Sphingosine-1-phosphate receptor 1 activation in contributes to neuropathic pain

Zhoumou 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 of Medicine, Tucson, AZ 85724; dDepartment of Pathology, Saint Louis University School of Medicine, St. Louis, MO 63104; eDepartment of Physiology and Pharmacology 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 of Pharmaceutical 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 December 3, 2018) Neuropathic pain afflicts millions of individuals and represents a discordant results from studies across models and types of neurop- major health problem for which there is limited effective and safe athies. This discordance presents a substantial barrier to cohesive therapy. Emerging literature links altered understanding of the function of S1PR subtypes in neuropathic pain, to nociceptive processing. However, the neuropharmacology of and thus the development of S1PR subtype-targeted strategies to sphingolipid signaling in the central nervous system in the context treat it. of chronic pain remains largely unexplored and controversial. We Emerging evidence implicates S1PR1 activation in the devel- now provide evidence that sphingosine-1-phosphate (S1P) gener- opment of chemotherapy (9, 10) and bone cancer-induced pain (11). ated in the dorsal horn of the spinal cord in response to nerve injury Moreover, intrathecal administration of a highly selective S1PR1 drives neuropathic pain by selectively activating the S1P receptor agonist, SEW2871 (12), caused the development of mechano- subtype 1 (S1PR1) in astrocytes. Accordingly, genetic and pharma- hypersensitivity in naïve animals (9). These findings suggest that cological inhibition of S1PR1 with multiple antagonists in distinct inhibiting S1PR1 with an antagonist would be beneficial to treating PHARMACOLOGY chemical classes, but not agonists, attenuated and even reversed neuropathic pain. However, studies in models of traumatic nerve neuropathic pain in rodents of both sexes and in two models of injury have attributed the analgesic effects of the S1PR modulator, traumatic nerve injury. These S1PR1 antagonists retained their FTY720 () (13), to S1PR1 agonism (14) or combined ability to inhibit neuropathic pain during sustained drug adminis- tration, and their effects were independent of endogenous opioid S1PR1/S1P3 agonism (15). Several hypotheses have been offered for circuits. Moreover, mice with -specific knockout of S1pr1 these apparently opposing mechanisms of action for S1PR1, in- did not develop neuropathic pain following nerve injury, thereby cluding differences in the neuropathic pain etiology (chemical toxicity identifying astrocytes as the primary cellular substrate of S1PR1 versus traumatic injury) (15). However, SEW2871 fails to attenuate activity. On a molecular level, the beneficial reductions in neuro- neuropathic pain arising from traumatic nerve injury (14, 15) or pathic pain resulting from S1PR1 inhibition were driven by interleu- kin 10 (IL-10), a potent neuroprotective and anti-inflammatory Significance cytokine. Collectively, our results provide fundamental neurobiolog- ical insights that identify the cellular and molecular mechanisms Understanding the cellular and molecular pathways of neuro- engaged by the S1PR1 axis in neuropathic pain and establish pathic pain is fundamental to discovering classes of non-opioid S1PR1 as a target for therapeutic intervention with S1PR1 antago- analgesics. Emerging evidence implicates altered sphingolipid nists as a class of nonnarcotic analgesics. metabolism and elevated levels of sphingosine-1-phosphate (S1P) in the spinal cord in the persistence of neuropathic pain, sphingosine-1-phosphate | S1P receptor subtype 1 | traumatic nerve but its neuropharmacology and pathways engaged are poorly injury-induced neuropathic pain | astrocytes | interleukin 10 understood. Using a multidisciplinary approach, we provide evi- dence that S1P drives neuropathic pain by selectively activating hronic neuropathic pain (1) constitutes a large unmet med- the S1P receptor subtype 1 (S1PR1) in astrocytes. Inhibiting