Sphingosine-1-phosphate receptor 1 activation inastrocytes contributes to neuropathic painZhoumou Chena, Timothy M. Doylea, Livio Luongob, Tally M. Largent-Milnesc, Luigino Antonio Giancottia, Grant Kolard,Silvia Squillacea,e, Serena Boccellab, John K. Walkera,f, Alexander Pendletonc, Sarah Spiegelg, William L. Neumannh,Todd W. Vanderahc, and Daniela Salveminia,1

aDepartment of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, MO 63104; bDepartment of Experimental Medicine,Division of Pharmacology, Università della Campania Luigi Vanvitelli, 81100 Naples, Italy; cDepartment of Pharmacology, University of Arizona College ofMedicine, Tucson, AZ 85724; dDepartment of Pathology, Saint Louis University School of Medicine, St. Louis, MO 63104; eDepartment of Physiology andPharmacology Vittorio Erspamer, Sapienza University of Rome, 00185 Rome, Italy; fDepartment of Chemistry, Saint Louis University, St. Louis, MO 63104;gDepartment of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298; and hDepartment ofPharmaceutical Sciences, School of Pharmacy, Southern Illinois University at Edwardsville, Edwardsville, IL 62026

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 28, 2019 (received for review December3, 2018)

Neuropathic pain afflicts millions of individuals and represents amajor health problem for which there is limited effective and safetherapy. Emerging literature links altered sphingolipid metabolismto nociceptive processing. However, the neuropharmacology ofsphingolipid signaling in the central nervous system in the contextof chronic pain remains largely unexplored and controversial. Wenow provide evidence that sphingosine-1-phosphate (S1P) gener-ated in the dorsal horn of the spinal cord in response to nerve injurydrives neuropathic pain by selectively activating the S1P receptorsubtype 1 (S1PR1) in astrocytes. Accordingly, genetic and pharma-cological inhibition of S1PR1 with multiple antagonists in distinctchemical classes, but not agonists, attenuated and even reversedneuropathic pain in rodents of both sexes and in two models oftraumatic nerve injury. These S1PR1 antagonists retained theirability to inhibit neuropathic pain during sustained drug adminis-tration, and their effects were independent of endogenous opioidcircuits. Moreover, mice with astrocyte-specific knockout of S1pr1did not develop neuropathic pain following nerve injury, therebyidentifying astrocytes as the primary cellular substrate of S1PR1activity. On a molecular level, the beneficial reductions in neuro-pathic pain resulting from S1PR1 inhibition were driven by interleu-kin 10 (IL-10), a potent neuroprotective and anti-inflammatorycytokine. Collectively, our results provide fundamental neurobiolog-ical insights that identify the cellular and molecular mechanismsengaged by the S1PR1 axis in neuropathic pain and establishS1PR1 as a target for therapeutic intervention with S1PR1 antago-nists as a class of nonnarcotic analgesics.

sphingosine-1-phosphate | S1P receptor subtype 1 | traumatic nerveinjury-induced neuropathic pain | astrocytes | interleukin 10

Chronic neuropathic pain (1) constitutes a large unmet med-ical need affecting 15–30 million people in the USA with an

annual economic burden of treatment that exceeds $600 billion(2). Neuropathic pain conditions are chronic, severe, debilitating,and exceedingly difficult to treat (3). Opioids are widely used totreat chronic pain but limited by severe side effects and strongabuse liability (4). Continued investigation into the molecularunderpinnings leading to neuropathic pain is essential for theidentification of nonnarcotic-based therapeutic approaches.Altered neuronal sphingolipid metabolism has been linked to

clinically evident neuropathic pain (5, 6). Recent evidence fromanimal studies by our groups and others suggests that neuropathicpain arises from the dysregulation of sphingolipid metabolism inthe dorsal horn of the spinal cord (DH-SC) and increasedsphingosine-1-phosphate (S1P) production (7). Release of S1Pinitiates autocrine or paracrine signaling by activating any of thefive known G protein-coupled S1P receptor subtypes (S1PR1-5)(8). However, the precise role of S1P and the identity and functionof its primary receptor(s) in neuropathic pain are not clear due to

discordant results from studies across models and types of neurop-athies. This discordance presents a substantial barrier to cohesiveunderstanding of the function of S1PR subtypes in neuropathic pain,and thus the development of S1PR subtype-targeted strategies totreat it.Emerging evidence implicates S1PR1 activation in the devel-

opment of chemotherapy (9, 10) and bone cancer-induced pain (11).Moreover, intrathecal administration of a highly selective S1PR1agonist, SEW2871 (12), caused the development of mechano-hypersensitivity in naïve animals (9). These findings suggest thatinhibiting S1PR1 with an antagonist would be beneficial to treatingneuropathic pain. However, studies in models of traumatic nerveinjury have attributed the analgesic effects of the S1PR modulator,FTY720 (Fingolimod) (13), to S1PR1 agonism (14) or combinedS1PR1/S1P3 agonism (15). Several hypotheses have been offered forthese apparently opposing mechanisms of action for S1PR1, in-cluding differences in the neuropathic pain etiology (chemical toxicityversus traumatic injury) (15). However, SEW2871 fails to attenuateneuropathic pain arising from traumatic nerve injury (14, 15) or

