment lowered serum Nab titers slightly but differences between groups were not statistically significant (Fig. 2C). 3.3. The effect of FTY720 on CSF measures: albumin, IgG, TMEV-Bab, and TMEV-NAb level Since antibody production within the CNS is a prominent feature of TMEV infection [17,18], and the anti-TMEV antibody response in the CNS is critical for control of virus in persistent infection [19,23], we assessed the effects of FTY720 on CNS antibody production using a variety of measures. These included the determination of concentrations of total IgG, TMEV-Bab, TMEV-Nab, and albumin concentrations in sera and CSF. Indices of antibody levels in sera and CSF were calculated from the above measurements. Serum and CSF were collected at necropsy, an average of 150 days post-infection.

Fig. 6. MNC counts in mouse organs. Organs analyzed included spleen (SP), whole blood (Blood) and spinal cord (SC), all collected at necropsy, an average of 150 days postinfection. Data showed average MNC counts in each spleen, each 50 ul whole blood, and each spinal cord. FTY-S and FTY-L animals were pooled in this analysis. Error bars represented Standard Deviation. N N 15 in each group. Data used in this analysis was representative of 2–4 experiments.

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Table 2 FTY720 and FTY720-P concentrations in the brain and blood of TIDD mice. TMEV-infected mice were treated intraperitoneally with 0.3 mg/kg/day (FTY-L) and 3.0 mg/kg/day (FTY-S) FTY720 from day 30 to necropsy. FTY720 (FTY) and FTY720-P (FTY-P) concentration data were shown as mean ± S.E. N N 15 in each group. Groups

FTY-L FTY-S

Daily dose

Level in blood

mg/kg

FTY (ng/ml)

FTY-P (ng/ml)

FTY-P/FTY ratio

Level in brain FTY (ng/g)

FTY-P (ng/g)

FTY-P/FTY ratio

FTY

FTY-P

0.3 3.0

7.9 ± 1.9 93.1 ± 21.0

44.7 ± 10.7 747.4 ± 169.4

5.7 8.0

423.3 ± 73.3 4203.6 ± 488.3

991.3 ± 132.1 9994.3 ± 955.6

2.3 2.4

53.7 45.1

22.2 13.3

There were generally lower levels of total IgG, TMEV-BAb, and TMEV-NAb in the CSF with FTY720 treatment, but only in quantitation of total IgG were the differences between FTY720- and PBS-treated mice significant. (p = 0.004 between FTY-S and PBS; p = 0.01 between FTY-L and PBS) (Fig. 3A–C). FTY720 treatment did not affect relative concentrations of albumin in serum and CSF. The CSF data, analyzed as indices, are shown in Table 1. The values of Qalb are a measure of BBB permeability, and mildly elevated Qalb values in infected mice indicated that there was slightly increased BBB permeability during TMEV infection, but this increased permeability was not affected by FTY720. QIgG values were significantly reduced after FTY720 treatment (p b 0.05). No significant differences were detected for other ratios and indices between FTY720 and PBS animals, but a trend of reduction was found in all the CSF immunoglobulin and specific antibody analyses, and this trend was dose-dependent.

3.4. Effect of FTY720 on IgG mRNA levels in tissues of TIDD mice Production of IgG within tissues was measured directly by realtime RT-PCR for IgG mRNA in the spinal cord compared to spleen and brain in FTY720- and PBS-treated mice. The tissues were collected at

Brain/blood ratio

necropsy, an average of 150 days post-infection. As in previous studies [17,18], the highest levels of IgG expression were found in the spinal cord, higher even than spleen (Fig. 4). Lower levels of expression were found in FTY720-treated mice relative to PBS-treated mice, but the difference was not significant. Substantial variability of the measure among individual animals, a previously recognized characteristic of this model [24], likely contributed to inability to demonstrate statistical significance of the differences. 3.5. FTY720 treatment did not affect disability progression in TIDD We evaluated disability over the course of infection in FTY720treated and PBS-treated mice with Rotarod testing, using neurological function index (NFI) values, which compare each animal's performance at any time to its peak performance. NFI values of animals from both groups decreased progressively beginning at approximately 6 weeks p.i. As in previous studies [17,18], 90% of infected animals had severe disability as defined by NFI values of less than 0.3 by 140 days p.i., and both groups had equal percentages of relatively unaffected mice with NFIs greater than 0.7, by that time. At any time point after infection, there was no significant difference in the progressive disability in FTYtreated mice relative to those given PBS (Fig. 5). 3.6. FTY720 treatment reduced numbers of circulating MNCs FTY720 suppresses lymphocyte egress from lymphoid tissues into the circulation, resulting in reduction of the peripheral lymphocyte count. We wished to confirm the bioactivity of FTY720 by measuring MNC count in peripheral and CNS organs after FTY720 treatment, including spleen, blood and spinal cord. MNC counts in the spleen and blood at necropsy, an average of 150 days post-infection, were significantly lower in FTY720-treated animals, decreased to 10% to 30% of values in PBS-treated mice (Fig. 6), consistent with previous reports in mice and humans [14,25]. However, this effect was less pronounced in the spinal cord. There were less MNCs in the cords of the FTY720-treated relative to the PBS-treated groups, but the difference was not significant (p = 0.10). 3.7. FTY720 concentrations in blood and brain were dose dependent We measured FTY720 concentrations in blood and brain samples that were taken during necropsy about 24 h after the last dose, i.e. trough levels since the drug was injected once a day. Both FTY720 and FTY720-P were found in the blood and brain in all treated animals, but the levels were considerably higher in the brain, measured in ng/g than those in blood, measured in ng/ml, with ratio of brain to blood being more than 10 (Table 2). Both in blood and brain, the levels of FTY720 and FTY720-P were much higher in FTY-S animals than in FTY-L ones.