S1PR1 Cical need affecting 15–30 million people in the USA with an attenuated and reversed neuropathic pain by engaging the annual economic burden of treatment that exceeds $600 billion potent anti-inflammatory cytokine, interleukin 10. Our find- (2). Neuropathic pain conditions are chronic, severe, debilitating, ings represent a paradigm shift in the understanding of S1PR1 and exceedingly difficult to treat (3). Opioids are widely used to signaling that has implications on the future development treat chronic pain but limited by severe side effects and strong of S1PR1 antagonists as a promising class of nonnarcotic abuse liability (4). Continued investigation into the molecular analgesics. underpinnings leading to neuropathic pain is essential for the Author contributions: T.W.V. and D.S. designed research; Z.C., T.M.D., L.L., T.M.L.-M., identification of nonnarcotic-based therapeutic approaches. L.A.G., G.K., S. Squillace, S.B., and A.P. performed research; J.K.W. and W.L.N. contributed Altered neuronal sphingolipid metabolism has been linked to new reagents/analytic tools; T.M.D., L.L., and T.M.L.-M. analyzed data; and T.M.D., L.L., clinically evident neuropathic pain (5, 6). Recent evidence from S. Spiegel, T.W.V., and D.S. wrote the paper. animal studies by our groups and others suggests that neuropathic Conflict of interest statement: D.S. is a cofounder of BioIntervene, Inc. that licensed re- pain arises from the dysregulation of sphingolipid metabolism in lated intellectual property from Saint Louis University. the dorsal horn of the spinal cord (DH-SC) and increased This article is a PNAS Direct Submission. sphingosine-1-phosphate (S1P) production (7). Release of S1P Published under the PNAS license. initiates autocrine or paracrine signaling by activating any of the 1To whom correspondence should be addressed. Email: [email protected]. five known G -coupled S1P receptor subtypes (S1PR1-5) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (8). However, the precise role of S1P and the identity and function 1073/pnas.1820466116/-/DCSupplemental. of its primary receptor(s) in neuropathic pain are not clear due to Published online May 8, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1820466116 PNAS | May 21, 2019 | vol. 116 | no. 21 | 10557–10562 Downloaded by guest on October 1, 2021 chemotherapy-induced neuropathic pain (9). A recent study has hinted at the possibility of functional S1PR1 antagonism in the anti- allodynic effects of FTY720 following traumatic nerve injury (16), but selective 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, such as FTY720, that target S1PR1 and identifying their cell substrate is crucial to understanding how S1P signaling via S1PR1 drives neuropathic pain. This could have immediate impact on the development of appropriate analgesic therapies that target S1PR1. For example, FTY720 is FDA approved for the treat- ment of relapsing–remitting (MS) and has a good 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 nerve injurymodels,species,sexes,andlaboratories, we present evidence unequivocally establishing that S1P activation of S1PR1 signaling in astrocytes is required for the development and maintenance of traumatic nerve injury-induced neuropathic pain. Our findings pro- vide foundational evidence to support the development of S1PR1 antagonistsratherthanagonistsasaclassofnonnarcoticanalgesics. Results Inhibiting S1PR1 Attenuates and Reverses Neuropathic Pain States. Several competitive and functional S1PR1 antagonists were Fig. 1. S1PR1 antagonism, but not agonism, attenuated and reversed evaluated in two well-characterized rodent models of neuro- neuropathic pain states. (A) In mice with CCI, administration of TASP0277308 pathic pain: constriction of the sciatic nerve (CCI) (18) and (3 mg/kg, ▲; n = 5) or FTY720 (1 mg/kg, ▼; n = 5), but not SEW2871 (20 spared nerve injury (SNI) (19). The competitive S1PR1 an- mg/kg, ■; n = 5) or their vehicle (●; n = 5), attenuated the development of – tagonists evaluated were NIBR-15 (20) and TASP0277308 (21). mechano-allodynia. (B