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1-phosphate receptor modulation suppresses pathogenic astrocyte activation and chronic progressive CNS

Veit 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 of MIT 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) (MS) is an autoimmune inflammatory demyelinating from the internalization of S1PR1 in T and B cells, blocking disease of the CNS that causes disability in young adults as a result of egress from lymph nodes and consequently limiting the irreversible accumulation of neurological deficits. Although there their recruitment to the CNS (19). FTY720 has also been shown are potent disease-modifying agents for its initial relapsing-remitting to modulate proinflammatory pathways in B and T cells (19–22). phase, these therapies show limited efficacy in secondary progressive In addition to these effects of FTY720 on the peripheral immune MS (SPMS). Thus, there is an unmet clinical need for the identification system, phosphorylated FTY720 crosses the blood-brain barrier of disease mechanisms and potential therapeutic approaches for (BBB) and is thus capable of interacting with CNS-resident SPMS. Here, we show that the sphingosine 1-phosphate receptor populations (19, 22, 23). (S1PR) modulator (FTY720) ameliorated chronic progres- In vitro observations suggest direct effects of FTY720 on as- sive experimental autoimmune encephalomyelitis in nonobese di- abetic mice, an experimental model that resembles several aspects of trocyte biology, , and remyelination, which SPMS, including neurodegeneration and disease progression driven impact mechanisms of disease pathogenesis relevant for the progressive stages of MS (20, 22–28). Indeed, animal studies by the innate immune response in the CNS. Indeed, S1PR modulation MEDICAL SCIENCES by FTY720 in murine and human astrocytes suppressed neurodegen- using acute models of RRMS suggest that FTY720 modulates eration-promoting mechanisms mediated by astrocytes, microglia, the activity of CNS-resident cell populations (28–31). However, and CNS-infiltrating proinflammatory monocytes. Genome-wide limited information is available on the effects of S1PR modu- studies showed that FTY720 suppresses transcriptional programs lation on SPMS and its experimental models of CNS chronic associated with the promotion of disease progression by astro- inflammation and progressive neurodegeneration. Thus, we in- cytes. The study of the molecular mechanisms controlling these vestigated the effects of FTY720 on the chronic progressive transcriptional modules may open new avenues for the devel- model of experimental autoimmune encephalomyelitis (EAE) in opment of therapeutic strategies for progressive MS. nonobese diabetic (NOD) mice, which resembles several aspects of SPMS (32). We found that FTY720 ameliorates EAE in NOD multiple sclerosis | | astrocytes | EAE | mice and decreases the production of proinflammatory and secondary progression neurotoxic mediators by mouse and human astrocytes. These findings identify potential targets for the modulation of local ultiple sclerosis (MS) is a chronic autoimmune disease of the CNS that, in most patients, initially presents with a M Significance relapsing-remitting course. This relapsing-remitting stage is often followed by a secondary progressive phase characterized by the progressive and irreversible accumulation of neurological defi- Secondary progressive multiple sclerosis (SPMS) inflicts severe cits. The available therapeutic approaches for relapsing-remitting and irreversible disability on the affected individuals. Astrocytes MS (RRMS) show limited efficacy in secondary progressive MS are thought to play a central role in the pathogenesis of SPMS. (SPMS), reflecting our insufficient understanding of the pathologic Here, we demonstrate that Sphingosine-1-receptor (S1PR) mod- mechanisms that drive disease progression in SPMS and primary ulation suppresses pathogenic astrocyte activation and disease progressive MS (1). Recent findings, however, suggest that the progression in an animal model of SPMS. Using functional in vitro innate immune response in the CNS promotes disease progression assays, we defined direct effects of S1PR modulation on murine in MS. Indeed, astrocytes (the most abundant cell population in and human astrocytes, as well as astrocyte-mediated effects on the mammalian CNS), microglia, and proinflammatory monocytes microglia and proinflammatory monocytes. Finally, in unbiased are thought to promote neurodegeneration, demyelination, and transcriptome-wide studies on human astrocytes, we identified scar formation (1–6). However, therapeutic strategies targeting candidate targets for the modulation of astrocyte function in these cell types remain elusive to date. SPMS. Collectively, this study sheds light on the pathogenesis of Sphingosine 1-phosphate (S1P) is a sphingosine-containing SPMS and evaluates the therapeutic value of S1PR modulation in generated from , which binds G protein-coupled an animal model of SPMS. receptors [Sphingosine 1-phospate receptors (S1PRs) 1–5] and 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., modulates the proliferation and trafficking of several cell types, 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. including immune cells. Consequently, S1PRs are considered can- analyzed data; and V.R. and F.J.Q. wrote the paper. didate therapeutic targets for inflammatory diseases, including MS, Conflict of interest statement: These studies were funded in part by a grant (to F.J.Q. , asthma, and polyneuritis, and also for hematologic and and J.A.). solid tumors, ischemic stroke, and wound healing (7–12). This article is a PNAS Direct Submission. FTY720 (fingolimod) is a modulator of S1P receptors 1, 3, 4, 1To whom correspondence should be addressed. Email: [email protected]. – and 5 with therapeutic effects on RRMS (13 18). The thera- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. peutic effects of FTY720 in RRMS are thought to result mainly 1073/pnas.1615413114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1615413114 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 I) CNS inflammation driven by astrocytes, microglia, and in- A C f

