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The Journal of Pharmacology and Experimental Therapeutics logoLink to The Journal of Pharmacology and Experimental Therapeutics
. 2020 Nov;375(2):349–357. doi: 10.1124/jpet.120.000105

GPR183-Oxysterol Axis in Spinal Cord Contributes to Neuropathic Pain

Kathryn Braden 1, Luigino Antonio Giancotti 1, Zhoumou Chen 1, Chelsea DeLeon 1, Nick Latzo 1, Terri Boehm 1, Napoleon D’Cunha 1, Bonne M Thompson 1, Timothy M Doyle 1, Jeffrey G McDonald 1, John K Walker 1, Grant R Kolar 1, Christopher Kent Arnatt 1, Daniela Salvemini 1,✉
PMCID: PMC7592849  PMID: 32913007

Abstract

Neuropathic pain is a debilitating public health concern for which novel non-narcotic therapeutic targets are desperately needed. Using unbiased transcriptomic screening of the dorsal horn spinal cord after nerve injury we have identified that Gpr183 (Epstein-Barr virus–induced gene 2) is upregulated after chronic constriction injury (CCI) in rats. GPR183 is a chemotactic receptor known for its role in the maturation of B cells, and the endogenous ligand is the oxysterol 7α,25-dihydroxycholesterol (7α,25-OHC). The role of GPR183 in the central nervous system is not well characterized, and its role in pain is unknown. The profile of commercially available probes for GPR183 limits their use as pharmacological tools to dissect the roles of this receptor in pathophysiological settings. Using in silico modeling, we have screened a library of 5 million compounds to identify several novel small-molecule antagonists of GPR183 with nanomolar potency. These compounds are able to antagonize 7α,25-OHC–induced calcium mobilization in vitro with IC50 values below 50 nM. In vivo intrathecal injections of these antagonists during peak pain after CCI surgery reversed allodynia in male and female mice. Acute intrathecal injection of the GPR183 ligand 7α,25-OHC in naïve mice induced dose-dependent allodynia. Importantly, this effect was blocked using our novel GPR183 antagonists, suggesting spinal GPR183 activation as pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify this receptor as a potential target for therapeutic intervention.

SIGNIFICANCE STATEMENT

We have identified several novel GPR183 antagonists with nanomolar potency. Using these antagonists, we have demonstrated that GPR183 signaling in the spinal cord is pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify it as a potential target for therapeutic intervention.

Introduction

Neuropathic pain conditions arising from nervous system injuries due to trauma, disease (i.e., diabetes), or neurotoxins (i.e., chemotherapy) are widespread and can be severe, debilitating, and difficult to treat (Finnerup et al., 2015). Opioids are widely used to treat chronic pain but are limited by severe side effects and strong abuse liability (Toblin et al., 2011). Therefore, there is a high priority for developing novel non–opioid-based analgesics.

We used an unbiased transcriptomic analysis in this study to identify novel pathways and therapeutic targets in the development of neuropathic pain. In a model of traumatic nerve injury–induced neuropathic pain, our findings identified G-protein coupled receptor 183 (GPR183)/Epstein-Barr virus–induced gene 2 (EBI2) as a potential target for neuropathic pain. GPR183 was originally identified in B cells as the most upregulated gene in response to Epstein-Barr virus infection (Birkenbach et al., 1993). This receptor has been found in multiple human tissues, including brain, but was found most abundantly in lymphoid organs and is most highly expressed on B cells (Rosenkilde et al., 2006). Similar patterns of expression have been found in rodents (Lein et al., 2007), and the human receptor sequence shares 88% homology with the rodent sequences, according to National Center for Biotechnology Information (NCBI) Blast (Boratyn et al., 2013). GPR183 is important for the positioning of immune cells, particularly B cells, within lymphoid tissues, such as the spleen, for the launching of T-cell–dependent antibody responses (Gatto et al., 2009; Pereira et al., 2009). GPR183 knockout mice are viable and have a normal gross phenotype: these mice have normal numbers of B cells and T cells, with no defect in B-cell localization within the spleen (Pereira et al., 2009). Besides its role in regulating immune cell migration, GPR183 has been linked to metabolic diseases, multiple sclerosis, and cancer; accordingly, GPR183 has been proposed to represent a potential target for several diseases, ranging from inflammation to cancer (Sun and Liu, 2015).

The primary endogenous ligand for GPR183 is the oxysterol 7α,25-dihydroxycholesterol (7α,25-OHC) (Hannedouche et al., 2011; Liu et al., 2011). This oxysterol is produced by the hydroxylation of cholesterol by cholesterol 25-hydroxylase (CH25H), resulting in its precursor, 25-hydroxycholesterol, which is subsequently hydroxylated by 25-hydroxycholesterol 7α-hydroxylase (CYP7B1) to form 7α,25-OHC (Mutemberezi et al., 2016). This oxysterol is rapidly degraded by hydroxyl-Δ-5-steroid dehydrogenase, 3 β- and steroid Δ-isomerase 7 to be further metabolized into bile acids (Mutemberezi et al., 2016). 7α,25-OHC has proven to be a potent agonist of GPR183 in vivo (Liu et al., 2011; Chalmin et al., 2015; Lu et al., 2017; Wanke et al., 2017).

