Laquinimod arrests experimental autoimmuneencephalomyelitis by activating the arylhydrocarbon receptorJoel Kayea,1, Victor Piryatinskya, Tal Birnbergb, Tal Hingalya, Emanuel Raymonda, Rina Kashia, Einat Amit-Romacha,Ignacio S. Caballeroc, Fadi Towficc, Mark A. Atora, Efrat Rubinsteina, Daphna Laifenfeldb, Aric Orbacha, Doron Shinara,Yael Marantza, Iris Grossmanb, Volker Knappertzd, Michael R. Haydene, and Ralph Laufera

aDiscovery & Product Development, Global Research & Development, Teva Pharmaceutical Industries Ltd., Netanya 42504, Israel; bPersonalized & PredictiveMedicine, Analytics and Big Data, Global Research & Development, Teva Pharmaceutical Industries Ltd., Netanya 42504, Israel; cImmuneering Corporation,Cambridge, MA 02412; dGlobal Clinical Development, Global Research & Development, Teva Pharmaceutical Industries Ltd., Netanya 42504, Israel; and eGlobalResearch & Development, Teva Pharmaceutical Industries Ltd., Netanya 42504, Israel

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved August 5, 2016 (received for review May 16, 2016)

Laquinimod is an oral drug currently being evaluated for the treat-ment of relapsing, remitting, and primary progressive multiplesclerosis and Huntington’s disease. Laquinimod exerts beneficial ac-tivities on both the peripheral immune system and the CNS withdistinctive changes in CNS resident cell populations, especially as-trocytes and microglia. Analysis of genome-wide expression datarevealed activation of the aryl hydrocarbon receptor (AhR) pathwayin laquinimod-treated mice. The AhR pathway modulates the differ-entiation and function of several cell populations, many of whichplay an important role in neuroinflammation. We therefore testedthe consequences of AhR activation in myelin oligodendrocyte glyco-protein (MOG)-induced experimental autoimmune encephalomyelitis(EAE) using AhR knockout mice. We demonstrate that the pronouncedeffect of laquinimod on clinical score, CNS inflammation, and demye-lination in EAEwas abolished in AhR−/− mice. Furthermore, using bonemarrow chimeras we show that deletion of AhR in the immune systemfully abrogates, whereas deletion within the CNS partially abrogatesthe effect of laquinimod in EAE. These data strongly support the ideathat AhR is necessary for the efficacy of laquinimod in EAE and thatlaquinimod may represent a first-in-class drug targeting AhR for thetreatment of multiple sclerosis and other neurodegenerative diseases.

aryl hydrocarbon receptor | EAE | laquinimod

Laquinimod is an oral drug that is currently in late-stage clinicaldevelopment for the treatment of relapsing remitting multiple

sclerosis (RRMS), primary progressive MS, and Huntington’sdisease. Current knowledge indicates that laquinimod exerts ac-tivities both on the peripheral immune system and within theCNS. Laquinimod, at the 0.6-mg/d dose, has demonstrated effi-cacy in phase II and III MS clinical trials, in which it reducedrelapse rate, disability progression, development of new and activeMRI lesions, and brain atrophy (1–3). The clinical efficacy profileof laquinimod is characterized by a dissociation of the moderatemagnitude of the effect on relapse reduction and its associatedinflammatory MRI findings and the disproportionally large effecton disability progression. Such an efficacy profile in patients withRRMS may relate to a distinctive intracerebral activity potentiallymediated via changes in CNS resident cell populations, potentiallyastrocytes and microglia.The influence of laquinimod on the immune system was studied

in experimental autoimmune encephalomyelitis (EAE) (4–12),an autoimmune disease mediated by proinflammatory myelin-reactive lymphocytes that cause CNS inflammation leading to de-myelination and axonal loss. Laquinimod has also been effective inthe treatment of other models of autoimmune diseases, specificallyexperimental autoimmune neuritis (13, 14), lupus nephritis (15),and colitis (16). A common characteristic of autoimmune diseasesis that autoantigen-reactive T cells must undergo several discretesteps to cause disease. Initial signals that direct T-cell activation

and differentiation are provided by antigen-presenting cells(APC), including monocytes, macrophages, and dendritic cells(DCs). It was reported that treatment of mice with laquinimod isassociated with alterations in the frequency of myeloid subpopulationsthat included a reduction in CD4+ DCs. Laquinimod treatment alsopromoted the development of anti-inflammatory type II monocytesand DCs (6, 7, 9), which are likely associated with its immunomod-ulatory activities. These activities include reduced production ofproinflammatory cytokines such as IL-17, reduced migration of lym-phocytes (4, 7), augmentation of regulatory T-cell numbers (5, 7), andproduction of brain-derived neurotrophic factor (5, 8). Although nomolecular target has been identified for laquinimod, it has been shownto modulate the T-cell response probably as a result of its effects onSTAT1, MAPK, and NF-κB signaling in APCs (reviewed in ref. 17).To further elucidate laquinimod’s immunomodulatory mechanisms ofaction, in this paper we analyzed gene expression levels modulated bylaquinimod versus vehicle-treated mice. We show that laquinimodinduces genes known to be associated with the aryl hydrocarbon re-ceptor (AhR). In the present study, we investigate whether laquini-mod suppresses EAE via the AhR pathway by testing its efficacy inmyelin oligodendrocyte glycoprotein (MOG)-induced EAE usingAhR knockout mice.