Significance

Understanding the cellular and molecular pathways of neuro-pathic pain is fundamental to discovering classes of non-opioidanalgesics. Emerging evidence implicates altered sphingolipidmetabolism and elevated levels of sphingosine-1-phosphate(S1P) in the spinal cord in the persistence of neuropathic pain,but its neuropharmacology and pathways engaged are poorlyunderstood. Using a multidisciplinary approach, we provide evi-dence that S1P drives neuropathic pain by selectively activatingthe S1P receptor subtype 1 (S1PR1) in astrocytes. Inhibiting S1PR1attenuated and reversed neuropathic pain by engaging thepotent anti-inflammatory cytokine, interleukin 10. Our find-ings represent a paradigm shift in the understanding of S1PR1signaling that has implications on the future developmentof S1PR1 antagonists as a promising class of nonnarcoticanalgesics.

Author contributions: T.W.V. and D.S. designed research; Z.C., T.M.D., L.L., T.M.L.-M.,L.A.G., G.K., S. Squillace, S.B., and A.P. performed research; J.K.W. and W.L.N. contributednew reagents/analytic tools; T.M.D., L.L., and T.M.L.-M. analyzed data; and T.M.D., L.L.,S. Spiegel, T.W.V., and D.S. wrote the paper.

Conflict of interest statement: D.S. is a cofounder of BioIntervene, Inc. that licensed re-lated intellectual property from Saint Louis University.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: daniela.salvemini@health.slu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1820466116/-/DCSupplemental.

Published online May 8, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1820466116 PNAS | May 21, 2019 | vol. 116 | no. 21 | 10557–10562

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chemotherapy-induced neuropathic pain (9). A recent study hashinted at the possibility of functional S1PR1 antagonism in the anti-allodynic effects of FTY720 following traumatic nerve injury (16), butselective S1PR1 antagonists and the cell substrate of this S1PR1 ac-tivity were not investigated.Targeting S1PR1 has clear benefits in treating neuropathic

pain. However, resolving the mechanism of action of agents, suchas FTY720, that target S1PR1 and identifying their cell substrateis crucial to understanding how S1P signaling via S1PR1 drivesneuropathic pain. This could have immediate impact on thedevelopment of appropriate analgesic therapies that targetS1PR1. For example, FTY720 is FDA approved for the treat-ment of relapsing–remitting multiple sclerosis (MS) and has agood safety profile (13), and second generation functional an-tagonists are in advanced clinical trials for other indications (17).Moreover, highly selective S1PR1 antagonists are now in ad-vanced preclinical studies for clinical application in various dis-ease states (17).Using a multidisciplinary genetic and pharmacological approach

with multiple S1PR1 antagonists and agonists across traumatic nerveinjury models, species, sexes, and laboratories, we present evidenceunequivocally establishing that S1P activation of S1PR1 signaling inastrocytes is required for the development and maintenance oftraumatic nerve injury-induced neuropathic pain. Our findings pro-vide foundational evidence to support the development of S1PR1antagonists rather than agonists as a class of nonnarcotic analgesics.

ResultsInhibiting S1PR1 Attenuates and Reverses Neuropathic Pain States.Several competitive and functional S1PR1 antagonists wereevaluated in two well-characterized rodent models of neuro-pathic pain: constriction of the sciatic nerve (CCI) (18) andspared nerve injury (SNI) (19). The competitive S1PR1 an-tagonists evaluated were NIBR-15 (20) and TASP0277308 (21).The functional S1PR1 antagonists evaluated included FTY720and the second generation S1PR1 modulators siponimod,ponesimod, and KRP-203, which are in advanced clinical trialsfor a variety of indications, including MS, psoriasis, and ar-thritis (17). Functional S1PR1 antagonists are typically S1PRagonists that cause the internalization and depletion of S1PR1at the plasma membrane to reduce extracellular S1PR1 sig-naling (13). For example, FTY720, when phosphorylated bysphingosine kinase 2 to its active counterpart, FTY720-P, actsas an S1P agonist at all S1PRs, except S1PR2 (13), but whenbound to S1PR1, FTY720 causes sustained depletion of thereceptor at the plasma membrane (13). The chemical structuresand respective S1PR activities for each agent used are docu-mented in SI Appendix, Table S1.Daily oral administration of either TASP0277308 or FTY720

