Sphingosine 1-phosphate receptor modulationsuppresses pathogenic astrocyte activation andchronic progressive CNS inflammationVeit Rothhammera, Jessica E. Kenisona, Emily Tjona, Maisa C. Takenakaa, Kalil Alves de Limaa, Davis M. Boruckia,Chun-Cheih Chaoa, Annabel Wilza, Manon Blainb, Luke Healyb, Jack Antelb, and Francisco J. Quintanaa,c,1

aAnn Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; bNeuroimmunology Unit,Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada QC H3A 2B4; and cBroad Institute ofMIT and Harvard, Cambridge, MA 02142

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved January 12, 2017 (received for review September 14, 2016)

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinatingdisease of the CNS that causes disability in young adults as a result ofthe irreversible accumulation of neurological deficits. Although thereare potent disease-modifying agents for its initial relapsing-remittingphase, these therapies show limited efficacy in secondary progressiveMS (SPMS). Thus, there is an unmet clinical need for the identificationof disease mechanisms and potential therapeutic approaches forSPMS. Here, we show that the sphingosine 1-phosphate receptor(S1PR) modulator fingolimod (FTY720) ameliorated chronic progres-sive experimental autoimmune encephalomyelitis in nonobese di-abetic mice, an experimental model that resembles several aspects ofSPMS, including neurodegeneration and disease progression drivenby the innate immune response in the CNS. Indeed, S1PR modulationby FTY720 in murine and human astrocytes suppressed neurodegen-eration-promoting mechanisms mediated by astrocytes, microglia,and CNS-infiltrating proinflammatory monocytes. Genome-widestudies showed that FTY720 suppresses transcriptional programsassociated with the promotion of disease progression by astro-cytes. The study of the molecular mechanisms controlling thesetranscriptional modules may open new avenues for the devel-opment of therapeutic strategies for progressive MS.

multiple sclerosis | sphingolipid metabolism | astrocytes | EAE |secondary progression

Multiple sclerosis (MS) is a chronic autoimmune disease ofthe CNS that, in most patients, initially presents with a

relapsing-remitting course. This relapsing-remitting stage is oftenfollowed by a secondary progressive phase characterized by theprogressive and irreversible accumulation of neurological defi-cits. The available therapeutic approaches for relapsing-remittingMS (RRMS) show limited efficacy in secondary progressive MS(SPMS), reflecting our insufficient understanding of the pathologicmechanisms that drive disease progression in SPMS and primaryprogressive MS (1). Recent findings, however, suggest that theinnate immune response in the CNS promotes disease progressionin MS. Indeed, astrocytes (the most abundant cell population inthe mammalian CNS), microglia, and proinflammatory monocytesare thought to promote neurodegeneration, demyelination, andscar formation (1–6). However, therapeutic strategies targetingthese cell types remain elusive to date.Sphingosine 1-phosphate (S1P) is a sphingosine-containing

lipid generated from ceramide, which binds G protein-coupledreceptors [Sphingosine 1-phospate receptors (S1PRs) 1–5] andmodulates the proliferation and trafficking of several cell types,including immune cells. Consequently, S1PRs are considered can-didate therapeutic targets for inflammatory diseases, including MS,psoriasis, asthma, and polyneuritis, and also for hematologic andsolid tumors, ischemic stroke, and wound healing (7–12).FTY720 (fingolimod) is a modulator of S1P receptors 1, 3, 4,

and 5 with therapeutic effects on RRMS (13–18). The thera-peutic effects of FTY720 in RRMS are thought to result mainly

from the internalization of S1PR1 in T and B cells, blockinglymphocyte egress from lymph nodes and consequently limitingtheir recruitment to the CNS (19). FTY720 has also been shownto modulate proinflammatory pathways in B and T cells (19–22).In addition to these effects of FTY720 on the peripheral immunesystem, phosphorylated FTY720 crosses the blood-brain barrier(BBB) and is thus capable of interacting with CNS-resident cellpopulations (19, 22, 23).In vitro observations suggest direct effects of FTY720 on as-

trocyte biology, neurodegeneration, and remyelination, whichimpact mechanisms of disease pathogenesis relevant for theprogressive stages of MS (20, 22–28). Indeed, animal studiesusing acute models of RRMS suggest that FTY720 modulatesthe activity of CNS-resident cell populations (28–31). However,limited information is available on the effects of S1PR modu-lation on SPMS and its experimental models of CNS chronicinflammation and progressive neurodegeneration. Thus, we in-vestigated the effects of FTY720 on the chronic progressivemodel of experimental autoimmune encephalomyelitis (EAE) innonobese diabetic (NOD) mice, which resembles several aspectsof SPMS (32). We found that FTY720 ameliorates EAE in NODmice and decreases the production of proinflammatory andneurotoxic mediators by mouse and human astrocytes. Thesefindings identify potential targets for the modulation of local

Significance

Secondary progressive multiple sclerosis (SPMS) inflicts severeand irreversible disability on the affected individuals. Astrocytesare thought to play a central role in the pathogenesis of SPMS.Here, we demonstrate that Sphingosine-1-receptor (S1PR) mod-ulation suppresses pathogenic astrocyte activation and diseaseprogression in an animal model of SPMS. Using functional in vitroassays, we defined direct effects of S1PR modulation on murineand human astrocytes, as well as astrocyte-mediated effects onmicroglia and proinflammatory monocytes. Finally, in unbiasedtranscriptome-wide studies on human astrocytes, we identifiedcandidate targets for the modulation of astrocyte function inSPMS. Collectively, this study sheds light on the pathogenesis ofSPMS and evaluates the therapeutic value of S1PR modulation inan animal model of SPMS.