Significance

Laquinimod is an oral drug currently being evaluated for thetreatment of relapsing, remitting, and primary progressive mul-tiple sclerosis as well as Huntington’s disease. It is thought thatlaquinimod has a primary effect on the peripheral innate immunesystem and also acts directly on resident cells within the CNS.However, the exact mechanism of action of laquinimod has notbeen fully elucidated. We investigated gene expression in laqui-nimod-treated mice and show induction of genes downstream toactivation of the aryl hydrocarbon receptor (AhR). In this paper,we examine the role of the AhR in laquinimod treatment of ex-perimental autoimmune encephalomyelitis and demonstrate thatAhR is the molecular target of laquinimod in this model.

Author contributions: J.K., V.P., T.B., E.A.-R., M.A.A., D.L., A.O., D.S., Y.M., I.G., V.K., andR.L. designed research; T.H., E. Raymond, and R.K. performed research; I.S.C. and F.T.contributed new reagents/analytic tools; J.K., V.P., T.B., T.H., E.A.-R., I.S.C., F.T., M.A.A., E. Rubinstein,D.L., A.O., D.S., M.R.H., and R.L. analyzed data; and J.K., V.K., and R.L. wrote the paper.

Conflict of interest statement: The authors are employees of Teva Pharmaceutical Indus-tries Ltd. or Immuneering Corporation.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The raw data are available at the National Center for BiotechnologyInformation (accession no. PRJNA319255).1To whom correspondence should be addressed. Email: joel.kaye@teva.co.il.

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ResultsTranscriptome Analysis Reveals That Laquinimod Treatment InducesGenes Associated with Activation of the AhR. To gain insight intolaquinimod’s immunomodulatory mechanisms of action, spleen geneexpression profiles were compared between laquinimod- and vehicle-treated EAE mice, 6 d postdisease induction. Our analysis showedthat 610 genes were differentially modulated between laquinimod-and vehicle-treated EAE mice. Of the 610 genes, 227 genes weredown-regulated by laquinimod treatment, including key Th17-relatedcytokine genes, specifically IL-17a, IL-17re, and IL-22Ra1 (Fig. 1),which were significantly reduced in laquinimod-treated mice [log2fold-changes of −2.91, −1.64, and −1.81, respectively; false-discoveryrate (FDR) values of 4.4e-4, 7e-3, and 2.7e-3]. These findings are inline with published data showing the beneficial effect of laquinimodon Th17 (4, 7). A total of 383 genes were up-regulated by laquini-mod in this analysis, and cytochrome P450 family member A1(Cyp1a1) and Ahrr, prototypical genes associated with the AhRpathway (18, 19), were among the highest fold-change genes inducedby laquinimod treatment in EAE mice (Fig. 2A). Analysis of laqui-nimod-induced genes in naive mice revealed a similar pattern (Fig.2B), indicating that activation of AhR was inherent to drug and in-dependent of disease state. However, many other AhR-associatedgenes, including Cyp1b1 (20), Tiparp (19), Ido1 and Ido2 (21, 22),Spint1, and Serpins (23) were induced by laquinimod in both naiveand EAE mice. Laquinimod-induced activation of the AhR pathwaywas confirmed by assessing gene expression levels of the AhR bio-marker, Cyp1a1 in mouse liver. As shown in Fig. 2C, the endogenousAhR ligand 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acidmethyl ester (ITE) (24) caused increases of up to 74- and 4.2-fold inhepatic Cyp1a1 and Cyp1a2 mRNA levels, respectively. Treatmentof mice with 25 mg/kg laquinimod caused 539- and 21-fold in-creases in hepatic Cyp1a1 and Cyp1a2 mRNA expression levels,respectively, compared with vehicle-treated mice. These data veri-fied the initial genomic findings and demonstrated that laquinimodconsistently induces the expression of genes downstream to theactivation of AhR.Additional experiments were performed to determine the

concentration dependence of the laquinimod effect by quantifyingthe level of mRNA changes in primary hepatocytes, the most AhR-responsive tissue. Treatment of human hepatocytes with laquinimodfor 24 h resulted in ∼180- and ∼80-fold induction of CYP1A1 andCYP1A2 mRNA, with an EC50 of 0.2 ± 0.04 μMand 0.3 ± 0.03 μM,respectively (Fig. 3A). Treatment with 100 μM omeprazole for 24 hwas used as a positive control and resulted in 1,487 ± 34 and 188 ±11 fold-induction of CYP1A1 and CYP1A2 mRNA. Treatment ofprimary mouse hepatocytes with laquinimod resulted in induction ofCyp1a1 mRNA with an EC50 of 0.06 ± 0.02 μM and 5.70 ± 0.20 μMat 4 and 24 h, respectively (Fig. 3B). Both the potency and magni-

tude of induction by laquinimod in mouse hepatocytes were larger at4 h than at 24 h, presumably due to metabolism following longerincubation. Omeprazole does not activate mouse AhR, so3-methylcholanthrene (3-MC) treatment (2 μM) for 24 h wasused as a positive control and resulted in a 116- ± 1.5-fold in-duction of Cyp1a1 mRNA. In mouse hepatocytes, basal levels ofCyp1a2 mRNA were high and were not significantly augmentedby treatment with either laquinimod or 3-MC.