Fig. 7. Histological analyses of FTY720 and vehicle treated mice at day 170 post-infection. Spinal cord tissues were obtained 170 days post-infection and examined histologically for inflammation (H&E, microglia/macrophages) and demyelination (solochrome cyanine). There was no significant difference between any of the quantitatively examined parameters (see Materials and methods for details). Similar data was obtained for day 114 post-infection but is not shown. n = 3/group. Data showed average ± standard deviation.

3.8. FTY720 did not significantly reduce neuroinflammation as determined by histology We examined neuroinflammation using a variety of markers. There was no significant difference in the percentage of spinal cord quadrants containing either perivenular or parenchymal infiltrating immune cells

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Fig. 8. Inflammatory cells were found in vehicle and FTY720 treated groups. Inflammatory cells were examined using H&E (A, B) and immunohistochemistry for CD3+ T cells (C, D). Both methods found immune cells in the parenchyma (arrows) and in perivenular cuffs (arrowheads) Macrophages and activated microglia were visualized using Iba-1 immunohistochemistry and found in both vehicle and FTY720 treated groups (E, F). Scale bars = 100 μm.

assessed by H&E (Fig. 7). Similar to MNC cell counts the FTY720 treatment groups at day 170 showed a decrease in perivenular T cells (vehicle 66.3 ± 23, FTY720 33.8 ± 16) and parenchyma infiltrating T cells (vehicle 125.2± 56, FTY720 60.1 ± 8). However, this did not reach statistical significance. Similar data were obtained at day 114 postinfection with T cells decreased in perivenular cuffs (vehicle 81.0 ± 31, FTY720 41.0 ± 5) and the parenchyma (vehicle 102.2 ± 17, FTY720 61.1 ± 16). Activated microglia/macrophages were examined using Iba1 and no significant difference was found at either day 114 or 170 postinfection (Fig. 8). Astrocytes were examined visually and appeared similar between groups (data not shown). Demyelination was examined using a solochrome cyanine technique in which myelin is stained blue (Fig. 9). There was no significant difference between FTY720 and vehicle treated groups. Axonal density did not appear to differ but was not quantified (Fig. 9). 4. Discussion FTY720 (fingolimod, Gilenya) is an oral therapy recently approved by the FDA for relapsing forms of multiple sclerosis [1,2]. Its mechanisms

of action are poorly understood, but may involve retention of T cells in lymphoid organs and direct effects on neural cells [7]. In this study we analyzed FTY720 in Theiler's murine encephalomyelitis virus-induced demyelinating disease (TIDD). TIDD faithfully models the clinical and pathological characteristics of multiple sclerosis, including progressive disability, spasticity, demyelination, axonal loss, inflammation, and antibody production within the CNS. The mechanisms responsible for CNS injury in this model are not clearly established, but appear to be primarily immune-mediated [26–28]. We wished to determine whether FTY720 would have an effect on measures of progressive CNS injury, the viral load, the pathology, and the antiviral immune response in TIDD. Our major goal was to determine whether FTY720 would have an effect on disability progression in this model. Progressive CNS injury is a hallmark of TIDD, and leads to progressive disability. Disability usually begins to develop in the second month post-infection, accelerates in the third month, and progresses steadily until 90% of mice are markedly weak by 4 months after infection. Rotarod testing has been demonstrated in this model of MS to be a reproducible and reliable means of assessing neurological disability [10,17]. In mice treated with FTY720 beginning at day 60, at about the time disability begins to develop,

L. Li et al. / Journal of the Neurological Sciences 308 (2011) 41–48

Fig. 9. Demyelination and axonal loss were present. Myelin loss was examined using solochrome histochemistry which stains myelin blue (A, B). In both the vehicle and FTY720 treated mice there was a loss of myelin (pale areas) in the lateral ventral white matter. Axons were stained black in the Bielschowsky's silver stain (C, D). There was a loss of axons in areas that were also demyelinated (arrows). Scale bar = 500 μm.