G) In mice with established neuropathic pain, mechano-allodynia was reversed by an acute oral administration (p.o.) The functional S1PR1 antagonists evaluated included FTY720 on day 10 post CCI of selective S1PR1 antagonists (B) TASP0277308 (0.3, ●; and the second generation S1PR1 modulators , 0.6, ■;1,▲ and 3 mg/kg, ▼; n = 6/group) or (C) NIBR-15 (0.3, ●;0.6,■;1,▲ , and KRP-203, which are in advanced clinical trials and 3 mg/kg, ▼; n = 6/group) or S1PR1 functional antagonists (D)FTY720(0.3, for a variety of indications, including MS, psoriasis, and ar- ●;0.6,■;1,▲ and 3 mg/kg, ▼; n = 6/group), (E) siponimod (0.5, ●;1.5,■;5, thritis (17). Functional S1PR1 antagonists are typically S1PR ▲ and 15 mg/kg, ▼; n = 5/group), (F) ponesimod (0.3, ●;0.6,■;1,▲ and agonists that cause the internalization and depletion of S1PR1 3mg/kg,▼; n = 6/group) or (G) KRP-203 (0.4, ●;1.3,■ and 4 mg/kg, ▲; n = 4/ at the plasma membrane to reduce extracellular S1PR1 sig- group). (H) In mice, an acute oral administration of NIBR-15 (3 mg/kg) on day naling (13). For example, FTY720, when phosphorylated by 10 post CCI reversed mechano-allodynia in the ipsilateral paw. This reversal = = sphingosine 2 to its active counterpart, FTY720-P, acts was not prevented (P 0.79) by naloxone (8 mg/kg; i.p.; n 5) given 30 min before NIBR-15 (n = 5). (I) CCI-induced mechano-allodynia (○; n = 5) was not as an S1P agonist at all S1PRs, except S1PR2 (13), but when attenuated by SEW2871 (20 mg/kg i.p., ●; n = 5). Data are expressed as bound to S1PR1, FTY720 causes sustained depletion of the mean ± SEM for (n) male mice and analyzed by (A–G and I) two-tailed, receptorattheplasmamembrane(13).Thechemicalstructures two-way ANOVA with Bonferroni comparisons or (H) two-tailed, Welch’s † and respective S1PR activities for each agent used are docu- corrected Student’s t test. *P < 0.05 vs. d 0, #P < 0.05 vs. Vehicle and P < mented in SI Appendix,TableS1. 0.05 vs. d 10. Daily oral administration of either TASP0277308 or FTY720 attenuated the development of mechano-allodynia in male mice = – μ with CCI (Fig. 1A). When given at time of peak mechano- ED50 0.61 mg/kg, 95%CI: 0.28 1.4 mg/kg or 1.02 mol/kg, allodynia (day 10), these selective and functional S1PR1 antago- 95%CI 0.46–2.3 μmol/kg). Similar effects were reached with nists also reversed mechano-allodynia in male mice (Fig. 1 B–G) three additional S1PR1 selective and functional antagonists (SI in a dose-dependent manner with similar effect in normal free- Appendix, Fig. S4 B–D). cycling female mice (SI Appendix,Fig.S1A). The ED50 (dose that The pharmacological mode of action of the functional provides 50% effect) value in male mice for each agent is reported S1PR1 antagonists stands in stark contrast to its endogenous in SI Appendix, Table S1. Contralateral paw withdrawal was not ligands (i.e., S1P) or SEW2871; both of which allow S1PR1 to SI Appendix B changed by any intervention ( , Figs. S1 and S2). The recycle back to the plasma membrane after its internalization reversal of CCI-induced neuropathic pain by competitive and and thus do not functionally antagonize S1PR1 (23). Accord- functional S1PR1 antagonists was corroborated by two additional ingly, S1PR1 activation with i.p. SEW2871 (20 mg/kg) did not laboratories using male mice and rats (SI Appendix,Fig.S3). attenuate the development of CCI-induced mechano-allodynia Moreover, the effects of S1PR1 antagonism, as exemplified by A NIBR-15, were independent of endogenous opioid pathways. In- in male mice (Fig. 1 ) nor reverse established mechano- I SI deed, intraperitoneal (i.p.) administration of the nonselective allodynia induced by CCI (Fig. 1 ) or SNI in male mice ( Appendix E μ-opioid antagonist, (-)naloxone (22) did not interfere with effects , Fig. S4 ). However, significant lymphopenia was ob- of NIBR-15 (Fig. 1H). served at these doses by day 7, confirming previous studies (12, 24) In the mouse SNI model, NIBR-15 reversed mechano- and lethargy was noted by day 1 post dosing that continued through allodynia in a dose-dependent fashion (SI Appendix, Fig. S4A; the dosing period.