% Vehicle flammatory monocytes in SPMS. 5 Vehicle 5

95 FTY720

FTY720 on o 4 4 **

** ssi

Results 3 re 3

2 eg 2 R res(Mean FTY720 Ameliorates Chronic Progressive EAE in NOD Mice. EAE in- 1 r 1 o Clinical Score (Mean ± SEM)

0 nea 0 duced in NOD mice by immunization with myelin oligodendrocyte lsc Li 0 20406080100120 a 0 20406080100120 – ic glycoprotein 35 55 (MOG – ) peptide recapitulates several n 35 55 Time after immunization (days) li Time after immunization (days) features of SPMS, including the progressive accumulation of c neurodegeneration and axonal loss and the chronic activation of B the innate immune system in the CNS (3, 33, 34). Thus, to assess 100 Vehicle the therapeutic potential of S1P receptor modulators in SPMS, we 90 FTY720 80 *

evaluated the effects of FTY720 on NOD EAE. vival (%) 70 r

Su 60 Following immunization with MOG35–55 in complete Freund’s 50 adjuvant (CFA) and pertussis toxin administration, NOD mice 0 20406080100120 initially develop an acute neurological event, which is followed Time after immunization (days) by a chronic progressive phase that starts at approximately day 25 (Fig. 1A). Thus, we initiated treatment with daily doses of Vehicle FTY720 C ** * ) 50 40 %

FTY720 (0.3 mg/kg body weight) or vehicle 40 d after NOD EAE ( 40 (%) 30 ion induction and monitored disease development until day 120. LFB t 30 20 FTY720 ameliorated NOD EAE during the progressive phase, na 20 eli

y 10 10 onal loss x m

as indicated by the reduction of clinical scores and mortality (Fig. A e 0 0 1 A and B). The beneficial effects of FTY720 in NOD EAE were D 720 also reflected in decreased demyelination and axonal loss (Fig. Silver VehicleFTY VehicleFTY720 1C). However, the amelioration of NOD EAE by FTY720 was not 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 progressive D ) 6 Vehicle NOD EAE without significant effects on the peripheral T-cell 5 FTY720 4 ns response. 3 2 unstimulated FTY720 Reduces Pathogenic CNS Innate Immune Activation. 1 The CNS fold Proliferation index

