Fingolimod may support neuroprotection via blockade of astrocyte nitric oxide

RESEARCH ARTICLE

Fingolimod May SupportNeuroprotection via Blockade of

Astrocyte Nitric Oxide

Emanuela Colombo, PhD,1 Marco Di Dario, BS,1 Eleonora Capitolo, BS,1

Linda Chaabane, PhD,1 Jia Newcombe, PhD,2 Gianvito Martino, MD,1 and

Cinthia Farina, PhD1

Objective: Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFjB path-way and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration.Here we investigated the role of the crosstalk between sphingosine-1-phosphate (S1P) and cytokine signaling cas-cades in astrocyte activation and inflammation-mediated neurodegeneration, and addressed the effects of fingolimodon astrocyte–neuron interaction and NO synthesis in vivo.Methods: Immunohistochemistry, immunofluorescence, and confocal microscopy were used to detect S1P receptors,interleukin (IL) 1R, IL17RA, and nitrosative stress in multiple sclerosis (MS) plaques, experimental autoimmuneencephalomyelitis (EAE) spinal cord, and the spinal cord of fingolimod-treated EAE mice. An in vitro model wasestablished to study the effects of S1P, IL1, and IL17 stimulation on NFkB translocation and NO production in astro-cytes, on spinal neuron survival, and on astrocyte–neuron interaction. Furthermore, fingolimod efficacy in blockingastrocyte-mediated neurodegeneration was evaluated.Results: We found coordinated upregulation of IL1R, IL17RA, S1P1, and S1P3 together with nitrosative markers inastrocytes within MS and EAE lesions. In vitro studies revealed that S1P, IL17, and IL1 induced NFjB translocationand NO production in astrocytes, and astrocyte conditioned media triggered neuronal death. Importantly, fingoli-mod blocked the 2 activation events evoked in astrocytes by either S1P or inflammatory cytokines, resulting in inhibi-tion of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of fingolimod to EAE micehampered astrocyte activation and NO production.Interpretation: A neuroprotective effect of fingolimod in vivo may result from its inhibitory action on key astrocyteactivation steps.

ANN NEUROL 2014;76:325–337

Sphingosine-1-phosphate (S1P) is a potent bioactive

sphingolipid regulating cellular processes such as

growth, survival, and differentiation.1 It is formed by

phosphorylation of sphingosine in a reaction catalyzed by

the sphingosine kinases SPHK1 and SPHK2. SPHK1 is

activated by numerous stimuli, including proinflamma-

tory cytokines, which promote SPHK1 translocation to

the plasma membrane, where it catalyzes the production

of S1P. On release, S1P activates specific G protein–

coupled receptors (S1P1–5) in an autocrine and/or para-

crine manner.2 This is an inside-out signaling pathway

that supports growth factor signal transduction, including

activation of nuclear transcription factor NFjB and nitric

oxide (NO) synthase.1 It is well established that the

S1P–S1P receptor axis controls trafficking and migration

of immune cells.1 S1P receptors may be expressed also

by cells in the central nervous system (CNS), where they

support neurogenesis, neurite outgrowth, astrocyte prolif-

eration, and migration.3–5 Importantly, in the CNS

inflammatory neurodegenerative disease multiple sclerosis

(MS),6 several elements of S1P signaling have dysregu-

lated expression.7,8 Fingolimod is an anti-inflammatory

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24217

Received Dec 23, 2013, and in revised form Jul 1, 2014. Accepted for publication Jul 1, 2014.

Address correspondence to Dr Farina, INSpe–Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, Via

Olgettina 58, 20132 Milan, Italy. E-mail: [email protected]

From the 1Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy; and 2NeuroResource, University

College London Institute of Neurology, London, United Kingdom.

VC 2014 American Neurological Association 325

drug approved as oral therapy for MS.9 This sphingosine

analogue is rapidly phosphorylated into its biologically

active form to become a potent modulator of S1P1,

S1P3, S1P4, and S1P5. Fingolimod decreases immune

cell trafficking out of secondary lymphoid organs by

inducing internalization and degradation of S1P receptors

and therefore preventing their migration into the CNS.9

In addition to the peripheral action, fingolimod may

cross the blood–brain barrier10 and target CNS cells

expressing S1P receptors, including astrocytes.11 On

CNS injury, astrocytes actively participate in scar forma-

tion and resolution, as they restrict lesion area, limit

inflammation, and promote tissue repair.12 Nevertheless,

recent reports have indicated that pathways leading to

NFjB activation and NO production in astrocytes con-

tribute to MS pathology.13–17 Notably, reactive astrocytes

are known to upregulate S1P1 and S1P3 in MS lesions8;