The Efficacy of Laquinimod in EAE Is Dependent on the AhR. Giventhat laquinimod activates AhR, we wanted to determine the roleof AhR in the efficacy of laquinimod in MOG-induced EAE. Wefound that AhR−/− mice were susceptible to EAE, albeit with

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Fig. 1. Laquinimod treatment down-regulates genes associated with the Th17pathway. These boxplots show the patterns of the expression of individualgenes from splenocyte samples taken from naive and EAE mice at day 6 post-induction (n = 6 per treatment). It shows the patterns of expression across threeconditions: EAE mice treated with vehicle, EAE mice treated with laquinimod,and naive mice treated with vehicle (n = 5). Laquinimod treatment significantlyreduced the expression of IL-17a [log2 fold change (FC): −2.91; FDR: 4.4e-4],Il17re (log2 FC: −1.64; FDR 7e-3), and Il22ra1 (log2 FC: −1.81; FDR: 2.7e-3).

Fbxo27Cdh1Stbd12210408F21RikAmz1Serpinb8Adamdec1TskuRusc2Cd93Ugt1a6aDpep2Gpt2Gpr82Dab2Msr1MgarpTrnp1Mmp27Ms4a7Slc27a2Pmp22Ltc4sFndc5AcppGsg1TiparpRP23−329A21.3Cxcl3Gm28548Ido1Syt15Kcnj16Aox3Crhr1Fgf3InhbbThrspLpar1Serpinb2RP23−326P16.3Slc9a3Cnga3Ppp1r3gGm26578Rims4Mb21d2Gm15759Wnt2GzmcPhactr1Ttc9Ctla2bOlfr334−ps1Ifitm7Serpinb10Muc6Dpep3Ido21700057G04RikCyp1b1Ppp1r3cPgfPdzph1PalmdOlfml2aMmp13Ppp1r14dPlet1Adamts9Mpzl2Col13a1Ntf3Eva1aSpint1Mcpt1NfascHic1Ripk4Adamts151810041L15RikScinDnah2GpnmbAmer3AhrrCyp1a1Wnt4

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Fig. 2. Transcriptome analysis reveals that laquinimod induces genes in the AhRpathway. (A) This heatmap was generated using the patterns of expression ofsplenocyte samples from EAE mice at day 6 postinduction. It shows the patterns ofexpression of 88 genes with higher levels of expression in EAE mice treated withlaquinimod (green, n = 6) than in EAE mice treated with vehicle (orange, n = 6).(B) This heatmap was generated using the patterns of expression of splenocytesamples fromnaivemice at 6 d postinduction. It shows the patterns of expression of45 genes with higher levels of expression in naive mice treated with laquinimod(red, n = 6) than in naive mice treated with vehicle (blue, n = 5). For both Aand B, the filters used to define statistical significance were a fold-change > 2and an FDR < 0.01. Rows were ordered from highest to lowest average fold-change. (C) In vivo induction of hepatic Cyp1a mRNA following 5-d treatmentwith 25 mg/kg laquinimod in C57BL/6 mice. This graph is taken from a rep-resentative experiment, which has been repeated at least four times.

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slightly less severe disease. Whereas WTmice developed EAE witha mean day of onset of 12.8 ± 1.3, AhR−/− mice developed EAEwith delayed onset (15.7 ± 4.5 d) and less severe disease (meanmaximal score of 3.5 ± 1.1, compared with 4.2 ± 0.7 in AhR−/− andWT, respectively). As previously reported, prophylactic daily oraltreatment with 25 mg/kg laquinimod inhibited MOG-induced EAEin WT C57BL/6 mice (Fig. 4). Laquinimod treatment reduceddisease incidence from 100 to 20% and decreased the severity ofdisease in mice that show clinical severity from 4.2 ± 0.7 to 0.3 ±

0.6 (93% inhibition, P < 0.0001). In contrast, laquinimod com-pletely lost its efficacy in AhR−/−mice (Fig. 4) that had comparabledisease severity (3.3 ± 1.7) and a profile undistinguishable fromvehicle-treated AhR−/− mice (3.5 ± 1.1).As previously shown (4), prophylactic treatment with laquini-

mod abrogated the extent of inflammation, demyelination, andacute axonal injury within the spinal cords from WT mice withMOG-induced EAE. The extent of damage in spinal cords fromWT and AhR−/− mice was assessed by histological analysis.