( 0 innate immune response plays a central role in the progressive 0 1 10 100 MOG (g/ml) phase of NOD EAE (1, 3, 5, 6, 35–39). Thus, we analyzed 35-55 the transcriptional profile of astrocytes, microglia, and proin- Fig. 1. FTY ameliorates chronic progressive EAE in NOD mice. EAE was induced flammatory monocytes isolated from vehicle- and FTY720-treated in NOD/ShiLtJ mice, which were treated with daily i.p. injections of FTY720 or mice 120 d after EAE induction using custom-made Nanostring vehicle in the secondary progressive phase of the disease starting from day 40 nCounter arrays (Table S1) (2). FTY720 administration down- after disease induction. (A) Clinical scores (Left) and linear regression analysis regulated the expression of proinflammatory and (Right)ofmiceundertreatmentwithFTY720orvehicle(n = 10 mice per group; chemokines in astrocytes, including Il6, chemokine (C-C motif) two-way ANOVA). (B)Kaplan–Meier survival analysis of mice in the experiment 2 (Ccl2), Ccl20, Ifng, Il23a, C-X-C motif chemokine 10 described in A by two-way ANOVA. (C) Histologic examination of transversal lumbar spinal cord sections isolated from FTY720- or vehicle-treated mice at day (Cxcl10), and Il1b, among others (Fig. 2A). Importantly, neuro- 120. (Left) representative sections stained for Luxol fast blue (LFB) for de- toxic mediators such as Tnfa and Nos2 as well as factors gov- myelination or Bielschowsky’s Silver stain (silver) for axonal loss. Representative erning proinflammatory polarization, including of three sections of three mice. (Right) Quantification of demyelination and colony stimulating factor 2 (Csf2)andIl12a, showed di- axonal loss in FTY720- or vehicle-treated mice (Student’s t test). (D) Proliferation minished expression under FTY720 treatment. Conversely, the assay from splenocytes isolated on day 120 of the experiment (n = 5; two-way expression of anti-inflammatory factors such as Cxcl12 (40) and ANOVA). Throughout, data are mean ± SEM and representative of two in- Il33 (41, 42) was up-regulated by FTY720 (Fig. 2A). dependent experiments (*P < 0.05 and **P < 0.01; ns, not significant). Microglia and CNS-infiltrating proinflammatory monocytes are thought to play an important role in the pathogenesis of SPMS and the progressive phase of NOD EAE (36, 37, 43, 44). We but the mechanisms involved are not completely understood. We thus analyzed the transcriptional profile of microglia and CNS- detected decreased expression of proinflammatory chemokines + + infiltrating CD11b CD45 Ly6Chi proinflammatory monocytes in (Ccl2), cytokines (Csf2, Il6, Tnfa), neurotoxic (Nos2, Tnfa), and vehicle- and FTY720-treated mice. We detected decreased expres- macrophage polarizing factors (Csf2) in highly purified primary sion of proinflammatory gene clusters associated with NOD EAE astrocyte cultures (Fig. S2) activated in the presence of FTY720 pathology (2, 3) in microglia and CNS-infiltrating proinflammatory in vitro. Conversely, FTY720 up-regulated the expression of monocytes (Fig. 2 B–D). Of note, the down-regulation of proin- neuroprotective Il10 (Fig. 3A). Thus, S1PR modulation in as- flammatory gene expression was stronger in microglia than in trocytes affects the expression of mediators thought to promote monocytes. Moreover, microglia displayed strong up-regulation of disease progression in MS and NOD EAE. factors expressed in alternatively activated microglia such as Csf2, Astrocytes display neurotoxic activities in the context of Chi3l3,andCd206 in FTY720-treated animals (Fig. 2B). In sum- chronic CNS inflammation and also induce and amplify patho- mary, FTY720 decreased the expression of proinflammatory, che- genic activities in microglia and monocytes recruited to the CNS moattractant, and neurotoxic molecules thought to mediate the (40, 45–51). Thus, to evaluate the relevance to disease patho- pathogenic role of astrocytes, microglia, and CNS-infiltrating genesis of the effects of FTY720 on the transcriptional program monocytes in MS and NOD EAE. of astrocytes, we analyzed the effects of FTY720 on the neuro- toxic potential of astrocytes and on their ability to control mi- Astrocytic S1PR Controls Astrocyte Neurotoxicity and Monocyte gration and monocyte polarization (2). Recruitment and Activation. S1PR1 expression in astrocytes con- Astrocytes promote neuronal death through the production of tributes to the protective effects of FTY720 in acute EAE (22), TNF-α, glutamate, lactate, and , among