however, functional implications for neurodegeneration

have not been described. Furthermore, several studies

have shown that cytokines and growth factors induce

inside-out S1P signaling,18 but the possibility of crosstalk

between the signaling cascades of S1P and inflammatory

cytokines such as interleukin (IL) 1 and IL17 in MS

remains largely unaddressed. Finally, despite the potential

impact of fingolimod treatment on CNS resident cells,

no study has so far investigated the effects on glia–neu-

ron interactions. Here we show that (1) lesions from MS

patients and animals with experimental autoimmune

encephalomyelitis (EAE) have upregulated and coordi-

nated expression of IL1R, IL17RA, S1P1, and S1P3 in

astrocytes; (2) S1P and cytokine signaling in astrocytes

leads to neuronal apoptosis; (3) although administration

of fingolimod does not hamper S1P/cytokine-mediated

neuronal loss in neuronal cultures, it blocks neurodegen-

eration induced by activated astrocytes; (4) S1P signaling

leads to NFjB translocation and NO production in

astrocytes, and is inhibited by fingolimod; (5) similarly,

fingolimod downregulates IL1 and IL17 signaling in

astrocytes; and (6) in vivo treatment with fingolimod

reduces astrocyte activation and NO production. These

data suggest that fingolimod administration to MS

patients may have neuroprotective effects by reducing

neurotoxic mediators released by astrocytes following

activation of S1P and/or inflammatory cytokine signaling

cascades.

Materials and Methods

Human Tissue SamplesSeven MS white matter brain samples containing plaques

(mean age 5 61.3 6 6.8 years) and 5 normal control white mat-

ter samples (mean age 5 63.8 6 22) were obtained postmortem

and conserved as snap-frozen tissue blocks at the Neuro-

Resource tissue bank, University College London Institute of

Neurology, London, United Kingdom, and used for immuno-

histochemical studies. Tissues were donated to the tissue bank

with informed consent using documentation approved by the

National Research Ethics Service Committee London–Central,

United Kingdom. This research (code HisPathCNS) was

approved by the in-house San Raffaele Ethical Committee for

human studies. Normal control white matter samples and MS

chronic inactive white matter plaques were from temporal, pari-

etal, and frontal ventricular or subventricular areas.

EAE Induction and TreatmentEAE was induced in 7-week-old female C57BL/6N mice (Har-

lan Laboratories, Udine, Italy), and animals were assessed for

clinical signs of EAE as previously described.13,19 Fingolimod

(FTY720, 3mg/kg body weight; Selleckchem, DBA Italia,

Milan, Italy) was given by oral gavage once daily after the

appearance of the first clinical sign of disease. Control mice

received administration of vehicle (0.9% NaCl physiological

solution). Experimenters were blind to the treatment regimen

for EAE mice. At day 22 postimmunization, mice were per-

fused with 4% paraformaldehyde, and spinal cords were

removed, frozen, and used for immunofluorescence studies. All

procedures involving animals were authorized by the local insti-

tutional animal ethics committee and by the Italian General

Direction for Animal Health at the Ministry for Health.

Immunohistochemistry and ImmunofluorescenceExperimentsTen-micrometer-thick serial sections were prepared from human

CNS tissue blocks or mouse spinal cords. Four naive, 5

fingolimod-treated, and 5 vehicle-treated mice derived from 2

independent EAE experiments were analyzed. At least 5 spinal

cord sections were labeled for each mouse. Immunohistochem-

istry and immunofluorescence experiments were performed as

previously described.13,20 The following antibodies were used:

rabbit a-glial fibrillary acidic protein (GFAP; catalog number

Z0334; Dako, Milan, Italy), rat a-GFAP (clone 2.2B10; catalog

number 345860; Calbiochem, Milan, Italy), rabbit a-MBP (cat-

alog number AB980; Millipore, Vimodrone, Italy), mouse

a-btubulin (clone TUJ1; catalog number MMS-435P; Covance,

DBA Italia, Milan, Italy), rabbit a-nitrotyrosine (catalog num-

ber 06–284; Chemicon, DBA Italia), rabbit a-iNOS (catalog

number 610332; BD Biosciences, Buccinasco, Italy), rabbit

a-EDG1 (H-60; catalog number sc-25489), rabbit a-EDG3

(H-70; catalog number sc-30024), rabbit a-IL17RA (H-168;

catalog number sc-30175), mouse a-IL1R (H-8; catalog num-

ber sc-393998; all from Santa Cruz Biotechnology, DBA Italia),

mouse a-IL1R (clone 35730; catalog number MAB269; R&D

Systems, Milan, Italy), and rabbit a-NFjB p65 subunit (catalog

number GWB-12B4DC; GenWay Biotech, San Diego, CA).

Apoptotic cells were detected with DeadEnd Fluorometric

TUNEL System (Promega, Milan, Italy). Immunohistochemis-

try images were acquired using a light microscope (Leica,

Milan, Italy). Fluorescence images were captured with Leica

TCS confocal laser-scanning microscopes. ImageProPlus 6.0

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326 Volume 76, No. 3

(Media Cybernetics, Silver Spring, MD) and ImageJ software

(http://rsbweb.nih.gov/ij/) were used for analysis. Stainings and

quantifications were blindly examined by an experimenter

unaware of the identity of individual sections.