Author contributions: V.R., J.E.K., J.A., and F.J.Q. designed research; V.R., J.E.K., M.C.T., K.A.d.L.,D.M.B., C.-C.C., A.W., M.B., L.H., and J.A. performed research; V.R., J.E.K., E.T., J.A., and F.J.Q.analyzed data; and V.R. and F.J.Q. wrote the paper.

Conflict of interest statement: These studies were funded in part by a Novartis grant (to F.J.Q.and J.A.).

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: fquintana@rics.bwh.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1615413114/-/DCSupplemental.

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CNS inflammation driven by astrocytes, microglia, and in-flammatory monocytes in SPMS.

ResultsFTY720 Ameliorates Chronic Progressive EAE in NOD Mice. EAE in-duced in NODmice by immunization with myelin oligodendrocyteglycoprotein 35–55 (MOG35–55) peptide recapitulates severalfeatures of SPMS, including the progressive accumulation ofneurodegeneration and axonal loss and the chronic activation ofthe innate immune system in the CNS (3, 33, 34). Thus, to assessthe therapeutic potential of S1P receptor modulators in SPMS, weevaluated the effects of FTY720 on NOD EAE.Following immunization with MOG35–55 in complete Freund’s

adjuvant (CFA) and pertussis toxin administration, NOD miceinitially develop an acute neurological event, which is followedby a chronic progressive phase that starts at approximately day 25(Fig. 1A). Thus, we initiated treatment with daily doses ofFTY720 (0.3 mg/kg body weight) or vehicle 40 d after NOD EAEinduction and monitored disease development until day 120.FTY720 ameliorated NOD EAE during the progressive phase,as indicated by the reduction of clinical scores and mortality (Fig.1 A and B). The beneficial effects of FTY720 in NOD EAE werealso reflected in decreased demyelination and axonal loss (Fig.1C). However, the amelioration of NOD EAE by FTY720 wasnot linked to alterations in the recall T-cell response to MOG35–55,or with changes in peripheral or CNS immune cell counts(Fig. 1D and Fig. S1). Thus, FTY720 ameliorated progressiveNOD EAE without significant effects on the peripheral T-cellresponse.

FTY720 Reduces Pathogenic CNS Innate Immune Activation. The CNSinnate immune response plays a central role in the progressivephase of NOD EAE (1, 3, 5, 6, 35–39). Thus, we analyzedthe transcriptional profile of astrocytes, microglia, and proin-flammatory monocytes isolated from vehicle- and FTY720-treatedmice 120 d after EAE induction using custom-made NanostringnCounter arrays (Table S1) (2). FTY720 administration down-regulated the expression of proinflammatory cytokines andchemokines in astrocytes, including Il6, chemokine (C-C motif)ligand 2 (Ccl2), Ccl20, Ifng, Il23a, C-X-C motif chemokine 10(Cxcl10), and Il1b, among others (Fig. 2A). Importantly, neuro-toxic mediators such as Tnfa and Nos2 as well as factors gov-erning proinflammatory macrophage polarization, includingcolony stimulating factor 2 (Csf2) and Il12a, showed di-minished expression under FTY720 treatment. Conversely, theexpression of anti-inflammatory factors such as Cxcl12 (40) andIl33 (41, 42) was up-regulated by FTY720 (Fig. 2A).Microglia and CNS-infiltrating proinflammatory monocytes are

thought to play an important role in the pathogenesis of SPMSand the progressive phase of NOD EAE (36, 37, 43, 44). Wethus analyzed the transcriptional profile of microglia and CNS-infiltrating CD11b+CD45+Ly6Chi proinflammatory monocytes invehicle- and FTY720-treated mice. We detected decreased expres-sion of proinflammatory gene clusters associated with NOD EAEpathology (2, 3) in microglia and CNS-infiltrating proinflammatorymonocytes (Fig. 2 B–D). Of note, the down-regulation of proin-flammatory gene expression was stronger in microglia than inmonocytes. Moreover, microglia displayed strong up-regulation offactors expressed in alternatively activated microglia such as Csf2,Chi3l3, and Cd206 in FTY720-treated animals (Fig. 2B). In sum-mary, FTY720 decreased the expression of proinflammatory, che-moattractant, and neurotoxic molecules thought to mediate thepathogenic role of astrocytes, microglia, and CNS-infiltratingmonocytes in MS and NOD EAE.

Astrocytic S1PR Controls Astrocyte Neurotoxicity and MonocyteRecruitment and Activation. S1PR1 expression in astrocytes con-tributes to the protective effects of FTY720 in acute EAE (22),

but the mechanisms involved are not completely understood. Wedetected decreased expression of proinflammatory chemokines(Ccl2), cytokines (Csf2, Il6, Tnfa), neurotoxic (Nos2, Tnfa), andmacrophage polarizing factors (Csf2) in highly purified primaryastrocyte cultures (Fig. S2) activated in the presence of FTY720in vitro. Conversely, FTY720 up-regulated the expression ofneuroprotective Il10 (Fig. 3A). Thus, S1PR modulation in as-trocytes affects the expression of mediators thought to promotedisease progression in MS and NOD EAE.Astrocytes display neurotoxic activities in the context of

chronic CNS inflammation and also induce and amplify patho-genic activities in microglia and monocytes recruited to the CNS(40, 45–51). Thus, to evaluate the relevance to disease patho-genesis of the effects of FTY720 on the transcriptional programof astrocytes, we analyzed the effects of FTY720 on the neuro-toxic potential of astrocytes and on their ability to control mi-gration and monocyte polarization (2).Astrocytes promote neuronal death through the production of