attenuated the development of mechano-allodynia in male micewith CCI (Fig. 1A). When given at time of peak mechano-allodynia (day 10), these selective and functional S1PR1 antago-nists also reversed mechano-allodynia in male mice (Fig. 1 B–G)in a dose-dependent manner with similar effect in normal free-cycling female mice (SI Appendix, Fig. S1A). The ED50 (dose thatprovides 50% effect) value in male mice for each agent is reportedin SI Appendix, Table S1. Contralateral paw withdrawal was notchanged by any intervention (SI Appendix, Figs. S1B and S2). Thereversal of CCI-induced neuropathic pain by competitive andfunctional S1PR1 antagonists was corroborated by two additionallaboratories using male mice and rats (SI Appendix, Fig. S3).Moreover, the effects of S1PR1 antagonism, as exemplified byNIBR-15, were independent of endogenous opioid pathways. In-deed, intraperitoneal (i.p.) administration of the nonselectiveμ-opioid antagonist, (-)naloxone (22) did not interfere with effectsof NIBR-15 (Fig. 1H).In the mouse SNI model, NIBR-15 reversed mechano-

allodynia in a dose-dependent fashion (SI Appendix, Fig. S4A;

ED50 = 0.61 mg/kg, 95%CI: 0.28–1.4 mg/kg or 1.02 μmol/kg,95%CI 0.46–2.3 μmol/kg). Similar effects were reached withthree additional S1PR1 selective and functional antagonists (SIAppendix, Fig. S4 B–D).The pharmacological mode of action of the functional

S1PR1 antagonists stands in stark contrast to its endogenousligands (i.e., S1P) or SEW2871; both of which allow S1PR1 torecycle back to the plasma membrane after its internalizationand thus do not functionally antagonize S1PR1 (23). Accord-ingly, S1PR1 activation with i.p. SEW2871 (20 mg/kg) did notattenuate the development of CCI-induced mechano-allodyniain male mice (Fig. 1A) nor reverse established mechano-allodynia induced by CCI (Fig. 1I) or SNI in male mice (SIAppendix, Fig. S4E). However, significant lymphopenia was ob-served at these doses by day 7, confirming previous studies (12, 24)and lethargy was noted by day 1 post dosing that continued throughthe dosing period.

Fig. 1. S1PR1 antagonism, but not agonism, attenuated and reversedneuropathic pain states. (A) In mice with CCI, administration of TASP0277308(3 mg/kg, ▲; n = 5) or FTY720 (1 mg/kg, ▼; n = 5), but not SEW2871 (20mg/kg, ■; n = 5) or their vehicle (●; n = 5), attenuated the development ofmechano-allodynia. (B–G) In mice with established neuropathic pain,mechano-allodynia was reversed by an acute oral administration (p.o.)on day 10 post CCI of selective S1PR1 antagonists (B) TASP0277308 (0.3, ●;0.6, ■; 1, ▲ and 3 mg/kg, ▼; n = 6/group) or (C) NIBR-15 (0.3,●; 0.6,■; 1,▲and 3 mg/kg,▼; n = 6/group) or S1PR1 functional antagonists (D) FTY720 (0.3,●; 0.6,■; 1,▲ and 3 mg/kg,▼; n = 6/group), (E) siponimod (0.5, ●; 1.5,■; 5,▲ and 15 mg/kg, ▼; n = 5/group), (F ) ponesimod (0.3, ●; 0.6, ■; 1, ▲ and3 mg/kg,▼; n = 6/group) or (G) KRP-203 (0.4,●; 1.3,■ and 4 mg/kg,▲; n = 4/group). (H) In mice, an acute oral administration of NIBR-15 (3 mg/kg) on day10 post CCI reversed mechano-allodynia in the ipsilateral paw. This reversalwas not prevented (P = 0.79) by naloxone (8 mg/kg; i.p.; n = 5) given 30 minbefore NIBR-15 (n = 5). (I) CCI-induced mechano-allodynia (○; n = 5) was notattenuated by SEW2871 (20 mg/kg i.p., ●; n = 5). Data are expressed asmean ± SEM for (n) male mice and analyzed by (A–G and I) two-tailed,two-way ANOVA with Bonferroni comparisons or (H) two-tailed, Welch’scorrected Student’s t test. *P < 0.05 vs. d 0, #P < 0.05 vs. Vehicle and †P <0.05 vs. d 10.

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The Spinal Cord Is a Primary Site of Action of S1PR1 Antagonists. TheDH-SC is a prominent center for the modulation of nociceptiveprocessing (25). S1P production and S1PR1 expression increasedin the DH-SC of male rats with CCI-induced neuropathic pain(Fig. 2A). Intrathecal administration of S1PR1-targeting siRNAattenuated CCI-induced expression of S1PR1 in the ipsilateralDH-SC (Fig. 2B) and significantly reversed mechano-allodynia(Fig. 2C). S1PR1-targeting and nontargeting siRNA had no ef-fects on contralateral paw behavior (SI Appendix, Fig. S5A).These results were validated by intrathecal delivery of S1PR1antagonists that blocked and reversed CCI-induced neuropathicpain (Fig. 2 D and E). In contrast, intrathecal administration ofSEW2871 did not attenuate or reverse CCI-induced neuropathicpain in male mice (Fig. 2 D and F). No effects on contralateralpaw behaviors were found (SI Appendix, Fig. S5 B–D).