Western BlotHuman and mouse astrocytes were homogenized in lysis buffer

containing 20mM Tris–HCl (pH 7.5), 150mM NaCl, 1% Tri-

ton X-100, 10% glycerol, 1% sodium dodecyl sulfate, 20mM

ethylenediaminetetraacetic acid, 1% NP40, 1mM phenylmetha-

nesulfonyl fluoride, and the protease inhibitory cocktail (Sigma-

Aldrich, Gallarate, Italy), and centrifuged at 13,000 3 g for 10

minutes at 4�C. Protein concentration was determined by

standard BCA assay (Thermo Fisher Scientific, Euroclone,

Milan, Italy). Proteins were separated on 12% Mini-

PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Milan,

Italy) and electroblotted onto nitrocellulose membranes (Bio-

Rad Laboratories). Blots were probed overnight with the follow-

ing antibodies: rabbit a-EDG1 (H-60; catalog number sc-

25489), rabbit a-EDG3 (H-70; catalog number sc-30024), rab-

bit a-IL17RA (H-168; catalog number sc-30175), and mouse

a-IL1R (H-8; catalog number sc-393998; all from Santa Cruz

Biotechnology) at 1:100 dilution. Finally, membranes were

incubated with IRDye 800 goat antirabbit or antimouse Ig

antibody (LI-COR Biosciences, Carlo Erba Reagents, Cornar-

edo, Italy), and signals were detected with the LI-COR infrared

imaging system (LI-COR Biosciences).

Stimulation and Analysis of Neuronal CulturesPrimary spinal neurons were obtained from 16-day-old Sprague

Dawley rat embryos as described21 and stimulated in vitro with

S1P (Tebu-bio, Magenta, Italy; 100nM, 1lM, 10lM, 50lM,

100lM), fingolimod (FTY720-phosphate; Tebu-bio, 100nM),

IL17A (Peprotech, Tebu-bio, 10ng/ml), IL1 (R&D Systems,

10ng/ml), or vehicle. Immunofluorescence experiments were

performed and analyzed as previously published.13 Images were

acquired using a Leica fluorescence microscope. ImageProPlus

6.0 and ImageJ software were used for analysis. Stainings and

quantifications were blindly examined by an experimenter

unaware of the identity of individual images.

Stimulation and Analysis of Astrocyte CulturesHuman primary fetal astrocytes were maintained and cultured

as described,13 and exposed to S1P, FTY720-P, IL17, IL1, or

vehicle. Astrocyte-conditioned medium (ACM) was collected as

published.13 Astrocytes were loaded with the nitric oxide dye

DAF-FM (Molecular Probes, Invitrogen, San Giuliano Mila-

nese, Italy), exposed to the stimuli or vehicle for 45 minutes,

and then fixed and stained for GFAP and 40,6-diamidino-2-

phenylindole (DAPI; Sigma-Aldrich). The number of NO-

producing cells was calculated as the percentage of total DAPI-

positive cells. To detect NFjB translocation into the nucleus,

astrocytes were treated for 15 minutes with stimuli, then fixed

in 4% paraformaldehyde, permeabilized with 0.2% Triton X-

100 in phosphate-buffered saline, and stained with a-NFjB or

isotype control.

Statistical AnalysisNormality of the distribution was assessed by Kolmogorov–

Smirnov statistics, and significance was measured by Student

t test with homeostatic variance for normal distribution or by

Mann–Whitney U test for non-normal distribution. For statisti-

cal evaluation of EAE clinical score the nonparametric Mann–

Whitney ranking U test was used. All probability values were

2-sided and subjected to a significance level of 0.05.

Results

IL1R and IL17RA Cytokine Receptors AreExpressed on Reactive Astrocytes in MS LesionsImmunohistochemistry and immunofluorescence experi-

ments in 5 normal control brain white matter samples

and 7 MS chronic inactive lesions (Fig 1A) were per-

formed to evaluate the expression of the receptors for

IL1 and IL17, the 2 inflammatory cytokines typically

found in innate and adaptive immune responses during

neuroinflammation. In contrast to normal control white

matter, where both IL1R and IL17RA were little

FIGURE 1: Interleukin (IL) 1R and IL17RA are stronglyexpressed by astrocytes in multiple sclerosis (MS) lesions.(A) Immunohistochemistry for myelin basic protein (MBP)and (B) double immunofluorescence stainings for IL1R or (C)IL17RA (green) and glial fibrillary acidic protein (GFAP; red)on serial sections from normal control white matter (left) orMS lesion (right). 40,6-Diamidino-2-phenylindole (DAPI) wasused for nuclear staining. The small panels represent singlestainings. Representative images of 7 analyzed MS lesionsand 5 normal control tissues are shown. Scale bars 5 20lm.

Colombo et al: Fingolimod Blocks Astrocyte NO

September 2014 327

http://rsbweb.nih.gov/ij/

expressed (see Fig 1B, C, left panels), MS lesions dis-

played robust upregulation of both receptors, and these

signals were clearly induced on GFAP-positive reactive

astrocytes (see Fig 1B, C, right panels).