TNF-α, glutamate, lactate, and reactive oxygen species, among

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Fig. 1. FTY ameliorates chronic progressive EAE in NODmice. EAE was inducedin NOD/ShiLtJ mice, which were treated with daily i.p. injections of FTY720 orvehicle in the secondary progressive phase of the disease starting from day 40after disease induction. (A) Clinical scores (Left) and linear regression analysis(Right) of mice under treatment with FTY720 or vehicle (n = 10 mice per group;two-way ANOVA). (B) Kaplan–Meier survival analysis of mice in the experimentdescribed in A by two-way ANOVA. (C) Histologic examination of transversallumbar spinal cord sections isolated from FTY720- or vehicle-treatedmice at day120. (Left) representative sections stained for Luxol fast blue (LFB) for de-myelination or Bielschowsky’s Silver stain (silver) for axonal loss. Representativeof three sections of three mice. (Right) Quantification of demyelination andaxonal loss in FTY720- or vehicle-treated mice (Student’s t test). (D) Proliferationassay from splenocytes isolated on day 120 of the experiment (n = 5; two-wayANOVA). Throughout, data are mean ± SEM and representative of two in-dependent experiments (*P < 0.05 and **P < 0.01; ns, not significant).

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others (1, 6, 49). Thus, we used a cell-based assay to evaluate theeffects of FTY720 on astrocyte-driven neurotoxicity. In this as-say, we activated astrocytes in vitro in the presence of FTY720 orvehicle for 24 h and then added new culture medium after ex-tensive washings. Following culture for additional 48 h, astro-cyte-conditioned medium (ACM) was collected. To evaluate theneurotoxic activity of ACMs, we exposed neuronal cells to theACMs and monitored neuronal death by quantifying lactatedehydrogenase (LDH) release. FTY720 treatment led to a smallbut significant reduction of ACM neurotoxic activity (Fig. 3B).During MS and EAE, proinflammatory monocytes are recruited

to the CNS, where they play a central role in promoting neuro-degeneration (2, 36, 37, 39). Thus, chemotactic factors produced byastrocytes play an important role in promoting disease progressionin MS (40, 52, 53). To address the effects of FTY720 on the re-cruitment of Ly6Chi proinflammatory monocytes by astrocytes weused an in vitro transwell migration assay. We found that FTY720decreased Ly6Chi inflammatory monocyte recruitment by ACM(Fig. 3C).Astrocytes also modulate myeloid cell activation and polariza-

tion in the CNS (1, 54). To test the effects of FTY720 on thecontrol of monocytes by astrocytes, we activated astrocytes in thepresence of FTY720 or vehicle for 24 h, washed them extensively,and established cocultures with sorted Ly6Chi inflammatorymonocytes. Following coculture for 24 h, the monocytes were rei-solated and their transcriptional profile was analyzed. Treatmentof astrocytes with FTY720 decreased the expression of proin-flammatory cytokines, chemokines, and neurotoxic molecules in

monocytes (Fig. 3D). Mouse and human activated microglia,however, did not exhibit significant changes in the expression ofproinflammatory cytokines or neurotoxic mediators upon exposureto FTY720 (Fig. S3 A and B). Also, neurotoxicity and induction ofmigration was not altered in supernatants of activated microgliaupon treatment with FTY720 (Fig. S3 C and D).FTY720 treatment led to a significant suppression of NF-κB

p65 nuclear translocation in activated astrocytes (Fig. S4A), con-comitant with decreased production of the proinflammatory andneurotoxic mediators IL-6, TNF-α, GM-CSF, CCL2, and nitricoxide (NO; Fig. S4 B and C). Indeed, in blocking experiments, weidentified CCL2 and IL-6 as the active components in ACMdriving monocyte migration in vitro, whereas TNF-α, GM-CSF,and IL-6 mediated astrocyte neurotoxic activity (Fig. S4 D and E).Collectively, these studies suggest that S1PR modulation in

astrocytes by FTY720 contributes to the amelioration of chronicprogressive NOD EAE.

FTY720 Modulates Activation of Human Astrocytes. To further in-vestigate the relevance of S1PR modulation in astrocytes to itspotential effects on MS, we analyzed the genome-wide tran-scriptional response of human astrocytes activated in vitro in thepresence of FTY720. We identified 1,221 transcripts that weredifferentially regulated under FTY720 treatment (Fig. 4 A andB). Ingenuity pathway analysis determined that FTY720 modu-lates the expression of transcriptional modules associated withmigratory pathways, antigen presentation, inflammasome activation,axonal guidance, and fatty acid α-oxidation (Fig. 4C). Interestingly,NF-κB signaling was significantly altered by FTY720 in human as-trocytes (Fig. 4C), recapitulating our findings of decreased NF-κB

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Fig. 2. FTY720 modulates activation of astrocytes, microglia, and proin-flammatory monocytes. Astrocytes, microglia, and proinflammatory mono-cytes were isolated by FACS sorting at day 120 of EAE, and RNA was subjectedto custom-made nCounter Nanostring arrays. (A and B) Fold change in mRNAexpression of the indicated genes from sorted astrocytes (A) and microglia (B)from FTY720- or vehicle-treated mice at day 120 of EAE as determined byNanoString analysis [fold change in relative expression as determined bylog2(FTY720/vehicle)]. Data are representative of two independent experi-ments of pooled astrocytes and microglia from three mice per group. (B and C)NanoString analysis of proinflammatory gene clusters (Table S2) from sortedmicroglia (C) and Ly6C1hi proinflammatory monocytes (D). Data are ratio ofcount numbers of cells from FTY720-treated to vehicle-treated mice, and arerepresentative of two independent experiments of pooled microglia andLy6Chi proinflammatory monocytes with three mice per group. Data aremean ± SEM (**P < 0.01 and ***P < 0.001, Student’s t test).