Fig. 3. In vitro verification of induction of CYP1A mRNA by laquinimod. (A) CYP1A1 (Left) and CYP1A2 (Right) fold-induction in human hepatocytes treatedwith laquinimod for 24 h compared with vehicle controls. Experiments were repeated at least four times with cells from different donors. (B) Cyp1a1 fold-induction in mouse hepatocytes treated with laquinimod for 4 (Left) or 24 h (Right). Experiments were repeated at least four times with different donors.

Fig. 4. Prophylactic treatment with laquinimod has no effect on clinical score in AhR−/− mice. The graph shows mean disease scores with SE from vehicle-treated (closed symbols) and laquinimod-treated (open symbols) WT or AhR−/− mice (n = 15 per group). The embedded table shows disease incidence, meanday of onset, and mean maximal score of disease. The data are representative of four independent experiments.

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Quantification of inflammatory demyelination as determinedin luxol-fast-blue (LFB)/periodic acid-Schiff (PAS) with H&Ecounterstaining revealed a significant protective effect of laqui-nimod in WT animals but not in AhR−/− animals (Fig. 5). Al-though widespread inflammatory demyelination was evidentin vehicle-treated WT mice, these histopathological alterationswere virtually absent in laquinimod-treated WT animals (P =0.0003). Unpaired t test with Welch’s correction showed nosignificant difference between vehicle-treated and laquinimod-treated AhR−/− animals (P = 0.31). Quantification of acute ax-onal damage in amyloid precursor protein (APP)-stained slidesrevealed extensive axonal damage in vehicle-treated mice. Asexpected, the number of APP spheroids was most intense inregions with white matter inflammation. Laquinimod was pro-tective in WT mice (P < 0.0001), but had no effect on acuteaxonal damage in AhR−/− mice (Fig. 5). Semiquantification ofthe extent of microglial and astrocytic activation was performedin Iba1- and GFAP-stained sections. Extensive microglial acti-vation and astrogliosis were present in vehicle-treated mice, andboth were significantly reduced by laquinimod in WT mice. Incontrast, laquinimod had no effect on either parameter in AhR−/−

mice (Fig. 5).Treatment with laquinimod has been shown to decrease Th1

and Th17 responses with a corresponding increase in CD4+

CD25+FoxP3+ regulatory T cells both in the periphery andwithin the CNS (4, 5, 7, 11). To investigate whether these im-munomodulatory effects of laquinimod are also mediated by theAhR pathway, we analyzed the production of Th1 and Th17 cy-

tokines and the frequency of regulatory T cells in spleens takenfrom WT and AhR−/− mice. Treatment with laquinimod in WTmice significantly increased the percentage of CD4+CD25+

FoxP3+ regulatory T cells (Fig. 6A) compared with vehicle-treatedmice (0.80 ± 0.06 vs. 0.37 ± 0.04; P < 0.05), whereas in AhR−/−

mice no difference was observed (0.33 ± 0.04 vs. 0.38 ± 0.06). Asexpected, laquinimod treatment resulted in decreased productionof IL-17, GM-CSF, and IFNγ in MOG-reactivated splenocytesfromWTmice, albeit only significantly for GM-CSF (P < 0.04). Incontrast, laquinimod treatment did not reduce MOG-specific IL-17, GM-CSF, and IFNγ production in splenocytes taken fromAhR−/− mice. In all mice, the recall response to purified proteinderivative (PPD) was not affected by laquinimod, and the level ofcytokine release was not statistically different between WT andAhR−/− mice (Fig. 6B). These data demonstrate that laquinimodexpands regulatory T cells and limits T-effector cells in an AhR-dependent manner in EAE.

AhR Deletion in the Immune System Is Necessary and Sufficient toNegate the Effect of Laquinimod in EAE. Current knowledge indi-cates that laquinimod exerts activities both on the peripheralimmune system and within the CNS. The data above clearly showthat AhR is required for the efficacy of laquinimod in EAE;however, it is not clear whether AhR is required in the periph-eral immune system, in the CNS, or in both. To address this, wemade chimeras by transplanting bone marrow (BM) cells intobusulfan-conditioned recipients: In one set of animals, AhR−/−

(CD45.2) BM cells were transplanted into congenic B6 CD45.1recipients to create mice with an AhR−/− peripheral immune

Fig. 5. Prophylactic treatment with laquinimod has no effect on inflammatory demyelination, acute axonal damage, or microglial and astroglial activation inAhR−/− mice. All stains were performed using n = 15 per treatment group. The inflammatory index was quantified using LFB/PAS and H&E staining of spinalcords taken from vehicle- or laquinimod-treated WT or AhR−/− mice. (Upper panels) A cross-section of the whole spinal cord using 4× objective (Nikon EclipseE200). (Lower panels) A higher magnification (40×) taken from the area marked by a rectangle. Quantification of acute axonal damage using APP stainingshowed a significant reduction (*P < 0.0001) in the number of APP+ spheroids in laquinimod-treated WT mice, but not in AhR−/− mice. Quantification ofmicroglial and astrocyte activation using Iba-1 or GFAP staining showed a significant reduction (*P < 0.0001) in the number of Iba-1 (black bars) and GFAP(striped bars) cells in laquinimod-treated WT mice, but not in AhR−/− mice.