10558 | www.pnas.org/cgi/doi/10.1073/pnas.1820466116 Chen et al. Downloaded by guest on October 1, 2021 The Spinal Cord Is a Primary Site of Action of S1PR1 Antagonists. The DH-SC is a prominent center for the modulation of nociceptive processing (25). S1P production and S1PR1 expression increased in the DH-SC of male rats with CCI-induced neuropathic pain (Fig. 2A). Intrathecal administration of S1PR1-targeting siRNA attenuated CCI-induced expression of S1PR1 in the ipsilateral DH-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 S1PR1 antagonists that blocked and reversed CCI-induced neuropathic pain (Fig. 2 D and E). In contrast, intrathecal administration of SEW2871 did not attenuate or reverse CCI-induced neuropathic pain in male mice (Fig. 2 D and F). No effects on contralateral paw 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 rat DH-SC ipsilateral to CCI at time of peak pain (Fig. 3A). Inhibiting S1PR1 with NIBR-15 (used as a prototype) blocked IL-1β and Fig. 3. The beneficial effects of S1PR1 antagonist are dependent on IL-10 enhanced IL-10 levels (Fig. 3 A and B). Moreover, the beneficial signaling. (A) At peak mechano-allodynia (day 7), IL-1β levels increased in the effects exerted by oral administration of other functional and spinal cord of rats and was attenuated 1 h after oral administration (p.o.) of = competitive S1PR1 antagonists were also associated with significant NIBR-15 (3 mg/kg; n 5/group). (B) IL-10 expression the spinal cord of rats did 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 of PHARMACOLOGY NIBR-15 (0.3 mg/kg/d, 7 d; n = 4) or FTY720 (0.3 mg/kg/d, 7 d; n = 5) in rats reversed mechano-allodynia. Intrathecal (i.th.) delivery of an anti-IL-10 an- tibody (αIL-10; 0.2 μgin5μl) 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 produced mechano-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 rapidly reversed 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 effects were 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 at peak 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 and analyzed 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. † d0, P < 0.05 vs. d 7, #P < 0.05 vs. daily baseline (BL), §P < 0.05 vs. 1 h.

Fig. 2. S1PR1 in the spinal cord mediates the development and mainte- increases in the levels of IL-10 (Fig. 3B). Noteworthy, pharmaco- 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 and logical and genetic approaches revealed that this increase in IL-10 D15 post CCI (peak pain), rats were treated with an intrathecal (i.th.) dose of was functionally relevant to the mechanism of action of S1PR1 nontargeting control siRNA (siNT; 2 μg; n = 4) or S1PR1-targeting siRNA inhibition. On day 7 post CCI, male rats received continuous s.c. (siS1PR1; 2 μg; n = 5). When measured on D16, S1PR1 expression was in- infusion of NIBR-15, FTY720, or their vehicle delivered over 7 creased in the DH-SC ipsilateral to CCI compared with the contralateral side d by an osmotic minipump. Continuous treatment with NIBR-15 of rats treated with siNT, but not those treated with siS1PR1. (C) In male rats, or FTY720 reversed mechano-allodynia throughout the entire μ intrathecal (i.th.) administration of S1PR1-targeting siRNA (siS1PR1; 2 g) period of the infusion (Fig. 3C), suggesting the lack of anti- on days 7 and 8 post CCI attenuated neuropathic pain (n = 5) compared with rats receiving nontargeting control siRNA (siNT; n = 5). (D) Compared with allodynic tolerance. Intrathecal injection of a neutralizing IL-10 vehicle (n = 8), daily i.th. administration of FTY720 (3 nmol, n = 6) or antibody, but not IgG control, on days 8, 13, and 15 reversed TASP0277308 (10 nmol, n = 6), but not SEW2871 (2 nmol/d, n = 5), prevented the anti-allodynic effects of NIBR-15 or FTY720, with peak effects the development of CCI-induced mechano-allodynia in male rats. (E) Acute reached at 1 h post injection (Fig. 3C). No effects were observed in i.th. administration of NIBR-15 (3 nmol, n = 6) or FTY720 (3 nmol, n = 6), but the contralateral paw (SI Appendix,Fig.S6A). Moreover, com- not vehicle (n = 6), on day 7 reversed CCI-induced mechano-allodynia in rats. pared with age- and sex-matched wild-type mice, S1PR1 antago- = (F) Acute intrathecal administration of SEW2871 (2 nmol; n 5) or its vehicle nists lost their ability to reverse mechano-allodynia in male IL-10 (n = 5) on D7 post CCI did not reverse mechano-allodynia in male mice. Data D are expressed as mean ± SEM for (n) male rats and analyzed by two-tailed, knockout (IL-10KO) mice (Fig. 3 ). The anti-allodynic responses Welch’s corrected Student’s t test (A and B) or two-tailed, two-way ANOVA to morphine, however, were not altered in IL-10KO mice com- with Bonferroni comparisons (C–F). *P < 0.05 vs. d 0; #P < 0.05 vs. siNT and paredwithcontrolmice(Fig.3E), which confirms that IL-10KO † P < 0.05 vs. d 7. mice could respond to IL-10-independent antinociceptive