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Fig. 3. FTY720 modulates proinflammatory activation of astrocytes. Pri-mary cultures of murine astrocytes were activated with LPS and treated withFTY720 or vehicle and analyzed for gene expression, neurotoxic and che-motaxic mediator production, as well as monocyte polarizing properties.(A) RNA expression of indicated genes from astrocytes isolated 24 h afteractivation and treatment from three biological replicates. (B) Neurotoxicityassay with supernatants of activated astrocyte cultures treated with FTY720or vehicle. (C) Migration assays with supernatants from activated FTY720- orvehicle-treated astrocytes using CD11b+Ly6C1hi monocytes as migratingcells. (D) qPCR analysis for expression of the indicated genes from sortedCD11b+Ly6C1hi proinflammatory monocytes that were cocultured with ac-tivated FTY720- or vehicle-treated astrocytes and reisolated thereafter forRNA analysis. Throughout, data are mean ± SEM and representative of threeindependent experiments with three biological replicates. P values werederived by Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

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p65 activation in mouse astrocytes in vitro (Fig. S4A). NF-κB plays acentral role in the control of astrocyte activities that promote in-flammation and neurodegeneration (2, 40). Thus, we performed anin-depth computational analysis of the NF-κB pathway, whichconfirmed that FTY720 down-modulates NF-κB signaling in humanastrocytes (activation z-score, −1.89; Fig. 4D).To evaluate the functional relevance of our transcriptional

analyses, we validated the expression of selected transcripts onindependent samples and examined the functional effects ofFTY720 on human astrocytes. FTY720 decreased the expressionof proinflammatory and neurotoxic mediators, including cyto-kines, chemokines, and degenerative mediators, while increasinganti-inflammatory IL10 in human astrocytes, as determined inquantitative PCR (qPCR) analyses from separate sets of samples(Fig. 5A). Moreover, ACMs from FTY720-treated human as-trocytes exhibited a marked reduction in neurotoxic and che-motactic properties, concomitant with an increased productionof neuroprotective mediators (Fig. 5 B–D). Human microglia,however, did not show a significant response to exposure toFTY720 (Fig. S3B).

Collectively, these findings reveal an overall neuroprotectiveeffect of FTY720 on human astrocytes mediated, at least in part,by the suppression of NF-κB signaling. In addition, these findingshighlight targets of FTY720 on astrocytes, which may guidenovel potential therapeutic interventions for the modulation ofastrocyte function during progressive MS.

DiscussionThe immunosuppressive and disease-ameliorating effects of FTY720 inRRMS are mostly attributed to the blockade of inflammatory cellmigration into the CNS (19, 28). Phosphorylated FTY720, however,crosses the BBB and acts on CNS-resident cells, including astrocytes(22, 24, 26). The effects of FTY720 in astrocytes, neurons, and oligo-dendrocytes have been previously documented in vitro (26, 27, 55, 56).

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Ingenuity Canonical Pathways p - valueGranulocyte Adhesion and Diapedesis 1,70E-09Agranulocyte Adhesion and Diapedesis 4,37E-09Communication between Innate and Adaptive Immune Cells 2,63E-07Role of Cytokines in the Communication between Immune Cells 6,46E-06T Helper Cell Differentiation 4,07E-05Pathogenesis of Multiple Sclerosis 1,78E-04Antigen Presentation Pathway 1,38E-03Inhibition of Matrix Metalloproteases 1,66E-03HMGB1 Signaling 1,91E-03Inflammasome pathway 2,51E-03IL-17 Signaling 5,25E-03Axonal Guidance Signaling 6,61E-03Leukocyte Extravasation Signaling 6,76E-03NF- B Signaling 9,77E-03IL-10 Signaling 1,23E-02HIF1 Signaling 1,78E-02Fatty Acid -oxidation 3,31E-02IL-8 Signaling 4,57E-02

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Fig. 5. FTY720 mitigates proinflammatory and neurotoxic properties of hu-man astrocytes. Human fetal astrocytes were activated and treated with FTY720or vehicle. (A) qPCR analysis of indicated genes expressed in activated astrocytesunder FTY720 or vehicle treatment 24 h after activation (n = 3 biological rep-licates). (B) Neurotoxicity assay with supernatants of activated human primaryastrocytes treated with FTY720 or vehicle (n = 3 biological replicates). (C) Foldchange in mRNA expression of the indicated genes in human activated astro-cytes treated with FTY720 or vehicle as determined by Affymetrix arrays [foldchange in relative expression as determined by log2(FTY720/vehicle)]. (D) Mi-gration assays with supernatants from activated FTY720- or vehicle-treatedhuman primary astrocytes using CD11b+Ly6C1hi proinflammatory monocytes asmigrating cells (n = 3 biological replicates). Throughout, data are mean ± SEMand representative of three independent experiments. P values were derived byStudent’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