The role of GPR183 in the central nervous system (CNS) is still under investigation, and its role in the context of pain is not known. At the cellular level, GPR183 has only been reported to be expressed in astrocytes within the CNS (Rutkowska et al., 2015). Other studies have found microglia are able to produce and release 7α,25-OHC, but it was not explored whether microglia respond to the GPR183 ligand (Mutemberezi et al., 2018). GPR183 is Gαi-coupled and when activated by 7α,25-OHC, it can inhibit adenylate cyclase activity, increase phosphorylation of extracellular signal–regulated kinase (ERK) and p38 and trigger serum response element activity (Rosenkilde et al., 2006; Benned-Jensen et al., 2011, 2013; Hannedouche et al., 2011; Liu et al., 2011). Activation of these pathways in CNS glia as well as in dorsal root ganglia neurons is crucial to the persistent pain sensitization and to pain chronification (Ji et al., 2009; Gomez et al., 2018).

Commercially available GPR183 antagonists are limited to a couple of compounds with chemical properties that question in vivo utility (Ardecky et al., 2010). To address this void, we embarked on drug discovery efforts to identify novel GPR183 antagonists for use in exploring the roles of GPR183 in neuropathic pain states. Our efforts led to the identification of several potent small-molecule selective GPR183 antagonists that were active in a rodent model of neuropathic pain caused by chronic constriction of the sciatic nerve (Bennett and Xie, 1988). These studies are the first step in investigating the role of GPR183 in neuropathic pain while providing the framework for future structure-activity relationship investigations in the development of highly selective GPR183 antagonists.

Materials and Methods

Experimental Animals

Male and female ICR mice (8 to 9 weeks old; starting weight of 25–40 g) or Sprague-Dawley rats (8 to 9 weeks old; starting weight of 250 g) from Envigo-Harlan Laboratories (Indianapolis, IN) were housed two to four per cage (rats) or 5–10 per cage (mice) in a controlled environment (12-hour light/dark cycle) with food and water available ad libitum. All experiments were performed with experimenters blinded to treatment conditions. All experiments were performed in accordance with the guidelines of the International Association for the Study of Pain and the National Institutes of Health and approvals from the Saint Louis University Animal Care and Use Committee. Experiments were performed in both male and female rodents; similar results were obtained in both sexes, so data were combined.

Test Compounds

7α,25-Dihydroxycholesterol (7α,25-OHC) was purchased from Avanti Polar Lipids (Alabaster, AL) and dissolved in DMSO as a 2 mM stock. For injections, 7α,25-OHC was diluted in saline. NIBR189 was purchased from Tocris Bioscience (Minneapolis, MN) and dissolved in DMSO as a 23 mM stock. For intrathecal injections, NIBR189 was diluted in saline. Fluoronated 7α,25-OHC analog (SLUPP-1492) was synthesized as described previously (Deng et al., 2016) (detailed methods below and continued in Supplemental Methods) and prepared as a 10 mM stock in DMSO. For injections, SLUPP-1492 was diluted in saline. SAE compounds were purchased from Enamine (Monmouth Jct., NJ); all compounds were purified via normal-phase chromatography and had purities of ≥95%. Stock solutions (100 mM) of the SAE compounds were prepared in DMSO. For intrathecal injections, SAE compounds were diluted in saline. Acute intrathecal injections of compounds were performed as described previously (Lu and Schmidtko, 2013); all compounds were administered intrathecally in a total volume of 5–10 μl.

Chronic Constriction Injury Model

Chronic constriction injury (CCI) of the left sciatic nerve of mice and rats was performed under general anesthesia as previously described (Yosten et al., 2020). Briefly, animals were anesthetized with 2% isoflurane/O2, and the left thigh was shaved and disinfected with Dermachlor solution. A small incision (1–1.5 cm) was made in the middle of the lateral aspect of the left thigh to expose the sciatic nerve. The nerve was loosely ligated around the diameter at three distinct sites (1 mm apart) using silk sutures (6.0, mice; 4.0, rats). The surgical site was closed with a skin clip and disinfected and treated with topical lidocaine (2%). Sham animals underwent the same procedure without nerve ligation. Peak allodynia developed by day 7 to day 10 after CCI surgery.

Behavioral Testing

Mechano-allodynia was measured as previously described (Yosten et al., 2020) using calibrated von Frey filaments (Stoelting; range in mice: 0.07–2.00 g; in rats: 2–26 g) using the Dixon up-and-down method (Dixon, 1991). Mechano-allodynia was defined as a significant (P < 0.05) reduction in mechanical paw withdrawal threshold (g) compared with baseline forces (before treatment).

Cold allodynia was measured as previously described using the acetone test (Xing et al., 2007; Yosten et al., 2020). Briefly, a small drop of acetone was applied to the hind paw of the animal using a flattened polyethylene tube and a syringe, and the response to the cold stimulus was scored (0, no response; 1, brisk withdrawal or flick of the paw; 2, repeated flicking of the paw; 3, repeated flicking and licking of the paw). The test was repeated three times with an interval of 5 minutes between each application for each paw and the scores for each paw were summed and reported as the response score (maximum of 9). When mechanical and cold allodynia were measured on the same animal, mechano-allodynia was measured first with at least 15 minutes before testing cold allodynia.