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system and AhR+/+ CNS; and in another set of animals B6CD45.1 BM cells were transplanted into AhR−/− recipients tocreate mice with an AhR+/+ peripheral immune system and anAhR−/− CNS. Chimerism was tracked using FACS analysis ofperipheral blood, and at 13 wk posttransplant recipient mice had>80% donor-derived blood cells. At that point, chimeric micewere induced with MOG EAE and treated with vehicle orlaquinimod. In chimeras with an AhR+/+ immune system,laquinimod treatment reduced disease severity from a meanclinical score of 2.5 ± 0.8–0.7 ± 0.8 (72% inhibition, Fig. 7),although the effect of laquinimod was not as efficacious as seenin WT C57BL/6 mice where it inhibited disease by 93% (Fig. 4).In contrast, laquinimod completely lost its efficacy in chimericmice with an AhR−/− immune system (Fig. 7) where diseaseseverity (2.9 ± 1.1) was comparable to vehicle-treated AhR−/−

mice (3.5 ± 0.5). These data indicate that AhR is fully requiredin the peripheral immune system for the effect of laquinimodand may be partially dependent on the expression of AhR for theeffect of laquinimod within the CNS.

DiscussionIn this paper we used transcriptome analysis to further elucidatethe mechanism of action (MoA) of laquinimod, and we dem-onstrate that laquinimod induces genes associated with the AhRpathway. The prototypical AhR genes Cyp1a1 and Ahrr wereamong those with the highest average fold-change in both naiveand EAE mice treated with laquinimod (Fig. 2 A and B). Weverified the in vivo induction of Cyp1a1 using qPCR of mRNAlevels from livers of treated mice (Fig. 2C) and demonstratedthat laquinimod is a potent inducer of Cyp1a in vitro (Fig. 3).Together, these findings demonstrate that laquinimod is an acti-vator of AhR, and the exact molecular events that result in acti-vation are currently being investigated. This raised the possibilitythat the therapeutic effect of laquinimod is dependent on AhRactivation. Indeed, we report here that the effect of laquinimodwas completely lost in MOG-induced EAE in AhR−/− mice, as

depicted by clinical score (Fig. 4) and histopathological findings(Fig. 5). These data indicate that deletion of AhR is necessary andsufficient to abrogate the effect of laquinimod in EAE.Treatment with laquinimod has been shown to shift the bal-

ance from pathogenic Th17 cells toward an increase in thenumber of CD4+CD25+FoxP3+ regulatory T cells both in theperiphery and in situ within the CNS (4, 5, 7). Our RNA-Seq datashowed 227 genes had decreased expression levels in laquinimod-treated mice, including key Th17-related cytokine genes (Fig. 1).The mechanism by which AhR signaling modulates T-regulatory(T-reg) biology includes shaping T-reg differentiation by dictatingthe state of FoxP3 promoter methylation and enhancing FoxP3expression; mediating methylation of the il17 promoter and de-creasing the expression of IL-17; and inducing tolerogenic DCsthat promote the generation of T regs (25–30). Although we donot have experimental evidence regarding the effect of laquinimodon FoxP3 promoter methylation, laquinimod has been shown toreduce IL-17 production (5, 7) and induce tolerogenic DCs (7, 9).Furthermore, we show that both the reduction of proinflamma-tory cytokines and the increase in CD4+CD25+FoxP3+ regulatoryT-cell numbers by laquinimod is AhR-mediated (Fig. 6). Consid-ering the link of AhR with other regulatory T-cell populations,specifically Tr1 cells (31–34), it will be interesting to investigatewhether laquinimod also modulates Tr1 cells. Various compoundsacting on the AhR pathway regulate adaptive immune responsesthrough effects on both APCs and T cells (29). In contrast, nodirect effects of laquinimod on T cells have been detected (7).Laquinimod, like other AhR activators, can induce the differen-tiation of APCs with a tolerogenic phenotype. The mechanismsresponsible for this tolerogenic phenotype may result from the up-regulation of indoleamine 2,3-dioxygenase expression, resulting inincreased synthesis of immunosuppressive kynurenines (21, 22).Indeed, our transcriptome data showed up-regulation of both Ido1and Ido2 (Fig. 2). Taken together, these data suggest that the ef-fects of laquinimod on the encephalitogenic T-cell response in EAEinvolve different cellular and potentially molecular mechanisms

Fig. 6. Laquinimod immunomodulation in EAE is AhR-dependent. (A) MOG EAE mice were treated daily with laquinimod (25 mg/kg, n = 6 per group) orvehicle (water, n = 6 per group), and 15 d after immunization, spleen cells were removed and evaluated by FACS for expression of CD25 and Foxp3 by CD4+

cells. (Left) A representative dot plot from a vehicle-treated mouse, where CD4+CD25+ cells were gated, and the percentage of FoxP3+ cells was calculatedfrom within that gate (Middle). The individual data from each animal demonstrates that laquinimod significantly increases the percentage of CD4+CD25+

FoxP3+ regulatory T cells in WT and not AHR−/− mice. Data are representative of two independent experiments. (B) The remaining spleen cells wererestimulated with PPD or MOG in vitro, and after 48 h the culture supernatants were analyzed for IL-17, GM-CSF, and IFNγ release. Under some conditions, thecytokine levels were below the level of detection and are marked as “BQL.” Laquinimod treatment reduced MOG-specific GM-CSF (P < 0.04), IL-17, and IFNγ.Data are representative of two independent experiments.