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Given the beneficial effects of FTY720 on organotypic cultures in vitroand its BBB permeability (27, 56), FTY720 has the potential tomodulate the local innate immune response in the CNS, thought tocontribute to disease progression in MS (1, 6, 49). However, the effectsof FTY720 on chronic CNS-innate immune responses in vivo, as wellas their relevance to the pathogenesis of disease progression inMS, arestill unknown.Our studies highlight the anti-inflammatory and neuroprotective

effects of S1PR modulation on mouse and human astrocytes attranscriptional and functional levels. Moreover, we evaluated thebiological relevance of these observations in the NOD EAE model(32). In the chronic phase of this EAE model, disease progression isdriven by the pathogenic activity of astrocytes, microglia, and in-flammatory monocytes, without a major contribution of the adap-tive immune system (e.g., T cells), thus recapitulating severalaspects of SPMS (3, 32, 57, 58). Our data show that FTY720ameliorates progressive CNS inflammation and neurodegeneration,most likely as a result of its CNS-intrinsic effects on astrocytes,microglia, and proinflammatory monocytes. Of note, our in vitrodata on microglia point to an indirect effect of FTY720 on microgliamediated by the modulation of astrocyte-derived factors that in-fluence microglia polarization and pathogenic activities.Five isoforms of S1P receptors are expressed by immune and

nonimmune cells relevant to autoimmune diseases, stroke, andcancer (11, 12, 59–62). Thus, S1P receptors constitute attractivetargets for therapeutic intervention. Indeed, S1PR modulationby FTY720 is efficacious in the treatment of RRMS (13, 14).S1PR modulation did not reach the primary endpoints in theINFORMS (oral fingolimod in primary progressive multiplesclerosis) trial in primary progressive MS (63), but amelioratedMRI parameters of disease activity (63). BAF312 (siponimod) isa BBB-permeable modulator of S1P1 and S1P5 receptors ef-fective in slowing down demyelination in vitro (64–66). Thesafety and therapeutic efficacy of BAF312 on RRMS has beenrecently demonstrated in terms of clinical and MRI parame-ters in the phase II BOLD (BAF312 on MRI lesion given oncedaily) study (67). The therapeutic effects of BAF312 on SPMS arecurrently being investigated in the EXPAND (exploring the effi-cacy and safety of siponimod in patients with secondary progressmultiple sclerosis) trial. Based on the effects of FTY720 on theCNS-innate immune response during NOD EAE, S1P receptormodulation by BAF312 is likely to modulate local CNS in-flammation in SPMS. Indeed, preliminary results of the EXPANDtrial suggest a protective effect of BAF312 on 3-mo confirmeddisease progression in SPMS (68). However, side-by-side compar-isons of the effects of FTY720 and BAF312 on adaptive and innateimmune cells are needed to determine whether the structural dif-ferences existing between these compounds affect their biologicactivities on specific cell populations relevant to SPMS. Indeed, it isworth noting that, whereas FTY720 modulates S1P1, S1P3, S1P4,and S1P5 receptors, BAF312 modulates S1P1 and S1P5receptors only.FTY720 is also reported to modulate sphingolipid metabo-

lism, which is thought to promote disease progression in NODEAE and MS (3, 19, 23, 69, 70). Indeed, we showed that inhib-itors of B4GALT6 and lactosyl ceramide production suppressdisease progression in NOD EAE by arresting local CNS-innateimmunity and neurodegeneration (3). The arrest of NODEAE obtained with B4GALT6 inhibitors is stronger than the

amelioration obtained with FTY720, thus calling for caution asto the therapeutic potency of FTY720 and its analog BAF312 inSPMS. Moreover, these findings suggest that the beneficial ef-fects of FTY720 on NOD EAE may reflect its moderate in-hibitory effects on sphingolipid metabolism.Finally, our unbiased genome-wide analyses identify additional

potential targets for the therapeutic modulation of astrocyte functionand the CNS-innate immune response, which may guide the devel-opment of novel therapeutics for the treatment of progressive MS.

Materials and MethodsMice. Female NOD/ShiLtJ mice and 1–3-d-old pups from C57BL/6J mice wereobtained from the Jackson Laboratory and were kept in a pathogen-freefacility at the Harvard Institutes of Medicine. All experiments were carriedout in accordance with guidelines prescribed by the institutional animal careand use committee at Harvard Medical School.

EAE Induction and Treatment. EAE was induced in 8-wk-old mice by s.c. im-munization with 150 μg MOG35–55 peptide emulsified in CFA (Difco Labo-ratories) per mouse, followed by administration of 200 ng pertussis toxin(List Biological Laboratories) on days 0 and 2 as described previously (2, 3).Starting from day 40 after disease induction, mice were treaty daily with i.p.injections of FTY720 0.3 mg/kg body weight or vehicle, respectively.

Generation of Astrocyte- and Microglia-Conditioned Medium for Migration andNeurotoxicity Assays. In vitro astrocyte and microglia cultures from WT pupswere treated with LPS (100 ng/mL) in the presence of FTY720 (1 μg/mL) orvehicle for 24 h, extensively washed, and supplemented with fresh culturemedium. Forty-eight hours later, supernatants were spun down and kept formigration and neurotoxicity assays at −80 °C.