Estrus Smears

Vaginal smears were taken for female mice and rats 5–7 days before experiment and after experiment until animals were sacrificed to confirm animals were cycling, and treatment did not alter their cycle. Cells were placed on a glass slide and allowed to dry, stained with Accustain (MilliporeSigma, Saint Louis, MO) for 45 seconds, and rinsed, as previously described (Byers et al., 2012). Fixed cells were viewed under a light microscope to determine their stage of estrus cycle. All animals displayed normal estrus cycles.

RNA-Sequencing

On day 10 after CCI, animals were perfused (1× PBS) under deep anesthesia, and lower lumbar spinal cord was harvested and placed in RNA-Later. Total RNA was isolated using RNeasy Plus Universal Mini kit (Qiagen, Germantown, MD) according to the manufacturer’s protocols. RNA-sequencing was performed in the Saint Louis University Genomics Core Facility. Total RNA samples were quality assessed using an Agilent Bioanalyzer RNA Nano chip and were determined to have an RNA integrity number of ≥9. Ribosomal RNA was depleted from total RNA using the Eukaryotic RiboMinus Core Module v2 (Life Technologies, Thermofisher, Waltham, MA), and libraries were constructed using the Ion Total RNA-seq v2 kit (Life Technologies, Thermofisher) according to the manufacturer’s protocols. Sequencing was performed on an Ion Torrent Proton with a mean read length of ∼140 nucleotides. Reads were aligned to the rat genome sequence (version rn6) using the Torrent Mapping Program aligner (Homer, 2011) map4 algorithm, requiring a minimum seed length of 20 nucleotides and allowing soft clipping at both 5′ and 3′ ends to accommodate spliced reads. The nucleotide coverage for all nonredundant exons was calculated and normalized to total exon coverage using BEDTools (Quinlan and Hall, 2010) and custom scripts in R (R Core Team, 2015). Expression values are given as total normalized nucleotide exon coverage per gene. Fold changes in gene expression and P values were calculated using R and Microsoft Excel.

RNAscope

On day 10 after CCI surgery, animals were perfused under deep anesthesia and with 4% paraformaldehyde, and lower lumbar spinal cord was harvested and postfixed in 4% paraformaldehyde. Spinal cord lumbar sections were cryosectioned at 10 µm and stained using the RNAscope technique. Probes for rat Gfap (NM_017009.2, Probe- Rn-Gfap-C2), Aif1 (NM_017196.3, Probe- Rn-Aif1-C3), Rbfox3 (NM_001134498.2, Probe- Rn-Rbfox3-C2), and Gpr183 (NM_001109386.1, Probe- Rn-Gpr183) were incubated with tissue strictly according to the Manual RNAscope Fluorescence Multiplex Protocol (v2) (Advanced Cell Diagnostics, Newark, CA). Sections were imaged as Z-stacks of the central lamina 1 and 2 region of the dorsal horn on a Lecia TCS SP8 confocal microscope using a 40× (Numerical aperture 1.30) (Leica Microsystems, Buffalo Grove, IL). RNAscope signal was dilated by three pixel diameters for large overview display purposes, or else signal was dilated by 0.5 pixel diameters. For analysis, RNAscope signal was dilated for each slice by 0.5 pixel diameters, and Gpr183 was isolated by thresholding the channel on positive signal, setting a selection area, and applying it to the appropriate lineage marker (GFAP, Aif1, or Rbfox3) within FIJI (Pub-Med Identification 22743772) using a custom macro (Supplemental Methods). Particle counts of the signals were made in the resulting overlapping region (which represented the major RNA pool of a particular cell) for each slice.

Oxysterol Quantification

Male Sprague-Dawley rats underwent CCI surgery on day 0 and were taken down on either day 0, day 5, or day 11 after surgery. Rats were perfused under deep anesthesia with 1× PBS, and ipsilateral dorsal horn spinal cord was harvested and flash-frozen in liquid nitrogen. Oxysterols were measured according to McDonald et al. (2012).

In Silico Modeling

An inactive homology model of GPR183 based largely upon the C-C chemokine receptor 5 (5UIW) was downloaded from the G protein-coupled receptors database (GPCRdb) (Pándy-Szekeres et al., 2018). Using Schrödinger, protein preparation was run to minimize the energy of the protein (Optomized potentials for liquid stimulations 3 [OPLS3] force field). Schrödinger Phase was then employed to build a pharmacophore hypothesis built around the binding of NIBR189 and GSK682753A in GPR183 using the automated build function within Phase for receptor-ligand complex pharmacophore. This included features such as aromatic, hydrophobic, H-bond acceptor/donator, and size exclusion spheres. Using a freely available data base from Enamine (Enamine_Diverse_REAL_drug-like_5M library), 5 million compounds were screened for their “likeness” to the properties of the NIBR189 and GSK682753A separately. The top 10,000 screening hits from each screen based upon their feature matching with the two pharmacophores were chosen for further screening. The 20,000 screening hits were then used in a high-throughput Grid-based ligand docking and energetics (GLIDE) docking, and the top 800 compounds (4%) with the lowest GlideScores (computational estimate of binding) were chosen for more precise docking studies. The top 800 compounds (4%) with the lowest GlideScores were subjected to standard precision docking using flexible ligand sampling in GLIDE using the default settings. The compounds were then sorted based upon their predicted Log(S) values [all the compounds were prescreened to have a Log(P) of less than 5]. Compounds with Log(S) values larger than −4 were then sorted based upon their GlideScores, and the top 16 commercially available compounds in that pool were then ordered from Enamine for initial in vitro screening.