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from other AhR ligands. Similar to other ligand-activatedtranscription factors, AhR-mediated biological responses havebeen shown to be compound, cell type, and species-dependent(35). Studies to delineate in which target cells and by what mech-anism laquinimod activates AhR are currently ongoing in ourlaboratory.Laquinimod inhibits astrogliosis in EAE (Fig. 5), and astrocytes

have been proposed to play an important role in the protectiveeffect of laquinimod in cuprizone-induced CNS demyelination(36). Laquinimod treatment reverses cuprizone-induced astro-gliosis and leads to decreased production of proinflammatoryfactors and reduced NF-κB activation in cultured murine andhuman astrocytes (36). Interestingly, cross-talk between AhR andNF-κB has been described in other cell types (37, 38), suggestingthat laquinimod-mediated AhR activation may lead to down-regulation of NF-κB in astrocytes and possibly other immune-related cells. In an attempt to elucidate the importance ofAhR in the peripheral immune system versus in resident CNScells like astrocytes, we performed a cross-over bone marrowtransplant experiment. In chimeras with an AhR−/− immunesystem, laquinimod completely lost efficacy (Fig. 7), indicatingthat the AhR in the immune system is sufficient and necessaryfor the effect of laquinimod. The exact population of immunecells that requires AhR is currently being investigated. In chi-meras with an AhR+/+ immune system, laquinimod treatmentinhibited disease by 72% (Fig. 7), although the effect oflaquinimod was not as strong as the almost complete inhibitionseen in WT C57BL/6 mice (Fig. 4). This finding supports theidea that laquinimod may be partially dependent on the ex-pression of AhR within the CNS, where lack of expression ofAhR in resident astrocytes may explain the less-than-expected

efficacy of laquinimod in chimeras with an AhR+/+ immune sys-tem. Indeed, our data would seem to corroborate a very recentpublication that reports that expression of AhR within astrocyteslimits CNS inflammation (39). Although our data strongly suggestthat the anti-inflammatory effects of laquinimod are mediated byAhR, the precise mechanism of its neuroprotective activities re-mains to be further investigated.In conclusion, we have demonstrated that laquinimod acti-

vates AhR, which is necessary for its therapeutic efficacy in theMOG-induced EAE model of MS. AhR has been known for itsability to mediate the biochemical, metabolic, and toxic effectsof environmental chemicals. Recently, a paradigm shift has oc-curred with the understanding that AhR has endogenous rolesand is an important regulator of cell development, differentia-tion, and function (40). AhR is an important regulator of thedevelopment and function of both innate and adaptive immunecells, mediated by the ability of AhR to respond to endogenousligands generated from the host cell, diet, and microbiota (41–44). This recent paradigm shift has opened new avenues of re-search in the possibility of targeting AhR to treat inflammatorydiseases in which a role of AhR has been found, including lupus(45) and colitis (46). Laquinimod may represent a first-in-classdrug targeting AhR for MS and other diseases with inflammatoryor neuroinflammatory components.

Materials and MethodsTest Compounds and Formulation. Laquinimod was synthesized at Teva Phar-maceutical Industries, Ltd. The compound was dissolved at 2.5 mg/mL in pu-rified water and administered orally by gavage in a volume of 0.2 mL. ITE waspurchased from Tocris Biosciences and was freshly prepared daily in corn oiland then administered intraperitoneally at a volume of 0.2 mL. Omeprazole

Fig. 7. Lack of AhR in the peripheral immune system is sufficient to negate the effect of laquinimod. Bone marrow chimeras were made from WT or AhR−/−

mice into preconditioned AhR−/− or WT recipients. MOG EAE was induced in the chimeric mice 13 wk posttransplant, and the graph shows mean disease scoreswith SE from vehicle-treated (closed symbols) and laquinimod-treated (open symbols) mice (n = 9 or 10 per group). The embedded table shows diseaseincidence, mean day of onset, and group mean score of disease. The data are representative of a single experiment.

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and 3-MC were purchased from Sigma-Aldrich. Busuflex (Busulfan) (6 mg/mL)was purchased from Otsuka America Pharmaceutical Inc.

Mice. Healthy C57BL/6 mice at 6–8 wk of age were obtained from the HarlanAnimal Breeding Center, Rehovot, Israel. AhR knockout (AhR−/−) mice on aC57BL/6 background (C57BL/6-Ahrtm1.2Arte) were obtained from Taconic.Congenic C57BL/6 B6.SJL-Ptprca Pepcb/BoyJ (B6 CD45.1) mice were obtainedfrom Jackson Laboratories. The mice were housed at 22–24 °C, and food andwater were available ad libitum. All experimental procedures conformed toaccepted ethical standards for use of animals in research and were in ac-cordance with Committee for the Care and Use of Experimental Animalguidelines and approved by the Teva Institutional Animal Care and UseCommittee.