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1615413114 Rothhammer et al. Downloaded by guest on September 29, 2021 monocytes (Fig. 3D). Mouse and human activated microglia, A Astrocytes B Microglia decreased by increased decreased by increased by however, did not exhibit significant changes in the expression of FTY720 FTY720 FTY720 FTY720 treatment treatment treatment Ccl7 treatment proinflammatory cytokines or neurotoxic mediators upon exposure Nos2 Cxcl13 Il6 Ccl5 Ccl20 Axl to FTY720 (Fig. S3 A and B). Also, neurotoxicity and induction of Ccl2 Ccr2 migration was not altered in supernatants of activated microglia Ccl1 Fosb Ccl1 Ifng Fos upon treatment with FTY720 (Fig. S3 C and D). Csf2 Cxcl11 Il23a H2-Aa FTY720 treatment led to a significant suppression of NF-κB Il21 Tnfa Il12a Ccl17 p65 nuclear translocation in activated astrocytes (Fig. S4A), con- Ccl17 Ccl4 H2-Ab1 Cxcl10 Ccl2 comitant with decreased production of the proinflammatory and Il1b Cxcl9 α Tnfa Csf2 neurotoxic mediators IL-6, TNF- , GM-CSF, CCL2, and nitric H2-Aa Chi3l3 H2-Ab1 Il10 oxide (NO; Fig. S4 B and C). Indeed, in blocking experiments, we Igf1 Cxcl12 Retnla identified CCL2 and IL-6 as the active components in ACM Il33 Cd206 α -4 -2 0 2 4 -3 -2 -1 0 1 2 3 driving monocyte migration in vitro, whereas TNF- ,GM-CSF, Fold change in relative expression Fold change in relative expression and IL-6 mediated astrocyte neurotoxic activity (Fig. S4 D and E). (FTY720 / Control Astrocytes, log ) (FTY720 / Control Astrocytes, log ) 2 2 Collectively, these studies suggest that S1PR modulation in C D astrocytes by FTY720 contributes to the amelioration of chronic Microglia Monocyte progressive NOD EAE. pro-inflammatory cluster pro-inflammatory cluster 1.2 1.2 *** ** e 1.0 1.0 FTY720 Modulates Activation of Human Astrocytes. To further in- 0.8 0.8 hang 0.6 0.6 vestigate the relevance of S1PR modulation in astrocytes to its ld change 0.4

0.4 o Fold c Fold 0.2 F 0.2 potential effects on MS, we analyzed the genome-wide tran- 0.0 0.0 scriptional response of human astrocytes activated in vitro in the

VehicleFTY720 VehicleFTY720 presence of FTY720. We identified 1,221 transcripts that were differentially regulated under FTY720 treatment (Fig. 4 A and Fig. 2. FTY720 modulates activation of astrocytes, microglia, and proin- B). Ingenuity pathway analysis determined that FTY720 modu- flammatory monocytes. Astrocytes, microglia, and proinflammatory mono- lates the expression of transcriptional modules associated with cytes were isolated by FACS sorting at day 120 of EAE, and RNA was subjected migratory pathways, antigen presentation, inflammasome activation, to custom-made nCounter Nanostring arrays. (A and B)FoldchangeinmRNA α expression of the indicated genes from sorted astrocytes (A)andmicroglia(B) axonal guidance, and -oxidation (Fig. 4C). Interestingly, κ MEDICAL SCIENCES from FTY720- or vehicle-treated mice at day 120 of EAE as determined by NF- B signaling was significantly altered by FTY720 in human as- NanoString analysis [fold change in relative expression as determined by trocytes (Fig. 4C), recapitulating our findings of decreased NF-κB

log2(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 sorted hi microglia (C) and Ly6C1 proinflammatory monocytes (D). Data are ratio of A Ccl2 Nos2 Csf2 Il6 Tnfa Il10 count numbers of cells from FTY720-treated to vehicle-treated mice, and are 30 *** 6 * 0.8 ** 10 * 5 * 0.08 ** ion

representative of two independent experiments of pooled microglia and ss 0.6 8 4 0.06

re 20 4 hi p 6 3 ld)

Ly6C proinflammatory monocytes with three mice per group. Data are o 0.4 0.04 ± < < ’ (f 10 2 4 2 mean SEM (**P 0.01 and ***P 0.001, Student s t test). 0.2 2 1 0.02

mRNA ex 0 0 0.0 0 0 0.00

e e e others (1, 6, 49). Thus, we used a cell-based assay to evaluate the Vehicl FTY720 Vehicl FTY720 Vehicl FTY720 VehicleFTY720 VehicleFTY720 VehicleFTY720 effects of FTY720 on astrocyte-driven neurotoxicity. In this as- BC 15 * 250 ** say, we activated astrocytes in vitro in the presence of FTY720 or rs)

be 200 10 vehicle for 24 h and then added new culture medium after ex- 150

tensive washings. Following culture for additional 48 h, astro- 5 num te 100 (% of max) 50 Neurotoxicity Neurotoxicity cyte-conditioned medium (ACM) was collected. To evaluate the 0 cells Migrated 0