Synthesis of SLUPP-1492

Material.

The 25-hydroxycholesterol was purchased from ChemShuttle (Hayward, CA); all other reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO), Alfa Aesar (Ward Hill, MA), or J.T. Baker (Radnor, PA) and used as received.

Instrumentation.

The purities of the final compounds were characterized by high-performance liquid chromatography using a gradient elution program (Ascentis Express Peptide C18 column, acetonitrile/water 5:95 → 95:5, 5 minutes, 0.05% trifluoroacetic acid) and UV detection (245 nM). Thin-layer chromatography was performed on Merck KGaA thin-layer chromatography silica gel 60 F254 plates. Visualization was accomplished by using phosphomolybdic acid solution followed by heat and by UV fluorescence (λmax 254 nm). 1H NMR was obtained on a Bruker 400 MHz instrument, and all chemical shifts are referenced to residual solvent peaks (details in Supplemental Methods).

Cell Line and Culture

The human leukemia (HL)-60 cells (American Type Culture Collection, Manassas, VA) were cultivated in RPMI 1640 media containing 10% heat-inactivated FBS, 1% penicillin, and 1% GlutaMax. Cells were passaged every 3 days and maintained at a cell concentration below 1 × 106 to prevent differentiation. The cells were incubated at 37°C under 5% CO2.

Calcium Mobilization Assays

Serial dilutions (5×) of compounds were prepared in 50:1 Hank's Balanced Salt Solution (HBSS)/HEPES. HL-60 cells (1 × 107) were incubated in 50:1 HBSS/HEPES containing 5 μM indo-1-AM (Thermo Fischer Scientific) and 0.05% pluronic acid for 0.5 hours at room temperature. Cells were centrifuged and washed with 50:1 HBSS/HEPES and resuspended in buffer. Cells were loaded onto a black 96-well Greiner Bio-One (Thermo Fischer Scientific) clear-bottom plate at 100,000 cells per well for a 5× dilution. For agonism assays, cells were immediately incubated for 15 minutes at 37°C inside a FlexStation 3 Multimode Plate Reader (Molecular Devices, Sunnyvale, CA). After the 15-minute incubation period, 5× of the compound was added, and fluorescence was read for 150 seconds at 37°C. This method was used to determine the EC80 value of 7α,25-OHC (EC80 = 200 nM). For antagonism assays, before the addition of agonist, 5× of the antagonist was added and incubated for 15 minutes at 37°C inside a FlexStation3. After allowing 15 minutes of equilibration, the determined EC80 of 7α,25-OHC (200 nM) was added, and fluorescence was read for 150 seconds at 37°C. Calcium mobilization was determined ratiometrically using λex 350 nm and λem 405/490 nm. Dose-response data were normalized to a 0.5% DMSO vehicle control and the maximum response. From the normalized data, nonlinear regression curves were then generated to calculate the appropriate EC50 and IC50 values. Each compound was run in triplicate (n = 3).

siRNA Knock-Down of GPR183

Small interfering RNA (siRNA) was purchased from Santa Cruz Biotechnology (Dallas, TX). HL-60 cells were centrifuged and counted with a hemocytometer to obtain 2.5 × 106 cells. Transfection of the siRNA was performed with Lipofectamine 2000 reagent (Invitrogen, Calsbad, CA) and Opti-MEM. Lipofectamine and siRNA (200 pmol) were incubated at room temperature for 15 minutes before addition to 5 mL of cell suspension in media. Transfected cells were incubated at 37°C under 5% CO2 for 48 hours. HL-60 control cells were treated with Lipofectamine 2000 reagent without siRNA. After 48 hours, cells were used in calcium mobilization assays.

Statistical Analysis

Data are expressed as means ± S.D. or S.E.M. for n biologic replicates and analyzed by paired or unpaired t test or one-way or two-way repeated measures ANOVA with Dunnett’s multiple comparisons. Sphericity was tested with Mauchly’s test, and Greenhouse-Geiser corrections were used when necessary. Significant differences were defined as P < 0.05. Statistical analyses were performed using GraphPad Prism (versions 5.00-8.1.1. for Windows; GraphPad Software, San Diego, CA; www.graphpad.com).

Results

GPR183 Upregulation in Spinal Cord after Nerve Injury.

A well characterized rodent model of neuropathic pain was used. In this model, constriction of the sciatic nerve produces robust mechanoallodynia that peaks within 7–10 days after injury and lasts for several weeks (Bennett and Xie, 1988). RNA-sequencing analysis of dorsal horn spinal cord tissues (DH-SC) harvested at peak mechano-allodynia (day 10 after CCI surgery; Fig. 1A) revealed a 2.8-fold (P = 7.59 × 10−14, False discovery rate [FDR] = 9.98 × 10−12) increase in Gpr183 expression in the DH-SC from rats with CCI compared with those that received sham surgery (Fig. 1B). To confirm this upregulation and identify where Gpr183 is expressed, we used RNAscope-based in situ hybridization performed in the spinal cord of rats with CCI. Gpr183 is expressed in the dorsal and ventral horn of the spinal cord (Supplemental Fig. 1). However, Gpr183 expression increased 2.4-fold (P = 0.024; paired t test) within lamina 1 and 2 of the DH-SC from mice with CCI ipsilateral to the nerve injury (Fig. 1, C and D). Further analyses revealed that although Gpr183 colocalized in microglia, astrocytes, and neurons (Fig. 2), its expression increased in microglia (6.4-fold; P = 0.0021) and astrocytes (2.5-fold; P = 0.0021) but not neurons (P = 0.4280) (Fig. 2) after CCI surgery.