Chen et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10559 Downloaded by guest on October 1, 2021 agents. No differences were detected in the contralateral paw behaviors 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 with intrathecal SEW2871 induces mechano-allodynia within 2 h (9). TASP0277308 prevented and reversed SEW2871-induced mechano-allodynia in male (Fig. 3 F and G) and female (SI Appendix, Fig. S7) mice and the effects of TASP0277308 were lost 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 to the development of neuropathic pain, we used transgenic mice with floxed S1pr1 recombined with a Gfap promoter-driven cre fl/fl (S1pr1 ;Gfap-cre) to knockout both S1pr1 alleles in GFAP-positive astrocytes in CNS tissues, including the spinal cord (26). Importantly, this S1pr1 deletion does not occur in the dorsal root ganglia in naïve mice (10) or mice with CCI (SI Appendix, Fig. S8). Compared with age- and sex-matched male S1pr1fl/fl and female control mice ( ), traumatic nerve injury- Fig. 4. Astrocytes are a primary cellular target of S1PR1 activity in the spinal induced neuropathic pain did not develop in astrocyte-specific cord. When measured on day 7, male (A; n = 13) and female (B; n = 13) S1pr1 knockout mice (Fig. 4 A and B). No effects were seen in astrocyte-specific S1pr1 knockout mice (S1pr1fl/fl;Gfap-cre) did not develop the contralateral paw behavior in either genotype of either sex the CCI-induced mechano-allodynia observed in ipsilateral paws of their sex- (SI Appendix, Fig. S9 A and B). These results suggest that matched controls (S1pr1fl/fl; A, n = 9; B, n = 14). (C) Female control mice (n = astrocyte-specific S1PR1 signaling in the CNS is necessary and 4), but not astrocyte-specific S1pr1 knockout (n = 3), mice developed sufficient for the development of mechano-allodynia associated mechano-allodynia by day 7. Intrathecal administration of anti-IL-10 anti- body (αIL-10) on day 7 precipitated mechano-allodynia within 1 h in with nerve injury. The acute nociceptive responses using the tail α S1pr1 astrocyte-specific S1pr1 knockout mice. The administration of IL-10 had no flick assay in astrocyte-specific knockout mice were similar effect on mechano-allodynia in control mice. (D and E) Working hypothesis: to control mice (tail flick latency of 5.2 ± 0.2 SEM seconds in Traumatic nerve injury induces increased S1P levels in the spinal cord from control mice compared with tail flick latency of 4.6 ± 0.3 SEM various sources (D). Activation of S1PR1 signaling on astrocytes activates seconds in knockout mice, n = 5 Welch’s corrected Student t test, NLRP3 inflammasome leading to release of IL-1β, which can then dampen IL- P = 0.17). These results are consistent with our previous findings 10 expression. Attenuation of IL-1β production following S1PR1 antagonism that S1PR1 functional or competitive antagonists have no effects may remove a brake on IL-10 production and engage IL-10-dependent sig- on animal responses to acute nociceptive tests (9). naling leading to an attenuation of the neuroinflammatory response and reduced neuronal excitability (E). Data are expressed as mean ± SEM for (n) Since inhibiting S1PR1 attenuated neuropathic pain in an IL- mice and analyzed by two-tailed, two-way ANOVA with Bonferroni com- S1pr1 † 10-dependent manner and astrocyte-specific knockout parisons. *P < 0.05 vs. d 0 and P < 0.05 vs. SEW2871+Veh+IgG. mice did not develop neuropathic pain, we hypothesized that astrocyte-specific S1PR1 exerts a suppressive activity on IL-10 production following CCI. When measured at the time of peak We also provided a mechanistic insight into the signaling pain on day 7, IL-10 transcripts and protein in the spinal cord of pathways that are engaged upon S1PR1 inhibition by demon- astrocyte-specific S1pr1 knockout and control mice with CCI strating that the beneficial effects of S1PR1 antagonists are were not detectable by PCR or Western blot analyses, thus IL-10 dependent. Neuroinflammation driven by increased suggesting that these approaches were not sensitive enough to proinflammatory cytokines in the spinal