Bone Marrow Transplantation. Ten congenic B6 CD45.1 or AhR−/− (CD45.2)recipient mice were preconditioned with three intraperitoneal injections of10 mg/kg busulfan on days −5, −3, and −1 before transplantation. Donormice were killed, and bone marrow cells were isolated from the femursand tibias of all four limbs. Following lysis of RBCs, 9–10 × 106 BM cellswere injected i.v. into the recipient mice. Peripheral blood was removed,lysed, and resuspended in FACS buffer and then stained using CD45.1-PEor CD45.2-APC (Miltenyi Biotec). After 13 wk, mice were induced withMOG EAE.

Induction of EAE and Clinical Evaluation. Mice were immunized in the flankswith 300 μg/mouse pMOG35–55 peptide (Novetide) in normal saline emul-sified in an equal volume of Complete Freund’s Adjuvant containing 500 μgper mouse Mycobacterium tuberculosis. Two injections of pertussis toxin(175 ng/0.2 mL per mouse, intraperitoneally) were given at the time of im-munization and 48 h later. Animals were scored for clinical signs of diseaseon a daily basis using the following scores: 0 = normal behavior, 1 = distallimp tail, 1.5 = complete limp tail, 2 = disturbed righting reflex, 3 = ataxia,4 = early paralysis (hind legs), 5 = full paralysis, and 6 = moribund or death.To calculate mean disease onset, animals that did not develop disease wereconsidered to have onset on day 31. Laquinimod was dosed orally at25 mg·kg·d beginning on the day of immunization until the end of the ex-periment (days 0–30). Placebo-treated mice were similarly administered avolume of 0.2 mL water orally 6 d a week. Each group contained between12 and 15 mice.

RNA Sequencing of Splenocytes from Naive and EAE Mice. Naive female C57BL/6mice or mice with MOG EAE were treated with vehicle or 25 mg/kglaquinimod for 25 d. After 6 d, spleens from six mice per treatment armwere removed, and RNA was isolated using the Qiagen miRNeasy Mini Kit.Globin mRNA-depleted RNA samples were converted into cDNA librariesusing the TruSeq Stranded mRNA Sample Prep Kit (Illumina, #RS-122-2103).Final cDNA libraries were analyzed for size distribution and, using anAgilent 2200 TapeStation (D1000 Screentape, Agilent # 5067–5582),quantitated by qPCR (KAPA Library Quant Kit, KAPA Biosystems # KK4824)and then normalized to 2 nM in preparation for sequencing. Sequencingwas performed using an Illumina TruSeq Paired-End Cluster Kit V4 (Illu-mina # PE-401-4001), and a clustered flowcell was generated using thenormalized cDNA libraries as templates. The cDNA templates were dena-tured using fresh 0.1 N NaOH, diluted to a final loading concentration of13 pM, and placed on an Illumina cBot (v1.5.12.0) for cluster generation.Templates were attached to the flowcell via a dense lawn of oligonucle-otides that bind to the sequencing adapters added during sample prepa-ration, which are extended and then denatured. The flowcell was thensequenced through 51 bases, paired end, with an 8-base index cycle on anIllumina HiSEq. 2000 (HiSeq Control Software v1.5.15.1). During sequenc-ing cycles, fluorescent reversible terminator dNTPs were added to theclusters with only a single base per target being incorporated. Followingimaging of the clusters, the terminator and fluorescent tags were cleavedso that the next base could be incorporated.

RNA Sequencing Analysis. Gene expression values were obtained by aligningthe sequencing reads to the mouse genome (GRCm38) using STAR (47) andusing featureCounts (48) to quantify the number of reads that aligneduniquely to genes specified in the GENCODE mouse transcriptome M7 (49).The resulting count matrix was filtered by removing genes with less than 20cumulative counts across splenocyte samples. The filtered matrix was nor-malized with Limma Voom (50) using the mouse type (EAE or naive) andtreatment (laquinimod or vehicle) variables to estimate the model coeffi-cients. Differential expression between EAE treated with laquinimod andEAE treated with vehicle was performed using Limma-moderated t tests (51).

The thresholds used to define statistical significance were a Benjamini-Hochberg–corrected (FDR) P < 0.05 and an absolute fold-change > 2.0. Theraw sequence data can be accessed with the accession no. PRJNA319255 atwww.ncbi.nlm.nih.gov/bioproject/319255.