Fig. 1.

Fig. 1.

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Gpr183 expression is upregulated in ipsilateral DH-SC from rats with CCI-induced neuropathic pain. (A) Mechano-allodynia in male rats after CCI or sham surgery (n = 4 per group); PWT (Paw Withdrawal Threshold). (B) Gpr183 expression in the ipsilateral DH-SC from male rats on day 10 post-CCI or sham surgery (n = 4). Full image is found in Supplemental Fig. 1. (C) Quantification and (D) representative image of Gpr183 expression in the spinal cord on day 10 CCI or sham surgery by RNAscope analyses. Image is stitched (x, y, and z; original magnification, 40×; axial depth of 6 μm) composite of spinal cord probed for Gfap (green), Gpr183 (red), and DAPI (4′,6-diamidino-2-phenylindole) (blue). Signal was dilated by three pixel diameters for display purposes; box = area of quantification; Ipsi = ipsilateral; and Contra = contralateral side to injury. Scale bar, 200 μm. Data are (A) means ± S.D. or (C) medians and analyzed by (A) two-way repeated measures ANOVA (RM-ANOVA) with Bonferroni comparisons or (C) two, tailed, paired Student’s t test. *P < 0.05 vs. sham or #P < 0.05 vs. Contra.

Fig. 2.

Fig. 2.

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Gpr183 expression is upregulated in microglia and astrocytes in ipsilateral (Ipsi) DH-SC from rats with CCI-induced neuropathic pain. (A–C) Representative images (ipsilateral) with magnified inset to show detail and (D–F) quantification of RNAscope signal for Gpr183 (yellow) and its colocalization with Aif1 [microglia; n = 10; magenta; (A and D)], Gfap [astrocytes; n = 10; magenta; (B and E)], or Rbfox3 [neurons; n = 8; magenta; (C and F)] in superficial DH-SC (regions are marked by white box in Fig. 1D). Signal for lineage markers (magenta) was dilated by 0.5 pixel diameters to create a selection region that was applied to quantify Gpr183 for that cell lineage. Scale bar, 50 μm. Data are medians and analyzed by two-tailed, paired Student’s t test. *P < 0.05 vs. contralateral (Contra).

Oxysterol Metabolism in Spinal Cord after Nerve Injury.

7α,25-OHC is the most potent ligand of GPR183, so if GPR183 were functionally involved in pain, we would expect a correlating increase in the ligand. So, we performed mass spectrometry to quantify the oxysterol content of the spinal cord at different time points after CCI surgery in rats (day 0 presurgery, day 5 and day 11 postsurgery). However, 7α,25-OHC was undetectable at all time points after CCI surgery (n = 8). We were able to detect its precursor, 25-hydroxycholesterol, but levels did not change over time (from 0.16 to 0.12 and 0.09 ng/mg on day 0, 5, and 11 respectively; one-way ANOVA P = 0.0662 for n = 8).

Failure of NIBR189 to Inhibit CCI-Induced Mechanoallodynia.

The commercially available small-molecule GPR183 antagonists are limited to NIBR189 (IC50 11 nM) (Gessier et al., 2014) and GSK682753A (IC50 = 0.2 μM) (Benned-Jensen et al., 2013). Since GSK682753A has poor microsomal and plasma stability (Ardecky et al., 2010) and is therefore not suitable for in vivo studies, we employed NIBR189 as our first attempt to explore the role of GPR183 in nerve injury–induced neuropathic pain. Intrathecal administration of NIBR189 on day 7 after CCI surgery in male mice failed to reverse mechano-allodynia at doses as high as 23 μM (Fig. 3). These doses were chosen based on previous literature using NIBR189 in vitro (Gessier et al., 2014; Preuss et al., 2014; Rutkowska et al., 2015). Since information regarding validation of NIBR189 as a specific GPR183 antagonist is limited to the original pharmacological characterization (Gessier et al., 2014), we undertook our own validation. We discovered that NIBR189 was unable to reliably inhibit 7α,25-OHC–induced calcium mobilization; we were unable to obtain an IC50 value for this compound. Because of the significant variations in dose responses, we observed single plate IC50 values that consistently had S.D. of multiple magnitudes larger/smaller. This variability was not resolved by different lots or manufacturers of NIBR189, different lots of cells, using different compound vehicles, changes in assay protocols, etc. Importantly, this variability was only observed for NIBR189 and none of the other agonists or antagonists used in this study. NIBR189 was previously claimed to be a competitive GPR183 antagonist reported to inhibit 7α,25-OHC binding to GPR183 with an IC50 of 11 nM (Gessier et al., 2014). Our findings do not support such a claim. In fact, we have found that it does not consistently inhibit 7α,25-OHC–induced signaling and, overall, has an unclear pharmacology that is highly variable. The reason for this discrepancy is at present unclear. Therefore, we underwent drug discovery efforts to identify more GPR183 antagonists that could be used for in vivo proof of concept studies.