Modulation of Neuroinflammation in Spinal Cord by S1PR1 Antagonists;Unraveling the Functional Contribution of Interleukin 10 (IL-10). Sig-nificant increases in the levels of the inflammatory and neuro-excitatory cytokine interleukin-1β (IL-1β) were found in the ratDH-SC ipsilateral to CCI at time of peak pain (Fig. 3A). InhibitingS1PR1 with NIBR-15 (used as a prototype) blocked IL-1β andenhanced IL-10 levels (Fig. 3 A and B). Moreover, the beneficialeffects exerted by oral administration of other functional andcompetitive S1PR1 antagonists were also associated with significant

increases in the levels of IL-10 (Fig. 3B). Noteworthy, pharmaco-logical and genetic approaches revealed that this increase in IL-10was functionally relevant to the mechanism of action of S1PR1inhibition. On day 7 post CCI, male rats received continuous s.c.infusion of NIBR-15, FTY720, or their vehicle delivered over 7d by an osmotic minipump. Continuous treatment with NIBR-15or FTY720 reversed mechano-allodynia throughout the entireperiod of the infusion (Fig. 3C), suggesting the lack of anti-allodynic tolerance. Intrathecal injection of a neutralizing IL-10antibody, but not IgG control, on days 8, 13, and 15 reversedthe anti-allodynic effects of NIBR-15 or FTY720, with peak effectsreached at 1 h post injection (Fig. 3C). No effects were observed inthe contralateral paw (SI Appendix, Fig. S6A). Moreover, com-pared with age- and sex-matched wild-type mice, S1PR1 antago-nists lost their ability to reverse mechano-allodynia in male IL-10knockout (IL-10KO) mice (Fig. 3D). The anti-allodynic responsesto morphine, however, were not altered in IL-10KO mice com-pared with control mice (Fig. 3E), which confirms that IL-10KOmice could respond to IL-10-independent antinociceptive

Fig. 2. S1PR1 in the spinal cord mediates the development and mainte-nance of neuropathic pain. (A) The levels of S1P in the spinal cord are in-creased by D7 in the DH-SC of rats with CCI (n = 5/group). (B) On D14 andD15 post CCI (peak pain), rats were treated with an intrathecal (i.th.) dose ofnontargeting control siRNA (siNT; 2 μg; n = 4) or S1PR1-targeting siRNA(siS1PR1; 2 μg; n = 5). When measured on D16, S1PR1 expression was in-creased in the DH-SC ipsilateral to CCI compared with the contralateral sideof rats treated with siNT, but not those treated with siS1PR1. (C) In male rats,intrathecal (i.th.) administration of S1PR1-targeting siRNA (siS1PR1; 2 μg)on days 7 and 8 post CCI attenuated neuropathic pain (n = 5) compared withrats receiving nontargeting control siRNA (siNT; n = 5). (D) Compared withvehicle (n = 8), daily i.th. administration of FTY720 (3 nmol, n = 6) orTASP0277308 (10 nmol, n = 6), but not SEW2871 (2 nmol/d, n = 5), preventedthe development of CCI-induced mechano-allodynia in male rats. (E) Acutei.th. administration of NIBR-15 (3 nmol, n = 6) or FTY720 (3 nmol, n = 6), butnot vehicle (n = 6), on day 7 reversed CCI-induced mechano-allodynia in rats.(F) Acute intrathecal administration of SEW2871 (2 nmol; n = 5) or its vehicle(n = 5) on D7 post CCI did not reverse mechano-allodynia in male mice. Dataare expressed as mean ± SEM for (n) male rats and analyzed by two-tailed,Welch’s corrected Student’s t test (A and B) or two-tailed, two-way ANOVAwith Bonferroni comparisons (C–F). *P < 0.05 vs. d 0; #P < 0.05 vs. siNT and†P < 0.05 vs. d 7.