Cytokine- and S1P-Activated Astrocytes InduceFast Neuronal DegenerationWe have shown recently that IL1 triggers degeneration

of primary spinal neurons both directly and via astro-

cyte activation.13 Here, we wondered whether IL17 may

exert the same effects. In a first set of experiments, spi-

nal neuron cultures were exposed to IL17 or IL1, as

control, and then evaluated for survival, apoptosis, and

integrity of the network. As shown in Figure 2A and B,

similarly to IL1, IL17 strongly reduced the number of

neurons in culture (39.3% reduction in IL17-exposed

cultures compared to control), and induced apoptosis

(18.1 6 1.7% in IL17-treated cultures vs 1.4 6 0.06%

FIGURE 2: Inflammatory cytokines or sphingosine-1-phosphate (S1P) induce neurite fragmentation and neuronal death. (A) Flu-orescence images showing representative stainings for b-tubulin (red), 40,6-diamidino-2-phenylindole (DAPI; blue), and terminaldeoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL; green) in neuronal cultures after24 hours of exposure to interleukin (IL) 1 or IL17 (10ng/ml). (B) Quantification of cells, percentage of apoptotic cells, andb-tubulin mean fluorescence intensity (MFI) in neuronal cultures exposed to IL1 or IL17. Cell numbers and b-tubulin MFI wereexpressed as a percentage of control (CTRL). (C) Fluorescence images showing representative stainings for b-tubulin (red),DAPI (blue), and TUNEL (green) in neuronal cultures after 24 hours of exposure to increasing concentrations of S1P. In A andC, arrows indicate TUNEL-positive nuclei. (D) The same parameters as in B in neuronal cultures treated with S1P. Data wereobtained from 2 to 3 independent experiments. In B and D, graphs show results from a representative experiment. All dataare represented as mean 6 standard deviation. Scale bars 5 50lm. *p < 0.05, **p < 0.01, ***p < 0.001 versus CTRL.NT 5 nontreated.

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in control cultures) and fragmentation of the neuronal

network (77% reduction of b-tubulin signal compared

to control). Interestingly, using the same in vitro cul-

tures, the bioactive lipid mediator S1P triggered limited

neuronal death at concentrations <10lM, and high

doses of the sphingolipid (50lM and 100lM) led to

massive cell loss, apoptosis, and neuronal fragmentation

(see Fig 2C, D).

In the second set of experiments, we studied the

effects of IL1, IL17, and S1P on astrocyte–neuron inter-

actions. First, we checked and confirmed by Western blot

and immunofluorescence staining that human astrocyte

cultures expressed IL1R, IL17RA, and the S1P receptors

S1P1 and S1P3 in vitro (Fig 3). The protein expression

of the receptors was also reproduced Fig 3A in mouse

astrocyte cultures as revealed by Western blot and immu-

nofluorescence (data not shown). Human astrocytes were

exposed to IL1, IL17, or S1P at a low dose (100nM, a

concentration in the lower ranges of that found in normal

human blood22), the medium was changed after 4 hours

to remove the stimuli, and the ACM was collected after a

further 24 hours of incubation and added to spinal neu-

ron cultures. As previously published,13 whereas superna-

tants from nontreated astrocyte cultures (sNT) did not

affect neuronal survival and network integrity, ACM

from IL1-treated astrocytes (sIL1) triggered a degenera-

tive response in neurons characterized by cell loss, apo-

ptosis, and neurite fragmentation. The same observations

were made in neuronal cultures exposed to supernatants

from IL17-treated astrocytes (sIL17; 32.7 6 2.8% apo-

ptosis vs 9.36 6 3.38% in control; 46% reduction of cell

number and 58.6% reduction of b-tubulin signal com-

pared to control). Furthermore, whereas 100nM S1P had

limited degenerative effects when given directly to neu-

rons, ACM from astrocytes activated with the same dose

(sS1P) induced neurodegeneration at levels comparable

to sIL1 and sIL17 media (30.5 6 2.8% apoptosis; 27%

reduction of cell number and 61% reduction of b-

tubulin signal compared to control). Overall, these obser-

vations indicate that astrocyte activation by inflammatory

cytokines or by a lipid mediator may drive neurodegener-

ative processes.

FIGURE 3: Astrocyte responses to inflammatory cytokines or sphingosine-1-phosphate (S1P) induce neurite fragmentation andneuronal death. (A) Human and mouse astrocytes express interleukin (IL) 1R, IL17RA, and S1P receptors as detected by West-ern blot. (B–E) Double immunofluorescence and confocal imaging for IL1R (B), IL17RA (C), S1P1 (D), and S1P3 (E; all in green)and glial fibrillary acidic protein (GFAP; red) in human astrocyte cultures. Small panels represent single stainings. Scalebars 5 20lm. (E) Quantification of cells, percentage of apoptotic cells, and b-tubulin mean fluorescence intensity (MFI) in neuro-nal cultures after exposure to astrocyte-conditioned medium (ACM). Stimuli or vehicle were given to astrocytes for 4 hoursand then removed. Cells received fresh medium and were cultured for a further 24 hours. Then, ACM was collected and addedto cultured neurons. Neurons were incubated with supernatants from nontreated (sNT), IL1-treated (sIL1; 10ng/ml), IL17-treated (sIL17; 10ng/ml), or S1P-treated astrocytes (sS1P; 100nM) for 24 hours. Data were obtained from 3 independentexperiments, and graphs show results from a representative experiment. All data are represented as mean 6 standard devia-tion. ***p < 0.001 versus control (CTRL). DAPI 5 40,6-diamidino-2-phenylindole; Tunel 5 terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling.