Monocyte Migration Assay. Splenic monocytes were purified fromWTmice byCD11b beads (Miltenyi) and sorted for F4/80+SSClowLy6C1hi (SSC, sidewaysscatter). These monocytes were seeded in the upper chamber of a 24-wellcell culture insert, with a 5-μm pore size (Corning), containing astrocyte- ormicroglia-conditioned medium (as detailed earlier). Migrating monocytes inthe lower chamber were quantified after 3 h by FACS.

Neurotoxicity Assay. N2A neuronal cells (CCL-131, American Type Culture Collec-tion) were grown in 96-well plates and preactivated with mouse IFN-γ (100 ng/mL;R&D Systems) for 24 h. Thereafter, medium was replaced, after extensive washingwith PBS solution, with astrocyte- or microglia-conditioned medium. Cytotoxicitywas measured by using LDH release (CytoTox 96 Nonradioactive Cytotoxicity Assay;Promega) after 24 h as suggested by the manufacturer’s protocol.

Monocyte Polarization Assays. Primary astrocyte cultures were activated in thepresence of FTY720 or vehicle for 24 h. Thereafter, activation medium wasremoved and primed astrocytes were washed extensively. CD11b+CD45+Ly6Chi

proinflammatory monocytes were FACS-sorted from spleens of naive WT miceand cocultured with primed astrocytes. After 24 h, monocytes were reisolatedand RNA was isolated, transcribed, and subjected to qPCR analysis.

EAE clinical scoring, mouse and human primary astrocyte and microgliacultures, isolation of cells from adult mouse CNS, subcellular fractionationand immunoblot analysis, T-cell proliferation, flow cytometry staining andacquisition, ELISA, detection of NO, histology, nCounter gene expression,qPCR, Affymetrix gene assay, heat-map generation, and Ingenuity pathwayand statistical analysis are provided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Howard L. Weiner for his suggestions. Thisworkwas supported by National Institutes of Health Grants AI075285 andAI093903(to F.J.Q.), National Multiple Sclerosis Society Grants RG4111A1 and JF2161-A-5 (toF.J.Q.), Novartis educational grants (to F.J.Q. and J.A.), a Canadian Institutes of HealthResearch Industry grant (to J.A.), Mallinckrodt Pharmaceuticals Educational GrantA219074 (to V.R.), and German Research Foundation Fellowship DFG RO4866 1/1.

1. Mahad DH, Trapp BD, Lassmann H (2015) Pathological mechanisms in progressivemultiple sclerosis. Lancet Neurol 14(2):183–193.

2. Rothhammer V, et al. (2016) Type I interferons and microbial metabolites of trypto-phan modulate astrocyte activity and central nervous system inflammation via thearyl hydrocarbon receptor. Nat Med 22(6):586–597.

3. Mayo L, et al. (2014) Regulation of astrocyte activation by glycolipids drives chronicCNS inflammation. Nat Med 20(10):1147–1156.

4. Butovsky O, et al. (2014) Identification of a unique TGF-β-dependent molecular andfunctional signature in microglia. Nat Neurosci 17(1):131–143.

5. Savarin C, et al. (2015) Astrocyte response to IFN-γ limits IL-6-mediated microglia acti-vation and progressive autoimmune encephalomyelitis. J Neuroinflammation 12(1):79.

6. Lassmann H, van Horssen J, Mahad D (2012) Progressive multiple sclerosis: Pathologyand pathogenesis. Nat Rev Neurol 8(11):647–656.

7. Tiper IV, East JE, Subrahmanyam PB, Webb TJ (2016) Sphingosine 1-phosphate sig-naling impacts lymphocyte migration, inflammation and infection. Pathog Dis 74(6):ftw063.

8. Garris CS, Blaho VA, Hla T, Han MH (2014) Sphingosine-1-phosphate receptor 1 sig-nalling in T cells: Trafficking and beyond. Immunology 142(3):347–353.

2016 | www.pnas.org/cgi/doi/10.1073/pnas.1615413114 Rothhammer et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 9,

202

0

9. Maceyka M, Spiegel S (2014) Sphingolipid metabolites in inflammatory disease.Nature 510(7503):58–67.

10. Brinkmann V (2009) FTY720 (fingolimod) in multiple sclerosis: Therapeutic effects inthe immune and the central nervous system. Br J Pharmacol 158(5):1173–1182.

11. White C, Alshaker H, Cooper C, Winkler M, Pchejetski D (2016) The emerging role ofFTY720 (fingolimod) in cancer treatment. Oncotarget 7(17):23106–23127.

12. Aoki M, Aoki H, Ramanathan R, Hait NC, Takabe K (2016) Sphingosine-1-phosphatesignaling in immune cells and inflammation: Roles and therapeutic potential.Mediators Inflamm 2016:8606878.

13. Calabresi PA, et al. (2014) Safety and efficacy of fingolimod in patients with relapsing-remitting multiple sclerosis (FREEDOMS II): A double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Neurol 13(6):545–556.

14. Kappos L, et al.; FREEDOMS Study Group (2010) A placebo-controlled trial of oralfingolimod in relapsing multiple sclerosis. N Engl J Med 362(5):387–401.

15. Hohlfeld R, Barkhof F, Polman C (2011) Future clinical challenges in multiple sclerosis:Relevance to sphingosine 1-phosphate receptor modulator therapy. Neurology 76(8,Suppl 3):S28–S37.

16. Hla T, Brinkmann V (2011) Sphingosine 1-phosphate (S1P): Physiology and the effectsof S1P receptor modulation. Neurology 76(8, suppl 3):S3–S8.