neurological function decreased progressively at the same rate in FTY- versus PBS-treated mice. Similar results were obtained if treatment was initiated at day 30, long before the development of disability, or day 85, after development of disability. The data thus indicate a lack of a robust ameliorative effect of FTY720 on disability progression at the tested doses in the TIDD model. It is possible that the mechanisms of neurodegeneration in the TIDD model differ from those in human MS. The Rotarod measures maximal functional capacity of mice. We did not use either activity boxes or activity wheels to test effects on spontaneous activity, but previous studies have demonstrated that these measures correlate well with Rotarod testing in the Theiler's model of MS [10]. Thus, the addition of these measures would have been unlikely to yield substantively different data. Both FTY- and PBS-treated groups were noted on evaluation to have similar levels of spastic weakness and tonic spasms and markedly impaired ability to move around their cages. The lack of an effect on clinical measures did not rule out the possibility of an effect on the histology of the disease. Thus, we also investigated whether months of treatment with FTY720 might impact pathology, specifically demyelination, axonal damage, or infiltration of the spinal cord with total mononucelar cells, macrophages or T cells. None of the above measures revealed any significant differences between FTY720- and PBS-treated mice. A major concern was whether FTY720 would increase viral load. Viral infections are a major cause of illness in newer therapies of MS, including a substantial incidence of progressive multifocal leukoencephalopathy (PML), a JC virus-induced potentially fatal infection in MS patients treated with natalizumab [29]. In our experiments, the viral load in the spinal cord, the usual reservoir of infection in TIDD, was not significantly increased by FTY720. In our studies, we used 300 and 3000 μg/kg/day of FTY720, doses in the range of those which had been shown to be effective in treating EAE in mice [13,14] or rats [30,31]. The drug exposures at these doses are at least 10-fold higher than those achieved with MS-therapeutic dosing in humans. High levels of both FTY720 and FTY720-P could be detected in CNS parenchyma, both at the 0.3 and 3.0 mg/kg doses, and

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were consistent with levels present in brain and blood during EAE experiments in rats [6]. The precise location of FTY720 within the CNS is unknown, but there is some evidence that tissue levels may predominantly reflect compound integrated into myelin sheaths. Progressive CNS injury in TIDD is associated with inflammation within the CNS, manifested as large numbers of MNCs within the CNS parenchyma. The pathogenesis of CNS damage in the chronic phase of the disease is thought to be immune-mediated. Macrophage, Th1, or Th17 functions were not tested in our experiments, although that would have been a logical next step had a clear therapeutic effect been documented. There was no significant difference in the areas demyelinated or axonal density in the FTY720 treated groups compared to vehicle treated groups. The absence of significant effects of FTY720 on disability progression in TIDD could be explained by its lack of ability to markedly lower the number of MNCs in the spinal cord. Our findings are in contrast to those of Fujino et.al. [32] in myelin basic protein-induced EAE, who found that FTY720 reduced lymphocyte and macrophage counts in the spinal cord. This discrepancy is likely explained by the fact that mechanisms of recruitment of mononuclear cells into the CNS chronically during TIDD are probably quite different from those in acute EAE, and are probably independent of high concentrations of FTY720 in the CNS. One population of mononuclear cells entering the CNS is B-cells, some of which ultimately become plasma cells secreting immunoglobulin within the CNS [33]. In TIDD, plasma cells within the CNS are numerous, primarily located in perivascular infiltrates and in the meninges, but also scattered throughout the parenchyma [34]. These cells are dependent on local production within the CNS of a variety of factors [35]. The finding of a lower level of CNS antibody production in FTY720- versus PBS-treated mice, as demonstrated by lower CSF IgG levels, lower spinal cord IgG mRNA levels, and lower TMEV-Nab indices, is consistent with a local suppressive effect of FTY720 on plasma cells within the CNS, possibly through interference with T cell help for antibody production [36]. We administered FTY720 at 30–85 days post-infection, because mice do not generally begin getting significant disability until after one and a half to two months after TMEV infection. The times of administration after initial infection were chosen to mimic timing of therapy in patients with MS, who do not initiate therapy until the first signs of disease. Timing of immune therapies relative to the induction of infection or disease is known to be critical in their effect in models of MS. For instance, B-cell depleting therapy can either exacerbate or suppress EAE depending upon when it is administered relative to the induction of the disease [37]. Thus, had FTY720 been administered prior to infection, there may have been different results, but this scenario does not mimic the clinic situation in MS. Another reason for not initiating treatment prior to infection is that infection with TMEV causes an early cerebritis that is usually mild, but can be exacerbated by immune therapies [38]. In conclusion, these studies demonstrate that FTY720 had relatively mild effects when treatment was initiated 30–85 days post-infection. Despite marked lowering of MNC counts in the blood and spleen, there was only a mild lowering of MNC counts in the spinal cord and of antibody production in the CNS. Disability progression and antiviral antibody levels in the serum were unaffected. Further studies of effects of FTY720 on TIDD, using different doses and different times relative to virus injection, are indicated.

Conflict of interest statement TS, CC and VB are or were employees of Novartis Pharma AG.

Acknowledgement We thank Novartis for funding these studies.

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