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Reactive Astrocytes Coexpress Cytokine andS1P Receptors in MS Lesions and in the SpinalCord of EAE MiceWe checked in vivo expression of cytokine and S1P recep-

tors in serial sections from MS lesions. Figure 4 shows

immunohistochemistry and double immunofluorescence

stainings of a representative MS sample. Loss of white

matter MBP and increased astrocyte GFAP immunoreac-

tivity were used to define lesion borders from the normal-

appearing white matter (NAWM). As already depicted in

Figure 1, reactive astrocytes in the MS lesion were charac-

terized by enhanced immunoreactivity for IL1R and

IL17RA, which, in contrast, were rarely detectable in

NAWM (see Fig 4B, C). In the same region, S1P1 and

S1P3 showed similar expression patterns. Whereas only

weak staining was detected in NAWM, they were strongly

upregulated on astrocytes in MS lesions (see Fig 4D, E),

indicating that reactive astrocytes may express both cyto-

kine and S1P receptors within MS lesions. We verified

that this observation was also seen in EAE. IL1R, and

IL17RA immunoreactivity was detected in the meninges

but not in the parenchyma of naive spinal cord. In con-

trast, upregulation of IL1R, IL17RA, and also of S1P1

and S1P3 was present in the parenchyma of EAE spinal

cord. Most importantly, both cytokine and S1P receptors

were strongly induced on GFAP-positive astrocytes

(Fig 5). Thus, in both human and mouse neuroinflamma-

tion, reactive astrocytes can potentially coexpress receptors

for inflammatory cytokines and for S1P.

Fingolimod Blocks Neurodegeneration Inducedby Astrocyte Responses to Cytokines and S1PAs S1P signaling amplifies growth factor signaling,18 coex-

pression of cytokine and S1P receptors by astrocyte raises the

possibility of a crosstalk between the 2 signaling cascades dur-

ing neuroinflammation. To test this hypothesis, we exposed

astrocytes or spinal neurons in vitro to distinct stimuli in the

presence of the S1P receptor modulator fingolimod. Spinal

neurons stimulated with 100nM S1P and fingolimod did not

show major changes in apoptosis rate and network integrity

compared to nontreated cultures (Fig 6). Furthermore, direct

exposure of neurons to inflammatory cytokines in the pres-

ence of fingolimod caused degeneration at levels comparable

to those detected in cultures exposed to cytokines alone, indi-

cating that blockade of S1P signaling in neurons does not

hinder apoptotic pathways triggered by inflammatory cyto-

kines. On the contrary, when spinal neurons were exposed to

ACM generated in the presence of fingolimod by cytokine-

or S1P-activated astrocytes, neurodegeneration was ham-

pered. Cell numbers, apoptosis, and b-tubulin signals in neu-

rons treated with sS1P/sIL1/sIL17 1 fingolimod were

comparable to those present in sNT cultures. The observation

that blockade of S1P signaling in astrocytes hampers neurode-

generation in all cases suggests that this pathway is essential

for astrocyte responses to the lipid mediator as well as to

inflammatory cytokines. For this reason, we verified the levels

of nuclear translocation of NFjB, a key transcription factor

in S1P and cytokine signaling1 in distinct astrocyte cultures.

FIGURE 4: Interleukin (IL) 1R, IL17RA, sphingosine-1-phosphate (S1P) 1, and S1P3 expression on astrocytes inmultiple sclerosis (MS) lesions. (A) Immunohistochemistryshowing myelin basic protein (MBP) staining in the normal-appearing white matter (NAWM) but not within the MSlesion. (B–F) Double immunofluorescence and confocalimaging for IL1R (B), IL17RA (C), S1P1 (D), S1P3 (E), or nitro-tyrosine (F; all in green) and glial fibrillary acidic protein(GFAP; red). 40,6-Diamidino-2-phenylindole was used fornuclear staining. Right panels show colocalization images.Stainings were performed on serial sections from the samespecimen. Representative images of 7 analyzed MS lesionsare shown. Scale bars 5 50lm.

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As shown in Figure 7A and B, several nuclei were positive for

NFjB when cells were stimulated with S1P, IL1, or IL17

(33 6 7%, 52 6 9%, 61 6 5% of nuclei after exposure to

S1P, IL1, or IL17, respectively). In contrast, when stimuli

were added to astrocyte cultures together with fingolimod,

NFjB nuclear translocation was blocked (0.76 6 0.45%,

7 6 5%, 5 6 1.2%, 6.5 6 1.6% of NFjB-positive nuclei

after exposure to vehicle or to S1P/IL1/IL17 1 fingolimod,

respectively). Furthermore, as neuronal degeneration may be

triggered by astrocyte NO,13 we investigated NO production

by astrocytes loaded with the fluorescent NO indicator DAF-

FM, and stimulated with S1P, IL1, or IL17. Differently from

resting cells, 45 to 60% of astrocytes produced NO upon

stimulation with S1P, IL1, or IL17 (see Fig 7C, D). Impor-

tantly, when fingolimod was given together with stimuli, only

10 to 20% of astrocytes produced NO. Thus, S1P signaling

in astrocytes regulates NFjB translocation and NO produc-

tion in response both to S1P and to inflammatory cytokines.