0 (absolu e l 20 neurotoxic activity of ACMs, we exposed neuronal cells to the 7 VehicleFTY72 Vehic FTY ACMs and monitored neuronal death by quantifying lactate D dehydrogenase (LDH) release. FTY720 treatment led to a small Ccl2 Nos2 Il6 Il12a Il23a 5 ** 1.0 * 2.0 *** 0.03 * 0.15 ** but significant reduction of ACM neurotoxic activity (Fig. 3B). ion 4 0.8 ss 1.5

e 0.02 0.10 r ) 3 0.6 During MS and EAE, proinflammatory monocytes are recruited d 1.0

(fol 2 0.4 0.01 0.05 to the CNS, where they play a central role in promoting neuro- 1 0.2 0.5

mRNAexp 0 0.0 0.0 0.00 0.00 degeneration (2, 36, 37, 39). Thus, chemotactic factors produced by e 0 e 0 le 72 72 720 astrocytes play an important role in promoting disease progression ehic VehicleFTY720 VehicleFTY720 Vehicl FTY Vehicl FTY V FTY in MS (40, 52, 53). To address the effects of FTY720 on the re- cruitmentofLy6Chi proinflammatory monocytes by astrocytes we Fig. 3. FTY720 modulates proinflammatory activation of astrocytes. Pri- used an in vitro transwell migration assay. We found that FTY720 mary cultures of murine astrocytes were activated with LPS and treated with decreased Ly6Chi inflammatory monocyte recruitment by ACM FTY720 or vehicle and analyzed for gene expression, neurotoxic and che- motaxic mediator production, as well as monocyte polarizing properties. (Fig. 3C). (A) RNA expression of indicated genes from astrocytes isolated 24 h after Astrocytes also modulate myeloid cell activation and polariza- activation and treatment from three biological replicates. (B) Neurotoxicity tion in the CNS (1, 54). To test the effects of FTY720 on the assay with supernatants of activated astrocyte cultures treated with FTY720 control of monocytes by astrocytes, we activated astrocytes in the or vehicle. (C) Migration assays with supernatants from activated FTY720- or + presence of FTY720 or vehicle for 24 h, washed them extensively, vehicle-treated astrocytes using CD11b Ly6C1hi monocytes as migrating hi cells. (D) qPCR analysis for expression of the indicated genes from sorted and established cocultures with sorted Ly6C inflammatory + hi monocytes. Following coculture for 24 h, the monocytes were rei- CD11b Ly6C1 proinflammatory monocytes that were cocultured with ac- tivated FTY720- or vehicle-treated astrocytes and reisolated thereafter for solated and their transcriptional profile was analyzed. Treatment RNA analysis. Throughout, data are mean ± SEM and representative of three of astrocytes with FTY720 decreased the expression of proin- independent experiments with three biological replicates. P values were flammatory cytokines, chemokines, and neurotoxic molecules in derived by Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Rothhammer et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 Vehicle FTY720 Ingenuity Canonical Pathways p - value Collectively, these findings reveal an overall neuroprotective A C Granulocyte Adhesion and Diapedesis 1,70E-09 Agranulocyte Adhesion and Diapedesis 4,37E-09 effect of FTY720 on human astrocytes mediated, at least in part, Communication between Innate and Adaptive Immune Cells 2,63E-07 κ Role of Cytokines in the Communication between Immune Cells 6,46E-06 by the suppression of NF- B signaling. In addition, these findings T Helper Cell Differentiation 4,07E-05 Pathogenesis of Multiple Sclerosis 1,78E-04 highlight targets of FTY720 on astrocytes, which may guide Antigen Presentation Pathway 1,38E-03 novel potential therapeutic interventions for the modulation of Inhibition of Matrix Metalloproteases 1,66E-03 HMGB1 Signaling 1,91E-03 astrocyte function during progressive MS. Inflammasome pathway 2,51E-03 IL-17 Signaling 5,25E-03 Axonal Guidance Signaling 6,61E-03 Discussion Leukocyte Extravasation Signaling 6,76E-03 NF- B Signaling 9,77E-03 The immunosuppressive and disease-ameliorating effects of FTY720 in IL-10 Signaling 1,23E-02 HIF1 Signaling 1,78E-02 RRMS are mostly attributed to the blockade of inflammatory cell Fatty Acid -oxidation 3,31E-02 IL-8 Signaling 4,57E-02 migrationintotheCNS(19,28).Phosphorylated FTY720, however, crosses the BBB and acts on CNS-resident cells, including astrocytes D (22, 24, 26). The effects of FTY720 in astrocytes, neurons, and oligo- dendrocytes have been previously documentedinvitro(26,27,55,56).

