GPR183-oxysterol axis in spinal cord contributes to neuropathic pain

Article abstract of DOI:10.1124/JPET.120.000105

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 7a,25-dihydrox-ycholesterol (7a,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 7a,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 7a,25-OHC in na?ve 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.

Full text of DOI:10.1124/JPET.120.000105

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GPR183-oxysterol axis in spinal cord contributes to neuropathic pain
3
Kathryn Braden 1,2, Luigino Antonio Giancotti 1,2, Zhoumou Chen 1,2, Chelsea DeLeon , Nick
3
1
1
4
1,2
Latzo , Terri Boehm , Napoleon D’Cunha , Bonne M. Thompson , Timothy M. Doyle
,
Jeffrey G. McDonald 4, John K. Walker 1,2, Grant R. Kolar 2,5, Christopher Kent Arnatt 2,3, Daniela
Salvemini 1,2
1 Department of Pharmacology and Physiology, Saint Louis University School of Medicine
2 Henry and Amelia Nasrallah Center for Neuroscience, Saint Louis University
3 Department of Chemistry, Saint Louis University
4 Center for Human Nutrition, University of Texas Southwestern Medical Center
5 Department of Pathology, Saint Louis University School of Medicine
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Running Title: GPR183-oxysterol contribution to neuropathic pain
Corresponding author: Daniela Salvemini, Saint Louis University School of Medicine, 1402
South Grand Boulevard, Saint Louis, MO 63104. Phone: 1-314-977-6430, Fax: 1-314-977-6411,
Email: daniela.salvemini@health.slu.edu, ORCID ID: 0000-0002-0612-4448.
Text pages: 20
Tables: 0
Figures: 6, 1 supplemental
References: 48
Abstract word count: 232
Intro word count: 691
Discussion word count: 1400
Non-standard abbreviations (A-Z): 25-OHC: 25-hydroxycholesterol, 7α,25-OHC: 7α,25-
dihydroxycholesterol, CCI: Chronic constriction injury, CH25H: cholesterol 25-hydroxylase,
CNS: Central nervous system, CYP7B1: 25-hydroxycholesterol 7α-hydroxylase, DH-SC: dorsal-
horn spinal cord, DMAP: 4-Dimethylaminopyridine, DRG: dorsal root ganglion, EAE:
experimental autoimmune encephalitis, EBI2: Epstein-Barr Virus-Induced gene 2, ERK:
Extracellular signal-related kinase, HSD3B7: hydroxyl-delta-5-steroid dehydrogenase, 3 beta-
and steroid delta-isomerase 7, i.th.: intrathecal, MS: multiple sclerosis, NMR: Nuclear magnetic
resonance, PWT: Paw withdrawal threshold, RNA-Seq: RNA-Sequencing, SAR: Structure-
activity relationship, SRE: Serum response element, TLC: Thin-layer chromatography
Recommended Section: Drug Discovery and Translational Medicine
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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 induced gene 2
[EBI2]) 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 50nM. 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 pro-nociceptive. 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 pro-nociceptive.
These studies are the first to reveal a role for GPR183 in neuropathic pain and identify it as a
potential target for therapeutic intervention.
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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 GPR183/EBI2 (Epstein-Barr virus-induced gene
2) 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 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).
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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 (25-OHC), 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-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-
isomerase 7 (HSD3B7) 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 (SRE)
activity (Rosenkilde et al., 2006; Benned-Jensen et al., 2011; Hannedouche et al., 2011; Liu et
al., 2011; Benned-Jensen et al., 2013). Activation of these pathways in CNS glia as well as in
dorsal root ganglia (DRG) 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
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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 (SAR) investigations in the
development of highly selective GPR183 antagonists.
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Materials and Methods:
Experimental Animals:
Male and female ICR mice (8-9 weeks old; 25-40g starting weight) or Sprague Dawley rats (8-9
weeks old; 250g starting weight) from Envigo-Harlan Laboratories (Indianapolis, IN) were
housed two to four per cage (rats) or five to ten 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 was combined.
Test Compounds:
7α,25-dihydroxycholesterol (7α,25-OHC) was purchased from Avanti Polar Lipids (Alabaster,
AL) and dissolved in DMSO as a 2mM stock. For injections 7α,25-OHC was diluted in saline.
NIBR189 was purchased from Tocris Bioscience (Minneapolis, MN) and dissolved in DMSO as
a 23mM 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 10mM
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%. 100mM Stock solutions of the SAE compounds were
prepared in DMSO. For intrathecal injections, SAE compounds were diluted in saline. Acute
intrathecal (i.th.) injections of compounds were performed as described previously (Lu and
Schmidtko, 2013), all compounds were administered intrathecally in a total volume of 5-10μL.
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Chronic constriction injury (CCI) model:
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, the left thigh was shaved and disinfected with Dermachlor solution. A small
incision (1-1.5cm) 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 3 distinct sites (1mm 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 D7-D10 following CCI surgery.
Behavioral Testing:
Mechano-allodynia was measured as previously described (Yosten et al., 2020) using calibrated
von Frey filaments (Stoelting; range in mice: 0.07-2.00g; in rats: 2-26g) 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 [PWT (g)] compared to 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 3 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.