Consequently, its modulation by fingolimod prevents neuro-

degeneration by blocking astrocyte activation triggered by

FIGURE 5: Cytokine and sphingosine-1-phosphate (S1P) receptors are upregulated in the spinal cord of experimental autoim-mune encephalomyelitis (EAE) mice. Double immunofluorescence and confocal imaging are shown for (A) interleukin (IL) 1R, (B)IL17RA, (C) S1P1, or (D) S1P3 (all in green) and glial fibrillary acidic protein (GFAP; red) in the white matter spinal cord of naive(left panels) or EAE mice (right panels). 40,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. Small panels repre-sent single stainings. Insets show enlarged images of double-positive astrocytes. Stainings were performed on serial sections.Four naive and 5 EAE mice derived from 2 independent EAE experiments were analyzed. Scale bars 5 50lm.


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FIGURE 6: Fingolimod blocks neurodegeneration induced by astrocyte responses to cytokines and sphingosine-1-phosphate(S1P). (A, B) Quantification of cell number, percentage of apoptotic cells, and b-tubulin mean fluorescence intensity (MFI) inneuronal cultures exposed to S1P (A) or interleukin (IL) 1 or IL17 (B) in the presence or absence of fingolimod. Cell numberand b-tubulin MFI were expressed as the percentage of control (CTRL). (C) Fluorescence images showing representative stain-ings for b-tubulin (red), 40,6-diamidino-2-phenylindole (DAPI; blue), and terminal deoxynucleotide transferase–mediated deoxy-uridine triphosphate nick-end labeling (TUNEL; green) in neuronal cultures after 24 hours of exposure to astrocyte-conditionedmedium (ACM). Arrows indicate TUNEL-positive nuclei. (D) The same parameters as in A and B in neuronal cultures treatedwith ACM: nontreated (sNT), IL1-treated (sIL1), IL17-treated (sIL17), or S1P-treated (sS1P) astrocytes 6 fingolimod. In A, graphsshow results from a representative experiment; bars represent standard deviation. In B and D, data are shown as mean 6 stan-dard error of the mean. Data were obtained from 2 or 3 independent experiments. Scale bars 5 50lm. *p < 0.05, **p < 0.01,***p < 0.001 versus CTRL.

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various detrimental stimuli. Otherwise, fingolimod treatment

does not hamper neuronal damage caused by direct exposure

to inflammatory cytokines.

Fingolimod Administration during EAE ReducesExpression of IL1R, IL17RA, and S1P Receptors,and Tyrosine NitrosylationWe performed EAE experiments to evaluate the expres-

sion of IL1R, IL17RA, and S1P receptors in the spinal

cord of immunized mice treated or not treated with fin-

golimod. EAE was induced in C57BL/6 wild-type mice

with myelin oligodendrocyte glycoprotein (MOG)35–55

peptide, and a group of animals was treated with

3mg/kg/day fingolimod by oral administration starting

the first day after disease onset, whereas the control

group received vehicle only. In this therapeutic setup,

fingolimod-treated animals initially experienced typical

EAE symptoms similar to vehicle-treated EAE mice

(Fig 8). At later time points, the fingolimod-treated

group clinically recovered from disease, whereas vehicle-

treated mice showed no signs of recovery. Histological

analyses revealed that at 22 days postinjection the astro-

cytosis typical of EAE, quantified as augmentation in

GFAP immunoreactivity in vehicle-treated EAE mice

compared to naive mice, was absent in EAE mice treated

with fingolimod, whose GFAP levels were comparable to

those of naive mice. Furthermore, accurate quantification

of IL1R, IL17RA, S1P1, and S1P3 signals in the spinal

cord white matter of naive and vehicle-treated EAE mice

indicated that in fingolimod-treated EAE mice the levels

of these receptors were as low as in naive mice. Consis-

tently, expression of the same receptors on astrocytes in

fingolimod-treated EAE mice was comparable to that of

naive mice. Finally, as in vitro data suggested a role for

S1P signaling in astrocyte NO production, we examined

the levels of inducible NO synthase (iNOS) and tyrosine

nitrosylation in fingolimod- and vehicle-treated EAE

mice. Whereas this was absent in the spinal cord of naive

animals, vehicle-treated EAE animals displayed strong

immunoreactivity for iNOS and nitrotyrosine, and these

signals were greatly present on astrocytes. Parallel analysis

of human brain tissue samples showed that tyrosine

nitrosylation reproduced the expression pattern of IL1R,

IL17RA, S1P1, and S1P3, being almost absent in control

white matter (not shown) and NAWM, but high in the

MS lesion (see Fig 4F). Notably, treatment of EAE mice

with fingolimod reduced iNOS and nitrotyrosine levels

in spinal cord white matter (see Fig 8G, H).