Histopathology of Spinal Cords from EAE. At the end of the EAE study, animalswere perfused with PBS solution containing 4% (vol/vol) paraformaldehyde.Thereafter, the entire vertebral column was carefully dissected, and isolatedvertebral columns were incubated overnight at 4 °C for the purpose of tissuepost fixation. For histological and immunohistochemical studies, spinal cordswere decalcified for 48 h (37 °C), and then samples were washed for 12 hunder running water to remove the decalcification solution. Spinal cordswere embedded in paraffin, and 5-μm-thick transverse sections were pre-pared and stained for myelination/inflammatory index, microglial andastrocytic activation, and acute axonal damage. Intact and damaged myelinplus inflammatory infiltrates were visualized using LFB/PAS stains andcounterstained with H&E. The extent of inflammatory demyelination wasquantified by assessing the inflammatory-demyelination index, which isdefined as the area covered by inflammatory demyelination in relation tothe entire white matter area of each slide. For the visualization and quan-tification of microglia/monocyte and astrocyte activation, paraffin-embed-ded sections were dewaxed, washed in PBS, and incubated overnight withthe respective primary antibody diluted in blocking solution. For the visu-alization of epitope-primary antibody complexes, HRP-coupled polymer sec-ondary antibodies were used (EnVision, Dako), and 3,3′-diaminobenzidine(DAB) was used as a chromogenic substrate. Quantification of the extentof microgliosis/monocytosis (anti-Iba1) and astrogliosis (anti-GFAP) wasperformed using a blinded staging approach. The following scoring systemwas used: 0 = normal cellular density; 1 = moderate increase in cellulardensity; 2 = intermediate increase in cellular density; 3 = high cellular density;and 4 = maximum cellular density. Anti-APP stains were performed to visu-alize acute axonal damage. To quantify the extent of acute axonal damage,the number of APP+ spheroids was counted in four randomly chosen fields ofthe white matter part of the corpus callosum irrespective of the presence orabsence of lesions. The number of positive spheroids was counted under high-power magnification and quantified as the number of spheroids per squaremillimeter.

CYP1A Induction in Primary Human and Murine Hepatocytes. Cryopreservedmurine (Bioreclamation In Vitro Technologies) and human (Celsis In VitroTechnologies) hepatocytes were thawed and plated on 24 multiwell platescoated with collagen type I substratum in William’s E medium (GIBCO-32551)supplemented with 5 μg/mL insulin, 0.1 μM dexamethasone, and 10% (vol/vol)FBS. Cells were for 3–4 h, and the medium was changed to Hepatocyte BasalMedium (HBM-Lonza CC-3199) supplemented with CC-4182 (Lonza). Plateswere maintained at 37 °C for 24 h before treatment with compounds. Pro-totypical CYP1A inducers 3-MC and omeprazole were dissolved in DMSO andadded to the culture medium at a final solvent concentration of 0.1% (vol/vol).After 4 or 24 h of treatment, total RNA was extracted from duplicate samplesusing an RNA extraction kit (RNeasy 96 kit, 74181, Qiagen). QuantitativemRNA analysis was performed by real-time qRT-PCR in the ABI Prism 7900 HTSequence Detection System (TaqMan, Perkin-Elmer-Applied Biosystem). EC50values were calculated by nonlinear regression using XLfit 4.2 (IDBS).

CYP1A mRNA Induction in Livers of WT Mice. Female C57BL/6 mice weretreated for 5 d with vehicle, laquinimod (25 mg/kg, orally), or ITE (10 mg/kg,i.p.). Mouse liver samples were taken 4 or 24 h after the final dose ofcompound, and tissue samples were lysed and total RNA purified. Single-stranded cDNA was prepared from RNA with the RT Master Mix using the AB7900HT Fast Real Time PCR System thermocycling program (Applied Biosys-tems). Probes for murine Beta-Actin (Mm01205647_g1), Cyp1a1 (Mm00487218_m1),and Cyp1a2 (Mm00487224_m1) were purchased from Thermo Fischer Sci-entific. The relative quantity of the target cDNA compared with that of thecontrol cDNA (β-actin) was determined by the ΔΔCt method. The results ofthis method are expressed as fold-change with respect to the target tran-script expression in the untreated control.

T-Cell Phenotyping in WT and AhR−/− Mice. Following EAE induction, WT andAhR−/− mice (n = 6 per group) were treated with vehicle or laquinimod for15 d. Spleens were removed under sterile conditions, crushed in PBS tocreate a suspension, and lysed with ammonium-chloride-potassium (ACK)lysis buffer, and the cell pellet was resuspended in FACS buffer or cellculture medium. Cells (1 × 106) were stained for CD4+CD25+Foxp3+ usingthe eBioscience mouse regulatory T-cell staining kit according to themanufacturer’s instructions. The percentage of FoxP3+ cells was calculated

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from within the CD4+CD25+ lymphocyte gate. The remaining cells wereresuspended in Stimulation Medium (RPMI; 2.5% FCS, Antibiotics,L-glutamine and β−mercaptoethanol) and seeded at 0.5 × 106 cells perwell in a 96-well, flat-bottom plate to a final volume of 0.25 mL/well. Cellswere exposed to medium, PPD (2 μg per well), or MOG35–55 (10 μg per well)for 48 h. Cell culture supernatants were then analyzed for cytokine levels

using the R&D mouse magnetic luminex kit LXSAMSM-14 according to themanufacturer’s instructions.

ACKNOWLEDGMENTS. We thank Prof. Markus Kipp of ProMyelo GmbH, whoperformed all of the histology analysis, and Dr. Annalise Di Marco from IRBMScience Park, who performed the AhR induction experiments in hepatocytes.

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