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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 D10 after CCI, animals were perfused (1X 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 (Qaigen; Germantown, MD) according to 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) 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 TMAP (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 non-redundant
exons was calculated and normalized to total exon coverage using BEDTools (Quinlan and Hall,
2010) and custom scripts in R (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.
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RNAScope®:
On D10 after CCI surgery animals were perfused under deep anesthesia and with 4%
paraformaldehyde and lower lumbar spinal cord was harvested and post-fixed in 4%
paraformaldehyde. Spinal cord lumbar sections were cryosectioned at 10 micrometers 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 40x (NA 1.30) (Leica
Microsystems, Buffalo Grove, IL). RNAscope signal was dilated by 3 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 (PMID 22743772) using a
custom macro (Supplementary 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 D0 and were taken down on either D0,
D5, or D11 after surgery. Rats were perfused under deep anesthesia with 1X PBS, ipsilateral
dorsal horn spinal cord was harvested and flash-frozen in liquid nitrogen. Oxysterols were
measured according to McDonald et. al. (McDonald et al., 2012).
In silico modeling:
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An inactive homology model of GPR183 based largely upon the Chemokine Receptor CCR5
(5UIW) was downloaded from the GPCRdb. (Pándy-Szekeres et al., 2017) Using Schrödinger,
protein preparation was run to minimize the energy of the protein (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
database 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 ten thousand screening hits from each screen based upon their feature
matching with the two pharmacophores were chosen for further screening. The twenty thousand
screening hits were then used in a high-throughput 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.
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Instrumentation: The purities of the final compounds were characterized by high-performance
liquid chromatography (HPLC) using a gradient elution program (Ascentis Express Peptide C18
column, acetonitrile/water 5/95 -> 95/5, 5 min, 0.05% trifluoroacetic acid) and UV detection (245
nM). TLC was performed on Merck KGaA TLC silica gel 60 F254 plates. Visualization was
accomplished by using phosphomolybdic acid solution followed by heat and by UV fluorescence
1
(λmax 254 nm). H nuclear magnetic resonance (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 fetal bovine serum (FBS), 1%
penicillin, and 1% GlutaMax. Cells were passaged every 3 days and maintained at a cell
concentration below 1x106 to prevent differentiation. The cells were incubated at 37C under 5%
CO2.
Calcium mobilization assays:
Serial dilutions (5X) of compounds were prepared in 50:1 HBSS/HEPES.HL-60 cells (1x107)
were incubated in 50:1 HBSS/HEPES containing 5 M indo-1-AM (Thermo Fischer Scientific;
Waltham, MA) 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; Waltham, MA) clear-bottom
plate at 100,000 cells/well for a 5X 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). Following the 15 minute incubation period, 5X of the compound was
added and fluorescence was read for 150 seconds at 37C. This method was utilized to
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determine the EC80 value of 7α,25-OHC (EC8o = 200 nM). For antagonism assays, before the
addition of agonist, 5X 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 7,25-OHC
(200 nM) was added, and fluorescence was read for 150 secs at 37C. Calcium mobilization
was determined ratiometrically using ex 350 nm and em 405/490 nm. Dose-response data were
normalized to a 0.5% DMSO vehicle control and the maximum response. From the normalized
data, non-linear 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:
siRNA was purchased from Santa Cruz Biotechnology (Dallas, TX). HL-60 cells were
centrifuged and counted with a hemocytometer to obtain 2.5x106 cells. Transfection of the
siRNA was performed with Lipofectamine 2000 reagent (Invitrogen; Calsbad, CA) and Opti-
MEM. Lipofectamine and siRNA (200pmol) 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 utilized in calcium mobilization assays.
Statistical analysis:
Data is expressed as mean ± SD or SEM for n biological 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)
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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 mechano-allodyina that peaks within 7-10 days after injury
and last for several weeks (Bennett and Xie, 1988). RNA-Seq analysis of dorsal horn spinal
cord tissues (DH-SC) harvested at peak mechano-allodynia (D10 after CCI surgery; Fig. 1A)
revealed a 2.8-fold (p=7.59x10-14, FDR=9.98x10-12) increase in Gpr183 expression in the DH-SC
from rats with CCI compared to 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 (Supplementary Fig. S1). 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. 1C-D). Further analyses revealed that while Gpr183 co-
localized 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.
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 pre-surgery, day 5 and day 11 post surgery). However, 7α,25-OHC was
undetectable at all time points after CCI surgery (n=8). We were able to detect its precursor, 25-
hydroxycholesterol (25-OHC) but levels did not change over time (from 0.16 ng/mg, to 0.12
ng/mg and 0.09 ng/mg on day 0, 5 and 11 respectively; one-way ANOVA p=0.0662 for n=8).
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Failure of NIBR189 to inhibit CCI-induced mechano-allodynia
The commercially available small molecule GPR183 antagonists are limited to NIBR189 (IC50
11nM) (Gessier et al., 2014) and GSK682753A (IC50=0.2μM) (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 23μM (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. Due to
significant variations in dose-responses, we observed single plate IC50 values that consistently
had standard deviations of multiple magnitudes larger/smaller. This viability 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 11nM (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.