Fig. 3.

Fig. 3.

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NIBR189 does not reverse CCI-induced mechanical allodynia. Acute intrathecal injections of NIBR189 (0.7, 2.3 μM n = 3; 23 μM n = 4) on day 7 post-CCI does not reverse mechanoallodynia in male mice. PWT (Paw Withdrawal Threshold) Data are means ± S.D.; two-way ANOVA with Dunnett’s multiple comparison; not significant P > 0.05 vs. day 7. i.th., intrathecal.

Discovery of Potent and Selective GPR183 Antagonists with Pharmacological Activity.

Knowing that both NIBR189 and GSK682753A have shown binding affinity to GPR183, they were docked within a GPR183 homology model to build a pharmacophore model. The pharmacophore model based upon the NIBR189-GPR183 and GSK682753A-GPR183 bound structures was built using the automated pharmacophore builder in Schrödinger Phase. This model consisted of aromatic, hydrophobic, H-bond acceptor/donator, and size exclusion spheres in three-dimensional space describing the key features needed for binding to GPR183 by those two compounds. Using an in silico approach, a library of 5 million compounds was screened for similarity to the GPR183 pharmacophore model. Compounds with the highest similarity scores were docked and ranked based upon their thermodynamics of binding (Fig. 4A). The top 16 commercially available compounds were purchased and then tested for GPR183-specific agonism and antagonism in a calcium mobilization assay (Fig. 4, B–D). Three of those compounds were able to antagonize 7α,25-OHC–induced calcium mobilization with IC50 values below 50 nM (Fig. 4, B and D). These compounds were unable to effect calcium mobilization in the HL-60 cells on their own. Results were confirmed to be GPR183-specific using siRNA to block protein expression. In these studies, 7α,25-OHC–induced calcium mobilization (Fig. 4E) and GPR183 antagonism (Fig. 4F) were both abolished using GPR183-specific siRNA.

Fig. 4.

Fig. 4.

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Novel GPR183 antagonists inhibit GPR183-dependent 7α,25-dihydroxycholesterol–induced calcium signaling in HL-60 cells. (A) Workflow of in silico modeling. (B) Structures, Enamine codes, and IC50 values of tested compounds. (C) Calcium mobilization dose response for HL-60 cells to 7α,25-OHC. Error bars represent means ± S.E.M. for n = 5. (D) Dose response of HL-60 cells to SAE-1, -10, and -14 inhibition of calcium mobilization induced by 7α,25-OHC (EC80 209 nM). Error bars represent means ± S.E.M. for n = 4. (E) Calcium mobilization dose response of HL-60 cells treated with or without Gpr183-targeting siRNA to 7α,25-OHC (n = 3). (F) Dose response of HL-60 cells treated with or without Gpr183-targeting siRNA to SAE-14 inhibition of calcium mobilization induced by 7α,25-OHC (EC80 209 nM). Data are means ± S.E.M. for n = 3.

GPR183 Antagonists Reverse CCI-Induced Mechanical Allodynia.

Using these lead compounds, we were able to assess whether GPR183 is functionally involved in neuropathic pain states. When administered in vivo to mice, these GPR183 antagonists (SAE-1, SAE-10, and SAE-14) were able to reverse CCI-induced mechanical allodynia in a time-dependent manner (Fig. 5). For our in vivo studies, we increased the concentration to 100× the in vitro IC50 of these compounds, as this is a reasonable approach when transitioning from in vitro to in vivo investigations. Future studies will include full dose responses with the most promising lead molecules that will emerge from our structure-activity relationship studies.

Fig. 5.

Fig. 5.

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GPR183 antagonists reverse nerve injury–induced allodynia in mice. Acute intrathecal (i.th.) injections of SAE-1 (800 nM), SAE-10 (1.4 μM), or SAE-14 (2.9 μM) were able to reverse CCI-induced mechano-allodynia on day 7 post-surgery in male and female mice (data combined) PWT (Paw Withdrawal Threshold). Data are means ± S.D. for n = 4 per group; two-way ANOVA with Dunnett’s multiple comparison *P < 0.05 vs. day 7.

GPR183 Signaling in Naïve Animals is Pronociceptive.

Our results using GPR183 antagonists in neuropathic pain models suggest that GPR183 signaling in the spinal cord contributes to the maintenance of neuropathic pain. We reasoned that if this was true, then intrathecal injections of the most potent GPR183 ligand, 7α,25-OHC, should recapitulate behavioral consequences of neuropathic pain states (i.e., allodynia). Indeed, intrathecal injections of 7α,25-OHC in mice induced a dose- and time-dependent mechano-allodynia (ED50 = 74 nM; Fig. 6A) and cold allodynia (Fig. 6B). Additionally, the use of a fluorinated analog of 7α,25-OHC, SLUPP-1492, induced similar results (Fig. 6, A and B). Importantly, pretreatment with one of our novel GPR183 antagonists (SAE-14) was able to block these effects of 7α,25-OHC in a dose-dependent manner (Fig. 6, C and D).

Fig. 6.

Fig. 6.