cord, notably IL-1β,is detect small changes from murine tissues. Accordingly, we tested critical to establishing and sustaining neuropathic pain (27). IL- our hypothesis by examining whether blocking IL-10 signaling 1β increases neuronal excitability that contributes to central sen- would reinstate the neuropathic pain phenotype in the astrocyte- sitization during neuropathic pain by inhibiting IL-10 release (28), specific S1pr1 knockout mice. When measured on day 7, enhancing presynaptic glutamate release, and increasing post- astrocyte-specific S1pr1 knockout female mice did not develop synaptic phosphorylation (27). Increased in- mechano-allodynia compared with control female mice (Fig. flammatory cytokine levels are modulated by anti-inflammatory/ 4C). However, astrocyte-specific S1pr1 knockout mice developed neuroprotective cytokines, such as IL-10 (29), to control the extent significant mechano-allodynia after an intrathecal injection of of the exaggerated inflammatory response (30, 31). Compelling the neutralizing IL-10 antibody (Fig. 4C). The neutralizing IL-10 evidence for the therapeutic efficacy of IL-10 in neuropathic pain antibody had no effect in control mice (Fig. 4C) or in the con- exists (30). We now show that S1PR1 antagonism inhibited IL-1β tralateral paws (SI Appendix, Fig. S9C). production and enhanced IL-10 expression in the spinal cord to attenuate neuropathic pain. Our pharmacological and genetic in- Discussion hibition of IL-10 also established that IL-10 signaling is necessary Several important findings have emerged from our studies. We for the beneficial effects of S1PR1 antagonism. Consistent with our unequivocally established that activation and not inhibition of findings in our nerve injury models, we found that S1PR1 antag- S1PR1 drives and maintains neuropathic pain. Consequently, onists also blocked and reversed S1PR1-induced mechano- S1PR1 antagonism and not agonism is required to inhibit the allodynia in normal mice, caused by direct intrathecal injection development of neuropathic pain and to reverse it once estab- of SEW287, through a similar IL-10-dependent mechanism. lished. It is noteworthy that S1PR1 antagonists did not lose their We also defined the cellular hub of S1PR1 activity by dis- beneficial effects during prolonged use nor did they engage en- covering that astrocyte-specific S1pr1 knockout mice do not de- dogenous opioid systems, suggesting that targeting S1PR1 is velop neuropathic pain, establishing that S1PR1 activation in unlikely to cause opioid-like abuse liability. astrocytes is required and sufficient to drive the behavior of this

10560 | www.pnas.org/cgi/doi/10.1073/pnas.1820466116 Chen et al. Downloaded by guest on October 1, 2021 neuropathic pain phenotype. This is consistent with our recent FTY720 attenuated MS-related pain (38), as did systemic delivery reports that the development of SEW2871-induced mechano- of the S1PR1 agonist SEW2871, but not the S1PR1 antagonist allodynia (32) and bortezomib-induced neuropathic pain (10) W146. The authors concluded, perhaps prematurely, that S1PR1 are completely lost in astrocyte-specific S1pr1 knockout mice, agonism and not antagonism is required for the beneficial effects suggesting that astrocytes are key to transducing the effects of of FTY720. Indeed, numerous studies have demonstrated that S1PR1 activation across pain etiologies. Astrocytes also appear both S1PR1 antagonists like FTY720 (17, 39) and agonists such as to be the predominant cell substrate for IL-10 signaling during SEW2871 (40) are protective in models of MS. Their protective neuroinflammation (33) and we found that blockade of IL-10 effects are exerted in part peripherally through lymphopenia signaling in the spinal cord was sufficient to restore the neuro- where these functional S1PR1 antagonists inhibit S1P/S1PR1 pathic pain phenotype in astrocyte-specific S1pr1 knockout mice. signaling in (39, 41, 42) and where S1PR1 agonists When taken together, these findings suggest that in nerve trauma, activate endothelial S1PR1 to tighten gap junctions and prevent astrocyte-specific S1PR1 signaling acts as a “rheostat” that favors egress of lymphocytes (43). Accordingly, both S1PR1 agonism and neuroinflammatory pathways at the expense of IL-10 signaling. antagonism can inhibit migration from lymph nodes The molecular mechanisms whereby activation and inhibition into the spinal cord to prevent or blunt neuroinflammatory pro- of S1PR1 in astrocytes modulate IL-10 signaling in the spinal cesses associated