B Vehicle FTY720 A CCL2 NOS2 IL6 5 * 0.0003 * 0.004 * 4 0.003 3 0.0002 0.002 2 (fold) 0.0001 1 0.001

mRNA expression 0 0.0000 0.000

720 VehicleFTY VehicleFTY720 VehicleFTY720

IL12A IL23A IL10 0.004 * 0.0006 * 0.0025 * on 0.003 0.0020 0.0004 0.0015 0.002 expressi 0.0010 (fold) 0.0002 0.001 Up 0.0005

Global Down Cell proliferation mRNA 0.000 0.0000 0.0000 0 – 0.5 + 0.5 720 72 VehicleFTY VehicleFTY720 VehicleFTY Fig. 4. Unbiased analyses reveal down-modulatory effects of FTY720 on hu- man astrocytes. Primary human fetal astrocytes were activated with IL-1β in the presence or absence of FTY720, and gene expression was assessed by BCAstrocytes decreased by increased by Affymetrix arrays. (A) Heat map of 16,799 expressed (detected at level 0.1 in at ** 80 FTY720 FTY720 least two of three samples) and 1,221 differentially regulated (B) genes (signal: 60 treatment treatment noise ratio) of activated FTY720- or vehicle-treated human primary astrocytes CSPG5 40 as assessed by Affymetrix assay. Data represent three replicates of three in- xicity oto NTF3 r dependent astrocyte cultures. Gene expression levels are row-centered and 20 PSPN (% max) of − + Neu log2-transformed, and saturated at levels 0.5 and 0.5 for visualization sat- 0 CNTF < isfying a false discovery rate (FDR) 0.1. (C) Ingenuity pathway analysis of the NRTN transcriptional profile of activated human primary astrocytes under treatment 720 VehicleFTY ARTN with FTY720 or vehicle. (D) Ingenuity pathway analysis diagram of NF-κBsig- BDNF naling pathways comparing FTY720 vs. vehicle treatment. Colors code for up- D NTF4 and down-regulation of individual members in red (up) and blue (down). GDNF 4,000 ** IL11 p65 activation in mouse astrocytes in vitro (Fig. S4A). NF-κBplaysa 3,000 LIF central role in the control of astrocyte activities that promote in- numbe rs) 2,000 CSPG4 NGF