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Intrathecal administration of 7α,25-dihydroxycholesterol and its analog induces allodynia in mice. (A) Acute intrathecal (i.th.) injection of 7α,25-OHC (24 nM, n = 6; 72 nM, n = 6 and 240 nM, n = 14) or its synthetic analog (SLUPP-1492; 615 nM, n = 8) induced mechano-allodynia in male and female mice (data combined) in a dose-dependent manner. (B) Intrathecal 7α,25-OHC (240 nM) and SLUPP-1492 (615 nM) also induced time-dependent development of cold allodynia (n = 6 per group). (C and D) Pretreatment with intrathecal SAE-14 (3 µM mechano n = 9/cold n = 8; 1 µM n = 9; 0.3 µM n = 9), but not vehicle (mechano n = 9/cold n = 6) dose dependently prevented the development of (C) mechano-allodynia and (D) cold allodynia induced by 7α,25-OHC (480 nM) in male and female mice. PWT (Paw Withdrawal Threshold), Veh (Vehicle), OHC (7α,25-OHC). Data are means ± S.D. and analyzed by two-way ANOVA with Dunnett’s comparison. *P < 0.05 vs. 0 hours; †P < 0.05 vs. Veh group.

Discussion

Our results suggest that spinal activation of GPR183 by the oxysterol 7α,25-OHC is pronociceptive and that blocking GPR183 in the spinal cord can attenuate mechano-allodynia after traumatic nerve injury. Furthermore, we have shown that GPR183 colocalizes with microglia and astrocytes within the dorsal horn spinal cord, suggesting that the receptor is specifically upregulated on these cell types. This is consistent with GPR183’s well characterized immune function in the peripheral immune system. GPR183 has been previously shown to be expressed on astrocytes (Rutkowska et al., 2015) and have some function in neuroinflammatory states (Kurschus and Wanke, 2018). However, the significance of an upregulation of this receptor on astrocytes and microglia in the context of neuropathic pain is not yet known but will be examined in future studies as we develop lead GPR183 antagonists.

The previously developed GPR183 antagonist, NIBR189, proved ineffective at reversing neuropathic pain states, and we were unable to independently validate its specificity toward GPR183. Previous studies have consistently shown that it is able to prevent in vitro migration of GPR183-expressing cells toward either exogenous 7α,25-OHC or media containing released GPR183 ligands (Preuss et al., 2014; Rutkowska et al., 2015, 2016b; Clottu et al., 2017; Wanke et al., 2017). However, only a few studies have investigated the functional effects of NIBR189 on the downstream signaling of GPR183 or in disease states. In human-derived macrophages, it was found that 7α,25-OHC–induced intracellular calcium mobilization could be inhibited by NIBR189, but an IC50 was not calculated (Preuss et al., 2014). A few studies found that NIBR189 is able to reduce 7α,25-OHC–induced changes in cell impedance of macrophages (Preuss et al., 2014) and astrocytes (Rutkowska et al., 2018). But this measurement is nonspecific and only implies calcium signaling or migration and does not hone in on the molecular consequences of 7α,25-OHC or NIBR189 treatment. In neonatal mouse cerebellar slices, it was shown that culture with either 7α,25-OHC or NIBR189 was able to reduce myelination by 20 days; the authors of this study postulate that both the agonist and antagonist have similar effects due to 7α,25-OHC’s ability to cause receptor internalization, thus having a functionally antagonistic effect (Rutkowska et al., 2017). However, the authors of that study did not do confirmatory experiments of this hypothesis other than to show internalization of the receptor by 7α,25-OHC and not NIBR189 (Rutkowska et al., 2017). It was previously shown that 7α,25-OHC is able to upregulate p-ERK, increase calcium flux, and induce migration of human astrocytes in vitro (Rutkowska et al., 2015). However only the upregulation of p-ERK was ameliorated by NIBR189 treatment, whereas the calcium flux and migration responses were reduced in GPR183 knockout cells (Rutkowska et al., 2015). Many of these in vitro studies were conducted by the same group that characterized NIBR189 and thus did not independently validate the specificity of this compound. In vivo studies using NIBR189 are limited, despite the original characterization claiming favorable pharmacokinetic properties (Gessier et al., 2014). Acute systemic treatment with NIBR189 in naïve mice resulted in a disruption of dendritic cell positioning within the T-cell zone of the spleen (Lu et al., 2017). In GPR183 knockout mice, the dendritic cells were still uniformly distributed within the T-cell zone; however, the authors did not comment on this discrepancy, as the GPR183 knockout mice had a significantly reduced number of dendritic cells, and not all tissues had enough cells for comparative analysis (Lu et al., 2017). In a disease model of orthotopic lung transplant, long-term NIBR189 treatment inhibited markers of chronic rejection, with similar results found in GPR183 knockout mice (Smirnova et al., 2019). This study is the only one to use NIBR189 in a disease model and to find the same results in the knockout model. Based on this and our results using this compound, we believe that NIBR189 is not suitable for in vivo use. Therefore, we propose that the SAE compounds described in this study could be used to further characterize the in vivo properties of GPR183.

In our studies, we were unable to detect 7α,25-OHC in the spinal cord after nerve injury, but this was not surprising, as this oxysterol is present in very small levels in biologic fluids, making its detection very difficult despite sophisticated bioanalytical methodologies (Griffiths et al., 2016). Nevertheless, it is well accepted that 7α,25-OHC is a very potent signaling molecule, able to exert profound effects at very low levels (Liu et al., 2011; Chalmin et al., 2015; Lu et al., 2017; Wanke et al., 2017).