Fig. 3. The beneficial effects of S1PR1 antagonist are dependent on IL-10signaling. (A) At peak mechano-allodynia (day 7), IL-1β levels increased in thespinal cord of rats and was attenuated 1 h after oral administration (p.o.) ofNIBR-15 (3 mg/kg; n = 5/group). (B) IL-10 expression the spinal cord of ratsdid not significantly change from baseline levels at peak mechano-allodynia(day 7), but increased 1 h after administration of NIBR-15, TASP0277308(TASP), FTY720, or ponesimod (3 mg/kg; p.o.; n = 6/group). (C) s.c. infusion ofNIBR-15 (0.3 mg/kg/d, 7 d; n = 4) or FTY720 (0.3 mg/kg/d, 7 d; n = 5) in ratsreversed mechano-allodynia. Intrathecal (i.th.) delivery of an anti-IL-10 an-tibody (αIL-10; 0.2 μg in 5 μl) on days 8, 13, and 15 attenuated the anti-allodynic effects of NIBR-15 (n = 5) and FTY720 (n = 5); nonspecific IgG(n = 5) had no effect on mechano-allodynia. (D and E) CCI producedmechano-allodynia in ipsilateral hind paws of IL-10KO (n = 5) mice and wild-type cohorts (WT; n = 5) by day 7. (D) Acute administration of TASP0277308(TASP; 3 mg/kg, i.p; n = 5) or FTY720 (3 mg/kg, i.p; n = 5) on day 7 rapidlyreversed CCI-induced hypersensitivity in WT mice but was ineffective in IL-10KO mice. (E) In contrast, morphine (1 mg/kg; s.c.; n = 5) analgesic effectswere similar in both mouse genotypes. (F and G) S1PR1 antagonist attenu-ates S1PR1-induced mechano-allodynia in an IL-10-dependent manner: (F)Mice were given a combined i.th. injection of SEW2871 (2 nmol) with vehicle(Veh) or TASP0277308 (TASP; 30 nmol) and αIL-10 (0.2 μg) or IgG (n = 5/group). (G) Mice were given an i.th. injection of SEW2871 (2 nmol) and atpeak mechano-allodynia (1 h) were given a second i.th. injection of vehicle(Veh) and IgG or TASP0277308 (TASP; 30 nmol) with αIL-10 or IgG (n = 5/group). Data are expressed as mean ± SEM for (n) male rats or mice andanalyzed by one-way ANOVA with Dunnett’s comparisons (A and B) or two-tailed, two-way ANOVA with Bonferroni comparisons (C–G). *P < 0.05 vs.d 0, †P < 0.05 vs. d 7, #P < 0.05 vs. daily baseline (BL), §P < 0.05 vs. 1 h.

Chen et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10559

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agents. No differences were detected in the contralateral pawbehaviors in wild-type or IL-10 knockout mice (SI Appendix,Fig. S6 B and C).We further validated that S1PR1 antagonism drives IL-10-

dependent anti-allodynic effects using our model of S1PR1-induced mechano-allodynia, where the activation of S1PR1 withintrathecal SEW2871 induces mechano-allodynia within 2 h (9).TASP0277308 prevented and reversed SEW2871-inducedmechano-allodynia in male (Fig. 3 F and G) and female (SIAppendix, Fig. S7) mice and the effects of TASP0277308 werelost in the presence of intrathecal IL-10 neutralizing antibody.

Astrocytes Are the Predominant Cellular Substrate for S1PR1 Activity.To investigate whether astrocyte-specific S1PR1 contributes tothe development of neuropathic pain, we used transgenic micewith floxed S1pr1 recombined with a Gfap promoter-driven cregene (S1pr1fl/fl;Gfap-cre) to knockout both S1pr1 alleles inGFAP-positive astrocytes in CNS tissues, including the spinalcord (26). Importantly, this S1pr1 deletion does not occur in thedorsal root ganglia in naïve mice (10) or mice with CCI (SIAppendix, Fig. S8). Compared with age- and sex-matched maleand female control mice (S1pr1fl/fl), traumatic nerve injury-induced neuropathic pain did not develop in astrocyte-specificS1pr1 knockout mice (Fig. 4 A and B). No effects were seen inthe contralateral paw behavior in either genotype of either sex(SI Appendix, Fig. S9 A and B). These results suggest thatastrocyte-specific S1PR1 signaling in the CNS is necessary andsufficient for the development of mechano-allodynia associatedwith nerve injury. The acute nociceptive responses using the tailflick assay in astrocyte-specific S1pr1 knockout mice were similarto control mice (tail flick latency of 5.2 ± 0.2 SEM seconds incontrol mice compared with tail flick latency of 4.6 ± 0.3 SEMseconds in knockout mice, n = 5 Welch’s corrected Student t test,P = 0.17). These results are consistent with our previous findingsthat S1PR1 functional or competitive antagonists have no effectson animal responses to acute nociceptive tests (9).Since inhibiting S1PR1 attenuated neuropathic pain in an IL-

10-dependent manner and astrocyte-specific S1pr1 knockoutmice did not develop neuropathic pain, we hypothesized thatastrocyte-specific S1PR1 exerts a suppressive activity on IL-10production following CCI. When measured at the time of peakpain on day 7, IL-10 transcripts and protein in the spinal cord ofastrocyte-specific S1pr1 knockout and control mice with CCIwere not detectable by PCR or Western blot analyses, thussuggesting that these approaches were not sensitive enough todetect small changes from murine tissues. Accordingly, we testedour hypothesis by examining whether blocking IL-10 signalingwould reinstate the neuropathic pain phenotype in the astrocyte-specific S1pr1 knockout mice. When measured on day 7,astrocyte-specific S1pr1 knockout female mice did not developmechano-allodynia compared with control female mice (Fig.4C). However, astrocyte-specific S1pr1 knockout mice developedsignificant mechano-allodynia after an intrathecal injection ofthe neutralizing IL-10 antibody (Fig. 4C). The neutralizing IL-10antibody had no effect in control mice (Fig. 4C) or in the con-tralateral paws (SI Appendix, Fig. S9C).