flammation and neurodegeneration (2, 40). Thus, we performed an olute 1,000

Migrated cells HBEGF in-depth computational analysis of the NF-κB pathway, which 0 (abs confirmed that FTY720 down-modulates NF-κB signaling in human 0 -0.4 -0.2 0.0 0.2 0.4 astrocytes (activation z-score, −1.89; Fig. 4D). Vehicle FTY72 Fold change in relative expression (FTY720 / Control Astrocytes, log ) To evaluate the functional relevance of our transcriptional 2 analyses, we validated the expression of selected transcripts on Fig. 5. FTY720 mitigates proinflammatory and neurotoxic properties of hu- independent samples and examined the functional effects of man astrocytes. Human fetal astrocytes were activated and treated with FTY720 FTY720 on human astrocytes. FTY720 decreased the expression or vehicle. (A) qPCR analysis of indicated genes expressed in activated astrocytes of proinflammatory and neurotoxic mediators, including cyto- under FTY720 or vehicle treatment 24 h after activation (n = 3 biological rep- kines, chemokines, and degenerative mediators, while increasing licates). (B) Neurotoxicity assay with supernatants of activated human primary anti-inflammatory IL10 in human astrocytes, as determined in astrocytes treated with FTY720 or vehicle (n = 3 biological replicates). (C)Fold quantitative PCR (qPCR) analyses from separate sets of samples change in mRNA expression of the indicated genes in human activated astro- (Fig. 5A). Moreover, ACMs from FTY720-treated human as- cytes treated with FTY720 or vehicle as determined by Affymetrix arrays [fold trocytes exhibited a marked reduction in neurotoxic and che- change in relative expression as determined by log2(FTY720/vehicle)]. (D)Mi- gration assays with supernatants from activated FTY720- or vehicle-treated motactic properties, concomitant with an increased production + hi – human primary astrocytes using CD11b Ly6C1 proinflammatory monocytes as of neuroprotective mediators (Fig. 5 B D). Human microglia, migrating cells (n = 3 biological replicates). Throughout, data are mean ± SEM however, did not show a significant response to exposure to and representative of three independent experiments. P values were derived by FTY720 (Fig. S3B). Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1615413114 Rothhammer et al. Downloaded by guest on September 29, 2021 Given the beneficial effects of FTY720 on organotypic cultures in vitro amelioration obtained with FTY720, thus calling for caution as and its BBB permeability (27, 56), FTY720 has the potential to to the therapeutic potency of FTY720 and its analog BAF312 in modulate the local innate immune response in the CNS, thought to SPMS. Moreover, these findings suggest that the beneficial ef- contribute to disease progression in MS (1, 6, 49). However, the effects fects of FTY720 on NOD EAE may reflect its moderate in- of FTY720 on chronic CNS-innate immune responses in vivo, as well hibitory effects on sphingolipid metabolism. as their relevance to the pathogenesis of disease progression in MS, are Finally, our unbiased genome-wide analyses identify additional still unknown. potential targets for the therapeutic modulation of astrocyte function Our studies highlight the anti-inflammatory and neuroprotective and the CNS-innate immune response, which may guide the devel- effects of S1PR modulation on mouse and human astrocytes at opment of novel therapeutics for the treatment of progressive MS. transcriptional and functional levels. Moreover, we evaluated the biological relevance of these observations in the NOD EAE model Materials and Methods (32). In the chronic phase of this EAE model, disease progression is Mice. Female NOD/ShiLtJ mice and 1–3-d-old pups from C57BL/6J mice were driven by the pathogenic activity of astrocytes, microglia, and in- obtained from the Jackson Laboratory and were kept in a pathogen-free flammatory monocytes, without a major contribution of the adap- facility at the Harvard Institutes of Medicine. All experiments were carried tive immune system (e.g., T cells), thus recapitulating several out in accordance with guidelines prescribed by the institutional animal care aspects of SPMS (3, 32, 57, 58). Our data show that FTY720 and use committee at Harvard Medical School. ameliorates progressive CNS inflammation and neurodegeneration, EAE Induction and Treatment. EAE was induced in 8-wk-old mice by s.c. im- most likely as a result of its CNS-intrinsic effects on astrocytes, munization with 150 μg MOG35–55 peptide emulsified in CFA (Difco Labo- microglia, and proinflammatory monocytes. Of note, our in vitro ratories) per mouse, followed by administration of 200 ng pertussis toxin data on microglia point to an indirect effect of FTY720 on microglia (List Biological Laboratories) on days 0 and 2 as described previously (2, 3). mediated by the modulation of astrocyte-derived factors that in- Starting from day 40 after disease induction, mice were treaty daily with i.p. fluence microglia polarization and pathogenic activities. injections of FTY720 0.3 mg/kg body weight or vehicle, respectively. Five isoforms of S1P receptors are expressed by immune and nonimmune cells relevant to autoimmune diseases, stroke, and Generation of Astrocyte- and Microglia-Conditioned Medium for Migration and (11, 12, 59–62). Thus, S1P receptors constitute attractive Neurotoxicity Assays. In vitro astrocyte and microglia cultures from WT pups μ targets for therapeutic intervention. Indeed, S1PR modulation were treated with LPS (100 ng/mL) in the presence of FTY720 (1 g/mL) or by FTY720 is efficacious in the treatment of RRMS (13, 14). vehicle for 24 h, extensively washed, and supplemented with fresh culture medium. Forty-eight hours later, supernatants were spun down and kept for S1PR modulation did not reach the primary endpoints in the −