Discovery of potent and selective GPR183 antagonists with pharmacological activity
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Knowing that both NIBR189 and GSK682753A have shown binding affinity to GPR183, they
were docked within a GPR183 homology model in order 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 Schrodinger Phase. This
model consisted of aromatic, hydrophobic, H-bond acceptor/donator, and size exclusion
spheres in 3-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 (Figs. 4B-D). Three
of those compounds were able to antagonize 7α,25-OHC-induced calcium mobilization with IC50
values below 50 nM (Fig. 4B, 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.
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 100x 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 SAR studies.
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GPR183 signaling in naïve animals is pro-nociceptive
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 i.th. injections of the most potent GPR183 ligand, 7α,25-OHC should
recapitulate behavioral consequences of neuropathic pain states (i.e., allodynia). Indeed, i.th.
injections of 7α,25-OHC in mice induced a dose and time-dependent mechano-allodynia
(ED50=74nM; Fig. 6A) and cold allodynia (Fig. 6B). Additionally, the use of a fluorinated analog
of 7α,25-OHC, SLUPP-1492, induced similar results (Figs. 6A-B). Importantly, pre-treatment
with one of our novel GPR183 antagonists (SAE-14) was able to block these effects of 7α,25-
OHC in a dose-dependent manner (Figs. 6C-D).
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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
following traumatic nerve injury. Furthermore, we have shown that GPR183 co-localizes 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 towards either exogenous 7α,25-OHC or media containing released
GPR183 ligands (Preuss et al., 2014; Rutkowska et al., 2015; Rutkowska et al., 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
which could be inhibited by NIBR189, but an IC50 was not calculated (Preuss et al., 2014). A few
studies found 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 non-specific 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
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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 while 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 biological fluids making its
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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 SRE (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 (MS). However, in mouse models of MS 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 to wild type mice (Wanke et al., 2017). However, when a transfer model of
EAE was used and GPR183-deficient Th17 cells were adoptively transferred to Rag-/- mice, they
had a delayed onset of disease (Wanke et al., 2017), similar to what was seen in Chalmin and
colleagues’ CH25H-/- mice (Chalmin et al., 2015). Therefore, it is likely that GPR183-oxysterol
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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 following traumatic nerve injury. Importantly, others have identified GPR183 as being
upregulated in the spinal cord of rats after a post-surgical 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 lead 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.
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/analytical tools: McDonald, Walker, Kolar
Performed data analysis: Braden, Giancotti, DeLeon, Thompson, Doyle, Kolar, Arnatt
Wrote or contributed to writing of the manuscript: Braden, Doyle, McDonald, Walker, Kolar,
Arnatt, Salvemini
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Footnotes*†
* 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, Salvemini D.
Identifying a role for GPR183 and 7α,25-dihydroxycholesterol in neuropathic pain. Poster, Henry and
Amelia Nasrallah Center for Neuroscience Research Symposium. Saint Louis, MO, November 1, 2019.
Braden K, Giancotti L, Chen Z, Salvemini D. GPR183 as a novel target for the treatment of neuropathic
pain. Poster, Society for Neuroscience Meeting. Chicago, IL, October 19-23, 2019.
†
All reprint requests should be addressed to: Daniela Salvemini, Saint Louis University School of
Medicine, 1402 South Grand Boulevard, Saint Louis, MO 63104. Email: daniela.salvemini@health.slu.edu
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‡
‡
These studies were supported by the Saint Louis University start-up funds of Dr. Salvemini and Dr.
Arnatt; the National Institute of Health T32 Training Grant (GM008306-01; KB); and in part by National
Institute of Health National Heart, Lung, and Blood Institute (HL020948: JGM).
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Figure 1: Gpr183 expression is upregulated in ipsilateral DH-SC from rats with CCI-
induced neuropathic pain. A) Mechano-allodynia in male rats following CCI or sham surgery
(n=4/group). 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 Fig. S1. 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 of 40x and axial depth of 6 μm)
composite of spinal cord probed for Gfap (green), Gpr183 (red) and DAPI (blue). Signal was
dilated by 3 pixel diameters for display purposes; box= area of quantification; ipsi=ipsilateral and
contra=contralateral side to injury. Scale bar= 200 μm. Data are A) mean ± SD or C) median
and analyzed by two-tailed, A) two-way repeated measures ANOVA (RM-ANOVA) with
Bonferroni comparisons or C) paired Student’s t test. *P<0.05 vs. Sham or # P<0.05 vs. Contra.
Figure 2: Gpr183 expression is upregulated in microglia and astrocytes in ipsilateral 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,D), Gfap (astrocytes; n=10;
magenta; B,E) or Rbfox3 (neurons; n=8; magenta; C,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 median and analyzed by two-tailed, paired Student’s t test. *P<0.05
vs. Contra.
Figure 3: NIBR189 does not reverse CCI-induced mechanical allodynia. Acute intrathecal
injections of NIBR189 (0.7μM, 2.3μM n=3; 23μM n=4) on D7 post CCI does not reverse
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