DiscussionSeveral important findings have emerged from our studies. Weunequivocally established that activation and not inhibition ofS1PR1 drives and maintains neuropathic pain. Consequently,S1PR1 antagonism and not agonism is required to inhibit thedevelopment of neuropathic pain and to reverse it once estab-lished. It is noteworthy that S1PR1 antagonists did not lose theirbeneficial effects during prolonged use nor did they engage en-dogenous opioid systems, suggesting that targeting S1PR1 isunlikely to cause opioid-like abuse liability.

We also provided a mechanistic insight into the signalingpathways that are engaged upon S1PR1 inhibition by demon-strating that the beneficial effects of S1PR1 antagonists areIL-10 dependent. Neuroinflammation driven by increasedproinflammatory cytokines in the spinal cord, notably IL-1β, iscritical to establishing and sustaining neuropathic pain (27). IL-1β increases neuronal excitability that contributes to central sen-sitization during neuropathic pain by inhibiting IL-10 release (28),enhancing presynaptic glutamate release, and increasing post-synaptic glutamate receptor phosphorylation (27). Increased in-flammatory cytokine levels are modulated by anti-inflammatory/neuroprotective cytokines, such as IL-10 (29), to control the extentof the exaggerated inflammatory response (30, 31). Compellingevidence for the therapeutic efficacy of IL-10 in neuropathic painexists (30). We now show that S1PR1 antagonism inhibited IL-1βproduction and enhanced IL-10 expression in the spinal cord toattenuate neuropathic pain. Our pharmacological and genetic in-hibition of IL-10 also established that IL-10 signaling is necessaryfor the beneficial effects of S1PR1 antagonism. Consistent with ourfindings in our nerve injury models, we found that S1PR1 antag-onists also blocked and reversed S1PR1-induced mechano-allodynia in normal mice, caused by direct intrathecal injectionof SEW287, through a similar IL-10-dependent mechanism.We also defined the cellular hub of S1PR1 activity by dis-

covering that astrocyte-specific S1pr1 knockout mice do not de-velop neuropathic pain, establishing that S1PR1 activation inastrocytes is required and sufficient to drive the behavior of this

Fig. 4. Astrocytes are a primary cellular target of S1PR1 activity in the spinalcord. When measured on day 7, male (A; n = 13) and female (B; n = 13)astrocyte-specific S1pr1 knockout mice (S1pr1fl/fl;Gfap-cre) did not developthe CCI-induced mechano-allodynia observed in ipsilateral paws of their sex-matched controls (S1pr1fl/fl; A, n = 9; B, n = 14). (C) Female control mice (n =4), but not astrocyte-specific S1pr1 knockout (n = 3), mice developedmechano-allodynia by day 7. Intrathecal administration of anti-IL-10 anti-body (αIL-10) on day 7 precipitated mechano-allodynia within 1 h inastrocyte-specific S1pr1 knockout mice. The administration of αIL-10 had noeffect on mechano-allodynia in control mice. (D and E) Working hypothesis:Traumatic nerve injury induces increased S1P levels in the spinal cord fromvarious sources (D). Activation of S1PR1 signaling on astrocytes activatesNLRP3 inflammasome leading to release of IL-1β, which can then dampen IL-10 expression. Attenuation of IL-1β production following S1PR1 antagonismmay remove a brake on IL-10 production and engage IL-10-dependent sig-naling leading to an attenuation of the neuroinflammatory response andreduced neuronal excitability (E). Data are expressed as mean ± SEM for (n)mice and analyzed by two-tailed, two-way ANOVA with Bonferroni com-parisons. *P < 0.05 vs. d 0 and †P < 0.05 vs. SEW2871+Veh+IgG.

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neuropathic pain phenotype. This is consistent with our recentreports that the development of SEW2871-induced mechano-allodynia (32) and bortezomib-induced neuropathic pain (10)are completely lost in astrocyte-specific S1pr1 knockout mice,suggesting that astrocytes are key to transducing the effects ofS1PR1 activation across pain etiologies. Astrocytes also appearto be the predominant cell substrate for IL-10 signaling duringneuroinflammation (33) and we found that blockade of IL-10signaling in the spinal cord was sufficient to restore the neuro-pathic pain phenotype in astrocyte-specific S1pr1 knockout mice.When taken together, these findings suggest that in nerve trauma,astrocyte-specific S1PR1 signaling acts as a “rheostat” that favorsneuroinflammatory pathways at the expense of IL-10 signaling.The molecular mechanisms whereby activation and inhibition