FIGURE 7: Fingolimod inhibits NFjB nuclear translocationand nitric oxide (NO) production in astrocytes exposed toinflammatory cytokines and sphingosine-1-phosphate (S1P).(A) Immunofluorescence staining for NFjB in astrocytesstimulated with S1P alone (upper panels) or in the presenceof fingolimod (lower panels). Cells were counterstained with40,6-diamidino-2-phenylindole (DAPI). Arrows indicate NFjB-positive nuclei. (B) Percentage of NFjB-positive nuclei inastrocyte cultures stimulated with S1P, interleukin (IL) 1, orIL17 in the presence or absence of fingolimod. (C) DAF-FM-loaded astrocytes were stimulated with IL1, IL17, or S1P inthe presence or absence of fingolimod. (D) Quantification ofthe percentage of NO-producing cells under the differentexperimental conditions. In A and C, representative fieldsare shown. Data are shown as mean 6 standard error of themean of 3 independent experiments. Scale bars 5 50lm.*p < 0.05, **p < 0.01, ***p < 0.001. CTRL 5 control.


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FIGURE 8: Therapeutic administration of fingolimod to experimental autoimmune encephalomyelitis (EAE) mice reduces cyto-kines and sphingosine-1-phosphate (S1P) receptor expression and nitric oxide production in vivo. (A) Effect of therapeuticadministration of fingolimod on EAE course. Drug or vehicle were administered the day after disease onset. Clinical expressionof MOG35–55 induced EAE in fingolimod-treated (n 5 7, black dots) or vehicle-treated (n 5 9, white dots) animals. Probabilityvalues refer to the significant difference between the 2 groups. Data from 2 EAE experiments were pooled, analyzed, andgiven as average 6 standard error of the mean. *p < 0.05. (B) Immunofluorescence for glial fibrillary acidic protein (GFAP) in spi-nal cord of naive mice and of vehicle or fingolimod-treated EAE mice at 22 days postinjection. Graph shows relative quantifica-tion. (C–H) Double immunofluorescence for interleukin (IL) 1R (C), IL17RA (D), S1P1 (E), S1P3 (F), inducible nitric oxide synthase(iNOS; G), or nitrotyrosine (H; green) and GFAP (red) in white matter spinal cord of vehicle-treated (left) or fingolimod-treated(right) EAE mice. 40,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. Quantification of IL1R, IL17RA, S1P1,S1P3, iNOS, and nitrotyrosine immunoreactivity in the spinal cord (left charts) and colocalization of these signals with GFAP(right charts) are shown. Four naive mice and 5 EAE mice per group from 2 independent EAE experiments were analyzed. Alldata are represented as mean 6 standard error of the mean. Scale bars 5 50lm. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

In this study, we implicated the crosstalk between the

sphingolipid S1P signaling and the IL1/IL17 cytokine

signaling cascades in astrocytes during neuroinflamma-

tion, and provided evidence on the role of S1P signaling

in inflammation-mediated neurodegeneration.

Published studies have shown that the proinflammatory

cytokines IL1 and IL17 are produced in CNS neuroinflam-

mation,23,24 that their receptors may be expressed on endo-

thelial cells in the CNS,25,26 and that blockade of IL17

signaling in astrocytes delays onset and reduces severity of

EAE.27 However, the contribution of distinct cell types and

pathways to neurodegeneration during CNS inflammation

and the relevance for the human disease remain largely

unknown. Here we show for the first time that the receptors

for IL1 and IL17 are strongly upregulated on astrocytes in

MS and EAE lesions, highlighting the astrocyte as an impor-

tant responder cell to the inflammatory milieu.

Intriguingly, along with these results, reactive astro-

cytes may respond to the lipid mediator S1P due to the

enhanced expression of S1P1 and S1P3 in demyelinated

areas. S1P signaling controls several physiological functions

in the CNS, including neurogenesis, axon growth, and

neurotransmitter release.28 Importantly, S1P concentra-

tions are high in blood (>100nM),22 and increased S1P

levels in the CNS promote scar formation in vivo.4 Fur-

ther, modulation of S1P signaling pathway has an impact

on neuronal performance, as its blockade via genetic engi-

neering or FTY720 administration reduces astrogliosis and

improves motor function in the mouse models of the neu-

rodegenerative Sandhoff disease and Rett syndrome.29,30

We can therefore envisage that during neuroinflammation

local increase of S1P concentration (due to the breakdown

of the blood–brain barrier) and/or higher responsiveness

to S1P (via enhanced expression of its receptors) may

induce neurodegeneration. To address this issue, we per-

formed a series of in vitro studies on the effects of S1P on

astrocyte–neuron interaction. Although 100nM S1P did

not induce morphological alterations of the network when

given directly to neurons, the same dose activated astro-

cytes to release factors triggering neuronal apoptosis, indi-

cating that astrocytes are crucial in driving S1P-induced

neurodegeneration. Similar neuronal loss was obtained

when astrocytes were stimulated with IL17 (this study)

and IL1 (this study and Colombo et al13), raising the pos-

sibility of a common final path in S1P and cytokine sig-

naling cascades.