17. Mehling M, Johnson TA, Antel J, Kappos L, Bar-Or A (2011) Clinical immunology ofthe sphingosine 1-phosphate receptor modulator fingolimod (FTY720) in multiplesclerosis. Neurology 76(8, suppl 3):S20–S27.

18. Chun J, Hartung HP (2010) Mechanism of action of oral fingolimod (FTY720) inmultiple sclerosis. Clin Neuropharmacol 33(2):91–101.

19. Lee CW, Choi JW, Chun J (2010) Neurological S1P signaling as an emerging mecha-nism of action of oral FTY720 (fingolimod) in multiple sclerosis. Arch Pharm Res33(10):1567–1574.

20. Brunkhorst R, Vutukuri R, Pfeilschifter W (2014) Fingolimod for the treatment ofneurological diseases-state of play and future perspectives. Front Cell Neurosci 8:283.

21. Willis MA, Cohen JA (2013) Fingolimod therapy for multiple sclerosis. Semin Neurol33(1):37–44.

22. Choi JW, et al. (2011) FTY720 (fingolimod) efficacy in an animal model of multiplesclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation.Proc Natl Acad Sci USA 108(2):751–756.

23. Kim HJ, et al. (2011) Neurobiological effects of sphingosine 1-phosphate receptormodulation in the cuprizone model. FASEB J 25(5):1509–1518.

24. Colombo E, et al. (2014) Fingolimod may support neuroprotection via blockade ofastrocyte nitric oxide. Ann Neurol 76(3):325–337.

25. Groves A, Kihara Y, Chun J (2013) Fingolimod: Direct CNS effects of sphingosine1-phosphate (S1P) receptor modulation and implications in multiple sclerosis therapy.J Neurol Sci 328(1-2):9–18.

26. Wu C, et al. (2013) Dual effects of daily FTY720 on human astrocytes in vitro: Rele-vance for neuroinflammation. J Neuroinflammation 10:41.

27. Miron VE, et al. (2010) Fingolimod (FTY720) enhances remyelination following de-myelination of organotypic cerebellar slices. Am J Pathol 176(6):2682–2694.

28. Imeri F, et al. (2016) Sphingosine kinase 2 deficient mice exhibit reduced experimentalautoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments.Neuropharmacology 105:341–350.

29. Foster CA, et al. (2009) FTY720 rescue therapy in the dark agouti rat model of ex-perimental autoimmune encephalomyelitis: Expression of central nervous systemgenes and reversal of blood-brain-barrier damage. Brain Pathol 19(2):254–266.

30. Anthony DC, Sibson NR, Losey P, Meier DP, Leppert D (2014) Investigation of immuneand CNS-mediated effects of fingolimod in the focal delayed-type hypersensitivitymultiple sclerosis model. Neuropharmacology 79:534–541.

31. di Nuzzo L, Orlando R, Nasca C, Nicoletti F (2014) Molecular pharmacodynamics ofnew oral drugs used in the treatment of multiple sclerosis. Drug Des Devel Ther 8:555–568.

32. Simmons SB, Pierson ER, Lee SY, Goverman JM (2013) Modeling the heterogeneity ofmultiple sclerosis in animals. Trends Immunol 34(8):410–422.

33. Ignatius Arokia Doss PM, Roy AP, Wang A, Anderson AC, Rangachari M (2015) Thenon-obese diabetic mouse strain as a model to study CD8(+) T cell function in re-lapsing and progressive multiple sclerosis. Front Immunol 6:541.

34. Dang PT, Bui Q, D’Souza CS, Orian JM (2015) Modelling MS: Chronic-relapsing EAE inthe NOD/Lt mouse strain. Curr Top Behav Neurosci 26:143–177.

35. Moreno M, et al. (2014) Conditional ablation of astroglial CCL2 suppresses CNS ac-cumulation of M1 macrophages and preserves axons in mice with MOG peptide EAE.J Neurosci 34(24):8175–8185.

36. Ajami B, Bennett JL, Krieger C, McNagny KM, Rossi FM (2011) Infiltrating monocytestrigger EAE progression, but do not contribute to the resident microglia pool. NatNeurosci 14(9):1142–1149.

37. Mildner A, et al. (2009) CCR2+Ly-6Chi monocytes are crucial for the effector phase ofautoimmunity in the central nervous system. Brain 132(pt 9):2487–2500.

38. Kim RY, et al. (2014) Astrocyte CCL2 sustains immune cell infiltration in chronic ex-perimental autoimmune encephalomyelitis. J Neuroimmunol 274(1-2):53–61.

39. Goldmann T, et al. (2013) A new type of microglia gene targeting shows TAK1 to bepivotal in CNS autoimmune inflammation. Nat Neurosci 16(11):1618–1626.

40. Rothhammer V, Quintana FJ (2015) Control of autoimmune CNS inflammation byastrocytes. Semin Immunopathol 37(6):625–638.

41. Chen H, et al. (2015) Interleukin-33 is released in spinal cord and suppresses experi-mental autoimmune encephalomyelitis in mice. Neuroscience 308:157–168.

42. Jiang HR, et al. (2012) IL-33 attenuates EAE by suppressing IL-17 and IFN-γ productionand inducing alternatively activated macrophages. Eur J Immunol 42(7):1804–1814.

43. Huang DR, Wang J, Kivisakk P, Rollins BJ, Ransohoff RM (2001) Absence of monocytechemoattractant protein 1 in mice leads to decreased local macrophage recruitmentand antigen-specific T helper cell type 1 immune response in experimental autoim-mune encephalomyelitis. J Exp Med 193(6):713–726.