The molecular mechanisms underpinning the analgesic effects provided by GPR183 antagonists will be explored in future studies as the chemistry of these antagonists evolves. However, a few possibilities are offered based upon our understanding of this system in other areas. GPR183 has mostly been characterized as a chemotactic receptor in the peripheral immune system (Sun and Liu, 2015), so the downstream effects of its signaling have not been elucidated. GPR183 is known to be coupled to Gαi and when activated can induce phosphorylation of ERK, p38, or serum response element (Rutkowska et al., 2016a). The activation of these signaling pathways in astrocytes or microglia within the spinal cord has been shown to be important for the initiation of neuroinflammation and the maintenance of chronic pain (Zhuang et al., 2005). Therefore, it is possible that 7α,25-OHC–mediated GPR183 activation on CNS glia could be contributing to neuropathic pain states through neuroinflammatory mechanisms. GPR183 has been previously investigated for a role in neuroinflammatory diseases, such as multiple sclerosis. However, in mouse models of multiple sclerosis, two groups found conflicting evidence, with one showing that knockout of the rate-limiting enzyme for 7α,25-OHC production (CH25H−/−) resulted in an exacerbated course of experimental autoimmune encephalitis (EAE) (Reboldi et al., 2014), and another found the same CH25H−/− mice to have a delayed course of EAE (Chalmin et al., 2015). In GPR183 knockout mice, there was no difference in EAE disease course compared with wild-type mice (Wanke et al., 2017). However, when a transfer model of EAE was used and GPR183-deficient T-helper cell 17 (Th17) cells were adoptively transferred to Recombination activating gene (Rag)−/− mice, they had a delayed onset of disease (Wanke et al., 2017), similar to what was seen in CH25H−/− mice from Chalmin et al. (2015). Therefore, it is likely that GPR183-oxysterol signaling contributes to neuroinflammatory states such as multiple sclerosis through recruitment of infiltrating immune cells to the CNS. Infiltration of immune cells has also been shown to be a contributing factor to neuropathic pain states, which can lead to enhancement of neuroinflammatory signaling and central sensitization (Grace et al., 2011). Future studies will reveal how oxysterols and GPR183 contribute to the recruitment of peripheral immune cells to the CNS after traumatic nerve injury. Importantly, others have identified GPR183 as being upregulated in the spinal cord of rats after a postsurgical model of pain (Raithel et al., 2018), supporting our data and further implicating GPR183 in pain. These findings further implicate GPR183 in disease states involving neuroinflammation, including neuropathic pain. This provides a platform to further investigate the chemistry of GPR183 within the CNS and leads to more viable tools for the pharmacological characterization of this receptor.

Acknowledgments

We would like to thank Dr. Dale Dorsett and the Saint Louis University Genomics Core for their assistance with the RNA Sequencing. We would also like to thank the NMR facility and the Department of Chemistry at Saint Louis University for use of the 400 MHz NMR.

Abbreviations

Aif1

allograft inflammatory factor 1

CCI

chronic constriction injury

CH25H

cholesterol 25-hydroxylase

CNS

central nervous system

DH-SC

dorsal horn spinal cord

EAE

experimental autoimmune encephalitis

ERK

extracellular signal–regulated kinase

Gfap

Glial fibrillary acidic protein

GPR183

G-protein coupled receptor 183

GLIDE

Grid-based ligand docking and energetics

HBSS

Hank's Balanced Salt Solution

HL

human leukemia

Rbfox3

RNA binding FOX-1 homolog 3

siRNA

small interfering RNA

7α,25-OHC

7α,25-dihydroxycholesterol

Authorship Contributions

Participated in research design: Braden, Arnatt, Salvemini.

Conducted experiments: Braden, Giancotti, Chen, DeLeon, Latzo, Boehm, D’Cunha, Thompson, Doyle, Kolar.

Contributed new reagents or analytic tools: McDonald, Walker, Kolar.

Performed data analysis: Braden, Giancotti, DeLeon, Thompson, Doyle, Kolar, Arnatt.

Wrote or contributed to the writing of the manuscript: Braden, Doyle, McDonald, Walker, Kolar, Arnatt, Salvemini.

Footnotes

These studies were supported by the Saint Louis University start-up funds of Dr. D.S. and Dr. C.K.A.; National Institute of Health National Institute of General Medical Sciences T32 Training Grant [T32-GM008306-01] (to K.B.); and in part by National Institute of Health National Heart, Lung, and Blood Institute [P01-HL020948] (to J.G.M.).

This work has been presented in part at the following meetings: Braden K, Giancotti L, Chen Z, DeLeon C, Latzo N, Doyle T, Kolar G, Walker J, Arnatt C, and Salvemini D. Identifying a role for GPR183 and 7α,25-dihydroxycholesterol in neuropathic pain. Henry and Amelia Nasrallah Center for Neuroscience Research Symposium. Saint Louis, MO, November 1, 2019.

Braden K, Giancotti L, Chen Z, and Salvemini D. GPR183 as a novel target for the treatment of neuropathic pain. Society for Neuroscience Meeting. Chicago, IL, October 19–23, 2019.

Inline graphicThis article has supplemental material available at jpet.aspetjournals.org.

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