Previous studies have implied the activation of

sphingosine kinases and thereby of the inside-out S1P

signaling by cytokines and growth factors such as tumor

necrosis factor a (TNFa) and platelet-derived growth

factor.18 Here we provide compelling evidence that func-

tional S1P signaling is necessary for astrocyte activation

by cytokines. Blockade of S1P signaling by fingolimod

completely impairs key astrocyte responses to IL1 and

IL17, thereby blocking astrocyte-mediated neurodegener-

ation. Importantly, in our in vitro experiments fingoli-

mod did not hamper cytokine signaling in neurons, as

neurons exposed to IL1 or IL17 in the presence of fingo-

limod underwent similar levels of cell death as the cul-

tures exposed to the cytokines in the absence of

fingolimod. These results indicate that fingolimod may

exert neuroprotective effects by impairing astrocyte acti-

vation, rather than directly rescuing neurons. An in vitro

study has demonstrated that in astrocytes fingolimod

inhibits S1P-induced cell proliferation and IL1-triggered

calcium flux, without changing IL6 and CXCL10 pro-

duction in response to IL1.31 These findings suggest that

in astrocytes fingolimod does not block completely the

activation machinery evoked by cytokines, but impairs

some specific functions. We identified NFjB activation

and NO production as crucial targets of fingolimod

action in astrocytes. As shown in several disease

models,17,32–34 NFjB pathway in astrocytes sustains scar

formation, neuroinflammation, and neurodegeneration;

thus, its blockade is beneficial and limits tissue damage.

Similarly, astrocyte-restricted ablation of IL17 signaling

impairs NFjB activation and ameliorates EAE.27 Regard-

ing NO synthesis in the CNS, basal NO levels regulate

homeostatic and physiological functions in the CNS.

Nevertheless, excessive NO synthesis under neuroinflam-

mation leads to formation of reactive nitrogen species

and cell death.35 We have recently provided evidence

that IL1-induced astrocyte activation contributes to neu-

ronal damage via NO production.13 Similarly, IL17 may

synergize with other proinflammatory cytokines, such as

IL1 and TNFa, to activate iNOS transcription and NO

production by astrocytes.36 Here we provide novel evi-

dence that IL17 alone or S1P may lead to NFjB nuclear

translocation and NO synthesis in glia cells and that this

is relevant for neurodegeneration. Transgenic mice with

selective removal of S1P1 from astrocytes develop a

milder EAE course,37 indicating that astrocyte responses

to S1P via S1P1 may support neuroinflammation. How-

ever, the effects on neurodegeneration were not eval-

uated, and astrocytes could potentially react to S1P via

other S1P receptors in that transgenic mouse model.

Notably, in our in vitro system, fingolimod hampered

NFjB activation and NO release in response to both

S1P and IL1/IL17, clearly indicating that these steps in

cytokine signaling depend on S1P signaling. Nitrosative

stress, detected as nitrosylation of proteins at the level of

tyrosine residues, is present in MS.13 Here, we show that


September 2014 335

tyrosine nitrosylation correlates with the upregulation of

the receptors for S1P, IL1, and IL17 on astrocytes,

strongly supporting the hypothesis that astrocyte

responses to these ligands generate nitrosative stress.

Astrocytes in the spinal cord of EAE mice showed strong

iNOS and nitrosylation signals together with S1P and

cytokine receptors. Fingolimod administration in a thera-

peutic setup reduced astrogliosis and expression of S1P1,

S1P3, IL1R, and IL17RA to the levels detected in naive

mice. Likewise, iNOS induction and nitrotyrosine depo-

sition were efficiently blocked by fingolimod treatment,

indicating that the drug inhibited NO synthesis in vivo.

In summary, our data demonstrate that astrocyte

responses to the inflammatory cytokines IL1 and IL17

and the lipid mediator S1P include NFjB pathway acti-

vation and NO release, and can cause neurodegeneration.

Importantly, cytokine-evoked NFjB translocation and

NO synthesis in astrocytes depend on S1P signaling and

are targeted by fingolimod, thereby fostering neuropro-

tection. Thus, in addition to the therapeutic effects in

MS due to the reduction of inflammatory cell influx into

the CNS, fingolimod may also exert neuroprotective

effects by restricting the release of neurotoxic mediators

from astrocytes.

Acknowledgment

This study was supported by the Fondazione Italiana

Sclerosi Multipla (grant number 2012/R/7), Amici Cen-

tro Sclerosi Multipla (Acesm), and Italian Ministry of

Health.

We thank D. Magni for preliminary in vitro experiments.

Authorship

C.F. conceived and designed the experiments; E.Co. per-

formed experiments; M.D.D. and E.Ca. performed in

vivo experiments; L.C. and G.M. contributed reagents

and analysis tools; J.N. provided postmortem brain sam-

ples and critical discussion; E.Co. and C.F. analyzed the

data and wrote the article.

Potential Conflicts of Interest

C.F.: grant, Merck Serono.

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Fingolimod may support neuroprotection via blockade of astrocyte nitric oxide

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