of S1PR1 in astrocytes modulate IL-10 signaling in the spinalcord are not known. We previously reported that functional orcompetitive S1PR1 antagonists do not increase IL-10 levels inspinal cord in noninjured rodents, suggesting that IL-10 signalingis engaged in response to S1PR1 inhibition only in neuro-inflammatory settings (9). However, we recently showed thatSEW2871-induced mechano-allodynia was driven by IL-1β for-mation through posttranslational processing by the leucine richrepeat and pyrin domain containing 3 (NLRP3) inflammasome,which was lost in mice with astrocyte-specific S1pr1 deletions(32). This suggests that astrocyte-specific S1PR1 signaling in-duces NLRP3-IL-1β activation (32). We therefore propose amodel whereby traumatic nerve injury increases the formation ofS1P within the spinal cord to activate S1PR1 in astrocytes toproduce IL-1β, which can dampen IL-10 formation (Fig. 4D).S1PR1 antagonism by blocking IL-1β production removes abrake on astrocyte production of IL-10 and guides these cellstoward an anti-inflammatory phenotype (Fig. 4E). In support, arecent in vitro study showed that FTY720 attenuated the pro-duction of LPS-induced proinflammatory mediators (e.g., IL-1β)in astrocytes, which was associated with increased production ofIL-10, while having little effect on LPS-activated microglia (34).In turn, IL-10 signaling is anticipated to attenuate neuro-inflammation in spinal cord and reduced neuronal excitabilitythrough several mechanisms, including limiting the neuro-excitatory effects of IL-1β (35, 36) (Fig. 4E).Finally, our findings help crystallize our understanding of

S1PR1 signaling in neuropathic pain. The pharmacological profileof S1PR1 modulators (i.e., FTY720, siponimod) tested in thisstudy was similar to that of competitive S1PR1 antagonists, but incomplete contrast to that of pure S1PR1 agonists (SEW2871),identifying functional S1PR1 antagonism as a major mechanism ofaction of these S1PR1 modulators. In light of our findings, thevarying reported effects of S1PR1 modulators across neuropathicpain models are likely due to the current limitations of S1PR1-targeted tools, approaches, or models, rather than the type of paininvoked. For example, in an earlier study, it was proposed that thebeneficial effects of FTY720 in a mouse SNI model were due tocombined S1PR1/S1P3 agonism (15). However, this pharmaco-logical characterization was limited by a lack of dose–responsestudies, use of only one S1PR1 or S1PR3 antagonist at high doses,the absence of genetic approaches to validate pharmacologicalresults, and a very narrow behavioral response range. Here, wefound that FTY720 exerted beneficial effects in traumatic nerveinjury with ED50 values similar to those obtained with competitiveS1PR1 antagonists. Moreover, the ED50 values for two S1PR3-sparing S1PR1 agonists, siponimod and KRP-203, were similar toFTY720 and other S1PR1 modulators that possess limited S1PR3activity, as well as competitive S1PR1 antagonists. These resultssuggest that engineering S1PR3 activity out of the molecule haslittle impact on the ability of the drug to reverse mechano-allodynia (14, 37) and argue against the prerequisite role forS1PR3 activation in the beneficial effects of FTY720 (15). Onerecent study in a model of central neuropathic pain reported that

FTY720 attenuated MS-related pain (38), as did systemic deliveryof the S1PR1 agonist SEW2871, but not the S1PR1 antagonistW146. The authors concluded, perhaps prematurely, that S1PR1agonism and not antagonism is required for the beneficial effectsof FTY720. Indeed, numerous studies have demonstrated thatboth S1PR1 antagonists like FTY720 (17, 39) and agonists such asSEW2871 (40) are protective in models of MS. Their protectiveeffects are exerted in part peripherally through lymphopeniawhere these functional S1PR1 antagonists inhibit S1P/S1PR1signaling in lymphocytes (39, 41, 42) and where S1PR1 agonistsactivate endothelial S1PR1 to tighten gap junctions and preventegress of lymphocytes (43). Accordingly, both S1PR1 agonism andantagonism can inhibit lymphocyte migration from lymph nodesinto the spinal cord to prevent or blunt neuroinflammatory pro-cesses associated with MS (44, 45) that could lead to reduced pain.Similar past discrepancies in pharmacological outcomes betweensystemic administration of FTY720 and W146 led to the conclu-sion that FTY720 did not act as a functional S1PR1 antagonist(46). This paradox was resolved by studies showing the discrep-ancies were due to the poor pharmacokinetic properties of W146(47). Subsequent studies with S1PR1 antagonists with improvedproperties confirmed that indeed S1PR1 antagonists mimic thepharmacological effects of S1PR1 agonists on lymphocyte re-distribution in vivo (17). More work is needed to define whetherS1PR1 antagonists are useful in MS-related pain.Collectively, our study suggests a central etiological contribu-

tion of astrocyte S1PR1 signaling in neuropathic pain, providesinsight on how S1PR1 antagonists exert their beneficial effects andsets the foundation to enable therapeutic efforts centered on thedevelopment of S1PR1 antagonists as nonnarcotic analgesics.