migration and neurotoxicity assays at 80 °C. MEDICAL SCIENCES INFORMS (oral fingolimod in primary progressive multiple sclerosis) trial in primary progressive MS (63), but ameliorated Monocyte Migration Assay. Splenic monocytes were purified from WT mice by + MRI parameters of disease activity (63). BAF312 () is CD11b beads (Miltenyi) and sorted for F4/80 SSClowLy6C1hi (SSC, sideways a BBB-permeable modulator of S1P1 and S1P5 receptors ef- scatter). These monocytes were seeded in the upper chamber of a 24-well fective in slowing down demyelination in vitro (64–66). The cell culture insert, with a 5-μm pore size (Corning), containing astrocyte- or safety and therapeutic efficacy of BAF312 on RRMS has been microglia-conditioned medium (as detailed earlier). Migrating monocytes in recently demonstrated in terms of clinical and MRI parame- the lower chamber were quantified after 3 h by FACS. ters in the phase II BOLD (BAF312 on MRI lesion given once Neurotoxicity Assay. N2A neuronal cells (CCL-131, American Type Culture Collec- daily) study (67). The therapeutic effects of BAF312 on SPMS are γ currently being investigated in the EXPAND (exploring the effi- tion) were grown in 96-well plates and preactivated with mouse IFN- (100 ng/mL; R&D Systems) for 24 h. Thereafter, medium was replaced, after extensive washing cacy and safety of siponimod in patients with secondary progress with PBS solution, with astrocyte- or microglia-conditioned medium. Cytotoxicity multiple sclerosis) trial. Based on the effects of FTY720 on the was measured by using LDH release (CytoTox 96 Nonradioactive Cytotoxicity Assay; CNS-innate immune response during NOD EAE, S1P receptor Promega) after 24 h as suggested by the manufacturer’s protocol. modulation by BAF312 is likely to modulate local CNS in- flammation in SPMS. Indeed, preliminary results of the EXPAND Monocyte Polarization Assays. Primary astrocyte cultures were activated in the trial suggest a protective effect of BAF312 on 3-mo confirmed presence of FTY720 or vehicle for 24 h. Thereafter, activation medium was disease progression in SPMS (68). However, side-by-side compar- removed and primed astrocytes were washed extensively. CD11b+CD45+Ly6Chi isons of the effects of FTY720 and BAF312 on adaptive and innate proinflammatory monocytes were FACS-sorted from spleens of naive WT mice immune cells are needed to determine whether the structural dif- and cocultured with primed astrocytes. After 24 h, monocytes were reisolated ferences existing between these compounds affect their biologic and RNA was isolated, transcribed, and subjected to qPCR analysis. activities on specific cell populations relevant to SPMS. Indeed, it is EAE clinical scoring, mouse and human primary astrocyte and microglia cultures, isolation of cells from adult mouse CNS, subcellular fractionation worth noting that, whereas FTY720 modulates S1P1, S1P3, S1P4, and immunoblot analysis, T-cell proliferation, flow cytometry staining and and S1P5 receptors, BAF312 modulates S1P1 and S1P5 acquisition, ELISA, detection of NO, histology, nCounter gene expression, receptors only. qPCR, Affymetrix gene assay, heat-map generation, and Ingenuity pathway FTY720 is also reported to modulate sphingolipid metabo- and statistical analysis are provided in SI Materials and Methods. lism, which is thought to promote disease progression in NOD EAE and MS (3, 19, 23, 69, 70). Indeed, we showed that inhib- ACKNOWLEDGMENTS. We thank Howard L. Weiner for his suggestions. This itors of B4GALT6 and lactosyl ceramide production suppress work was supported by National Institutes of Health Grants AI075285 and AI093903 (to F.J.Q.), National Multiple Sclerosis Society Grants RG4111A1 and JF2161-A-5 (to disease progression in NOD EAE by arresting local CNS-innate F.J.Q.), Novartis educational grants (to F.J.Q. and J.A.), a Canadian Institutes of Health immunity and neurodegeneration (3). The arrest of NOD Research Industry grant (to J.A.), Mallinckrodt Pharmaceuticals Educational Grant EAE obtained with B4GALT6 inhibitors is stronger than the A219074 (to V.R.), and German Research Foundation Fellowship DFG RO4866 1/1.

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