with MS (44, 45) that could lead to reduced pain. cord are not known. We previously reported that functional or Similar past discrepancies in pharmacological outcomes between competitive S1PR1 antagonists do not increase IL-10 levels in systemic administration of FTY720 and W146 led to the conclu- spinal cord in noninjured rodents, suggesting that IL-10 signaling sion that FTY720 did not act as a functional S1PR1 antagonist is engaged in response to S1PR1 inhibition only in neuro- (46). This paradox was resolved by studies showing the discrep- inflammatory settings (9). However, we recently showed that ancies were due to the poor pharmacokinetic properties of W146 SEW2871-induced mechano-allodynia was driven by IL-1β for- (47). Subsequent studies with S1PR1 antagonists with improved mation through posttranslational processing by the leucine rich properties confirmed that indeed S1PR1 antagonists mimic the repeat and pyrin domain containing 3 (NLRP3) inflammasome, pharmacological effects of S1PR1 agonists on lymphocyte re- S1pr1 which was lost in mice with astrocyte-specific deletions distribution in vivo (17). More work is needed to define whether (32). This suggests that astrocyte-specific S1PR1 signaling in- S1PR1 antagonists are useful in MS-related pain. β duces NLRP3-IL-1 activation (32). We therefore propose a Collectively, our study suggests a central etiological contribu- model whereby traumatic nerve injury increases the formation of tion of astrocyte S1PR1 signaling in neuropathic pain, provides

S1P within the spinal cord to activate S1PR1 in astrocytes to insight on how S1PR1 antagonists exert their beneficial effects and PHARMACOLOGY β D produce IL-1 , which can dampen IL-10 formation (Fig. 4 ). sets the foundation to enable therapeutic efforts centered on the β S1PR1 antagonism by blocking IL-1 production removes a development of S1PR1 antagonists as nonnarcotic analgesics. brake on astrocyte production of IL-10 and guides these cells toward an anti-inflammatory phenotype (Fig. 4E). In support, a Methods recent in vitro study showed that FTY720 attenuated the pro- Study Approval. All experiments were performed in accordance with the duction of LPS-induced proinflammatory mediators (e.g., IL-1β) International Association for the Study of Pain, the National Institutes of in astrocytes, which was associated with increased production of Health, and the European Community (E.C. L358/1 18/12/86) guidelines on IL-10, while having little effect on LPS-activated microglia (34). laboratory animal welfare and approved by the Saint Louis University In- In turn, IL-10 signaling is anticipated to attenuate neuro- stitutional Animal Care, Animal Ethics Committee of The Second University of inflammation in spinal cord and reduced neuronal excitability Naples (Naples), and University of Arizona Animal Care and Use Committee. through several mechanisms, including limiting the neuro- – – excitatory effects of IL-1β (35, 36) (Fig. 4E). Experimental Animals. Male Sprague Dawley rats (200 220 g) or male and – – Finally, our findings help crystallize our understanding of female 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) S1PR1 signaling in neuropathic pain. The pharmacological profile with food and water available ad libitum. All animals were randomly assigned of S1PR1 modulators (i.e., FTY720, siponimod) tested in this to experimental groups based on sex and genotype. All experimenters were study was similar to that of competitive S1PR1 antagonists, but in blinded to treatment conditions and genotype. Data were unblinded during complete contrast to that of pure S1PR1 agonists (SEW2871), data analysis. identifying functional S1PR1 antagonism as a major mechanism of Astrocyte-specific S1pr1 knockout mice. Astrocyte-specific S1pr1 knockout action of these S1PR1 modulators. In light of our findings, the mouse colonies are descendants of original homozygous S1pr1fl/fl,Gfap-Cre varying reported effects of S1PR1 modulators across neuropathic breeder mice kindly gifted to us by Jerold Chun, The Scripps Research In- pain models are likely due to the current limitations of S1PR1- stitute, La Jolla, CA (26). Littermates were used in all experiments. These mice targeted tools, approaches, or models, rather than the type of pain have been morphologically and functionally characterized and do not have invoked. For example, in an earlier study, it was proposed that the any reported abnormal developmental issues (26). S1pr1 knockout and control mice had similar