44. Izikson L, Klein RS, Charo IF, Weiner HL, Luster AD (2000) Resistance to experimentalautoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2.J Exp Med 192(7):1075–1080.

45. SofroniewMV (2015) Astrocyte barriers to neurotoxic inflammation. Nat Rev Neurosci16(5):249–263.

46. Olabarria M, Putilina M, Riemer EC, Goldman JE (2015) Astrocyte pathology in Al-exander disease causes a marked inflammatory environment. Acta Neuropathol130(4):469–486.

47. Chung WS, Allen NJ, Eroglu C (2015) Astrocytes control synapse formation, function,and elimination. Cold Spring Harb Perspect Biol 7(9):a020370.

48. Lundgaard I, Osório MJ, Kress BT, Sanggaard S, Nedergaard M (2014) White matterastrocytes in health and disease. Neuroscience 276:161–173.

49. Lassmann H (2014) Mechanisms of white matter damage in multiple sclerosis. Glia62(11):1816–1830.

50. Linne ML, Jalonen TO (2014) Astrocyte-neuron interactions: From experimental re-search-based models to translational medicine. Prog Mol Biol Transl Sci 123:191–217.

51. Brosnan CF, Raine CS (2013) The astrocyte in multiple sclerosis revisited. Glia 61(4):453–465.

52. Ascherio A, Munger KL, Lünemann JD (2012) The initiation and prevention of mul-tiple sclerosis. Nat Rev Neurol 8(11):602–612.

53. Lopes Pinheiro MA, et al. (2016) Immune cell trafficking across the barriers of thecentral nervous system in multiple sclerosis and stroke. Biochim Biophys Acta 1862(3):461–471.

54. Sheridan GK, Murphy KJ (2013) Neuron-glia crosstalk in health and disease:Fractalkine and CX3CR1 take centre stage. Open Biol 3(12):130181.

55. Janssen S, et al. (2015) Effect of FTY720-phosphate on the expression of in-flammation-associated molecules in astrocytes in vitro. Mol Med Rep 12(4):6171–6177.

56. Sheridan GK, Dev KK (2012) S1P1 receptor subtype inhibits demyelination and reg-ulates chemokine release in cerebellar slice cultures. Glia 60(3):382–392.

57. Basso AS, et al. (2008) Reversal of axonal loss and disability in a mouse model ofprogressive multiple sclerosis. J Clin Invest 118(4):1532–1543.

58. Farez MF, et al. (2009) Toll-like receptor 2 and poly(ADP-ribose) polymerase 1 pro-mote central nervous system neuroinflammation in progressive EAE. Nat Immunol10(9):958–964.

59. Farez MF, Correale J (2016) Sphingosine 1-phosphate signaling in astrocytes: Impli-cations for progressive multiple sclerosis. J Neurol Sci 361:60–65.

60. Healy LM, Antel JP (2016) Sphingosine-1-phosphate receptors in the central nervousand immune systems. Curr Drug Targets 17(16):1841–1850.

61. Nazari M, Keshavarz S, Rafati A, Namavar MR, Haghani M (2016) Fingolimod (FTY720)improves hippocampal synaptic plasticity and memory deficit in rats following focalcerebral ischemia. Brain Res Bull 124:95–102.

62. Tsai HC, Han MH (2016) Sphingosine-1-phosphate (S1P) and S1P signaling pathway:Therapeutic targets in autoimmunity and inflammation. Drugs 76(11):1067–1079.

63. Lublin F, et al.; INFORMS study investigators (2016) Oral fingolimod in primary pro-gressive multiple sclerosis (INFORMS): A phase 3, randomised, double-blind, placebo-controlled trial. Lancet 387(10023):1075–1084.

64. O’Sullivan C, Schubart A, Mir AK, Dev KK (2016) The dual S1PR1/S1PR5 drugBAF312 (siponimod) attenuates demyelination in organotypic slice cultures.J Neuroinflammation 13:31.

65. Pan S, et al. (2013) Discovery of BAF312 (siponimod), a potent and selective S1P re-ceptor modulator. ACS Med Chem Lett 4(3):333–337.

66. Briard E, Rudolph B, Desrayaud S, Krauser JA, Auberson YP (2015) MS565: A SPECTtracer for evaluating the brain penetration of BAF312 (siponimod). Chem Med Chem10(6):1008–1018.

67. Kappos L, et al. (2016) Safety and efficacy of siponimod (BAF312) in patients withrelapsing-remitting multiple sclerosis: Dose-blinded, randomized extension of thephase 2 bold study. JAMA Neurol 73(9):1089–1098.

68. Novartis Media Relations (2016) Novartis announces positive phase III resultsshowing efficacy of BAF312 in patients with secondary progressive MS. Available athttps://www.novartis.com/news/media-releases/novartis-announces-positive-phase-iii-results-showing-efficacy-baf312-patients. Accessed September 1, 2016.

69. Proia RL, Hla T (2015) Emerging biology of sphingosine-1-phosphate: its role inpathogenesis and therapy. J Clin Invest 125(4):1379–1387.

70. Asle-Rousta M, Kolahdooz Z, Oryan S, Ahmadiani A, Dargahi L (2013) FTY720 (fin-golimod) attenuates beta-amyloid peptide (Aβ42)-induced impairment of spatiallearning and memory in rats. J Mol Neurosci 50(3):524–532.

71. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of imageanalysis. Nat Methods 9(7):671–675.

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