MethodsStudy Approval. All experiments were performed in accordance with theInternational Association for the Study of Pain, the National Institutes ofHealth, and the European Community (E.C. L358/1 18/12/86) guidelines onlaboratory animal welfare and approved by the Saint Louis University In-stitutional Animal Care, Animal Ethics Committee of The Second University ofNaples (Naples), and University of Arizona Animal Care and Use Committee.

Experimental Animals. Male Sprague–Dawley rats (200–220 g) or male andfemale CD1 mice (20–30g) from Envigo were housed 2–4 per cage (for rats)and 5 per cage (for mice) in a controlled environment (12 h light/dark cycle)with food and water available ad libitum. All animals were randomly assignedto experimental groups based on sex and genotype. All experimenters wereblinded to treatment conditions and genotype. Data were unblinded duringdata analysis.Astrocyte-specific S1pr1 knockout mice. Astrocyte-specific S1pr1 knockoutmouse colonies are descendants of original homozygous S1pr1fl/fl,Gfap-Crebreeder mice kindly gifted to us by Jerold Chun, The Scripps Research In-stitute, La Jolla, CA (26). Littermates were used in all experiments. These micehave been morphologically and functionally characterized and do not haveany reported abnormal developmental issues (26). S1pr1 knockout and controlmice had similar weight gain, general behavior, motor skills, and reproductionrates. All mice were bred, genotyped, and ear tagged for identification asdescribed previously (10).IL-10 knockout mice. IL-10 knockout mice (B6.129P2-IL-10tm1Cgn/J) and wild-type control mice (C57BL/6) were obtained from The Jackson Laboratory. Allsurgical approaches and experiments using IL-10 knockout mice followedsterile laboratory techniques in accordance with the Saint Louis UniversityAnimal Care Committee.

Defining Estrous Cycle Stage.Given the multiday nature of the design in freelycycling females, a vaginal smear was taken after the last behavioral timepoint and stage of estrous defined by cytology as described (48). All animalsdisplayed a normal 4–5 d estrous cycle.

Rodent Models of Traumatic Nerve Injury-Induced Neuropathic Pain. Two well-characterized rodent models were used for our studies: chronic constrictioninjury (CCI) to the sciatic nerve (18), and spared nerve injury (SNI) or shamsurgery was performed on mice according to the method of Decosterd andWoolf (19).

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Test Compounds. Fingolimod (FTY720) and SEW2871 were purchased fromCayman Chemical. NIBR-15 was synthesized by us according to the publishedroute (20). According to methods previously described, ponesimod (49) andTASP0277308 (21) were synthesized by Shanghai ChemPartner Co; whereasKRP-203 (50) was synthesized from Atomax Chemicals. Siponimod (BAF312)(51) was synthesized from MedChemExpress LLC. Certificate of analysisconfirmed purity of each test agent of >95%. Morphine sulfate was a kind giftfrom Mallinckrodt Pharmaceuticals. Sheep anti-rat IL-10 IgG antibody was agenerous gift from Linda Watkins, University of Colorado Boulder, Boulder,CO. Control sheep serum IgG was obtained from Sigma Aldrich. All solutions ofagents were made fresh each day immediately before administration.

Behavioral Testing. Mechano-allodynia was determined by probing theplantar aspect of the hind pawwith calibrated von Frey filaments (mice: 0.04–2.00 g; rats: 1.4–26 g; Stoelting) according to the “up-and-down” method(52) and calculating the paw withdrawal threshold (PWT, g), as previously

described (53). Acute thermal anti-nociception was measured using the tailflick latency test (54), which measures the withdrawal latency of the tailfrom a noxious radiant heat source with baseline latencies of 2–3 s and acutoff time of 10 s to prevent tissue injury.

Additional details for all methods can be found in SI Appendix.

ACKNOWLEDGMENTS. We thank Drs. R. Samson and G. Yosten (Saint LouisUniversity) for their assistance with the vaginal smear assays and Dr. A. J.Lechner (Saint Louis University) for his valuable input and careful editorialreview of our work. This study was funded by Saint Louis University startupfunds (to D.S. and J.K.W.), NIH National Institute of General Medical SciencesGrant R01GM043880 (to S. Spiegel), Southern Illinois University EdwardsvilleSchool of Pharmacy Research Grant (to W.L.N.), University of Arizona Startupfunds (to T.W.V.), and by the Washington University Institute of Clinical andTranslational Sciences, which is, in part, supported by the NIH/National Centerfor Advancing Translational Sciences (NCATS), Clinical and Translational Sci-ence Awards Grant UL1 TR002345 (to D.S.).

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