weight gain, general behavior, motor skills, and reproduction beneficial effects of FTY720 in a mouse SNI model were due to rates. All mice were bred, genotyped, and ear tagged for identification as combined S1PR1/S1P3 agonism (15). However, this pharmaco- described previously (10). logical characterization was limited by a lack of dose–response IL-10 knockout mice. IL-10 knockout mice (B6.129P2-IL-10tm1Cgn/J) and wild- studies, use of only one S1PR1 or S1PR3 antagonist at high doses, type control mice (C57BL/6) were obtained from The Jackson Laboratory. All the absence of genetic approaches to validate pharmacological surgical approaches and experiments using IL-10 knockout mice followed results, and a very narrow behavioral response range. Here, we sterile laboratory techniques in accordance with the Saint Louis University found that FTY720 exerted beneficial effects in traumatic nerve Animal Care Committee. injury with ED50 values similar to those obtained with competitive Defining Estrous Cycle Stage. Given the multiday nature of the design in freely S1PR1 antagonists. Moreover, the ED50 values for two S1PR3- sparing S1PR1 agonists, siponimod and KRP-203, were similar to cycling females, a vaginal smear was taken after the last behavioral time FTY720 and other S1PR1 modulators that possess limited S1PR3 point and stage of estrous defined by cytology as described (48). All animals displayed a normal 4–5 d estrous cycle. activity, as well as competitive S1PR1 antagonists. These results suggest that engineering S1PR3 activity out of the molecule has Rodent Models of Traumatic Nerve Injury-Induced Neuropathic Pain. Two well- little impact on the ability of the drug to reverse mechano- characterized rodent models were used for our studies: chronic constriction allodynia (14, 37) and argue against the prerequisite role for injury (CCI) to the sciatic nerve (18), and spared nerve injury (SNI) or sham S1PR3 activation in the beneficial effects of FTY720 (15). One surgery was performed on mice according to the method of Decosterd and recent study in a model of central neuropathic pain reported that Woolf (19).

Chen et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10561 Downloaded by guest on October 1, 2021 Test Compounds. Fingolimod (FTY720) and SEW2871 were purchased from described (53). Acute thermal anti-nociception was measured using the tail Cayman Chemical. NIBR-15 was synthesized by us according to the published flick latency test (54), which measures the withdrawal latency of the tail route (20). According to methods previously described, ponesimod (49) and from a noxious radiant heat source with baseline latencies of 2–3sanda TASP0277308 (21) were synthesized by Shanghai ChemPartner Co; whereas cutoff time of 10 s to prevent tissue injury. KRP-203 (50) was synthesized from Atomax Chemicals. Siponimod (BAF312) Additional details for all methods can be found in SI Appendix. (51) was synthesized from MedChemExpress LLC. Certificate of analysis > confirmed purity of each test agent of 95%. Morphine sulfate was a kind gift ACKNOWLEDGMENTS. We thank Drs. R. Samson and G. Yosten (Saint Louis from Mallinckrodt Pharmaceuticals. Sheep anti-rat IL-10 IgG antibody was a University) for their assistance with the vaginal smear assays and Dr. A. J. generous gift from Linda Watkins, University of Colorado Boulder, Boulder, Lechner (Saint Louis University) for his valuable input and careful editorial CO. Control sheep serum IgG was obtained from Sigma Aldrich. All solutions of review of our work. This study was funded by Saint Louis University startup agents were made fresh each day immediately before administration. funds (to D.S. and J.K.W.), NIH National Institute of General Medical Sciences Grant R01GM043880 (to S. Spiegel), Southern Illinois University Edwardsville School of Pharmacy Research Grant (to W.L.N.), University of Arizona Startup Behavioral Testing. Mechano-allodynia was determined by probing the funds (to T.W.V.), and by the Washington University Institute of Clinical and – plantar aspect of the hind paw with calibrated von Frey filaments (mice: 0.04 Translational Sciences, which is, in part, supported by the NIH/National Center 2.00 g; rats: 1.4–26 g; Stoelting) according to the “up-and-down” method for Advancing Translational Sciences (NCATS), Clinical and Translational Sci- (52) and calculating the paw withdrawal threshold (PWT, g), as previously ence Awards Grant UL1 TR002345 (to D.S.).

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