Cytochrome P450 26A1 Contributes to the Maintenance of Neuropathic Pain.

The cytochrome P450 proteins (CYP450s) have been implicated in catalyzing numerous important biological reactions and contribute to a variety of diseases. CYP26A1, a member of the CYP450 family, carries out the oxidative metabolism of retinoic acid (RA), the active metabolite of vitamin A. Here we report that CYP26A1 was dramatically upregulated in the spinal cord after spinal nerve ligation (SNL). CYP26A1 was mainly expressed in spinal neurons and astrocytes. HPLC analysis displayed that the content of all-trans-RA (at-RA), the substrate of CYP26A1, was reduced in the spinal cord on day 7 after SNL. Inhibition of CYP26A1 by siRNA or inhibition of CYP26A1-mediated at-RA catabolism by talarozole relieved the SNL-induced mechanical allodynia during the maintenance phase of neuropathic pain. Talarozole also reduced SNL-induced glial activation and proinflammatory cytokine production but increased anti-inflammatory cytokine (IL-10) production. The RA receptors RARα, RXRß, and RXRγ were expressed in spinal neurons and glial cells. The promoter of Il-10 has several binding sites for RA receptors, and at-RA directly increased Il-10 mRNA expression in vitro. Finally, intrathecal IL-10 attenuated SNL-induced neuropathic pain and reduced the activation of astrocytes and microglia. Collectively, the inhibition of CYP26A1-mediated at-RA catabolism alleviates SNL-induced neuropathic pain by promoting the expression of IL-10 and suppressing glial activation. CYP26A1 may be a potential therapeutic target for the treatment of neuropathic pain.

Spinal CBX2 contributes to neuropathic pain by activating ERK signaling pathway in male mice.

The deregulated spinal cord proteins induced by nerve injury are the key to neuropathic pain. Integrated transcriptome and translatome analyses can screen out deregulated proteins controlled by only post-transcriptional regulation. By comparing RNA sequencing (RNA-seq) and ribosome profiling sequencing (Ribo-seq) data, we identified an upregulated protein, chromobox 2 (CBX2), with its mRNA level unchanged in the spinal cord after peripheral nerve injury. CBX2 was mainly distributed in the spinal cord neurons. Blocking the SNL-induced increase of spinal CBX2 attenuated the neuronal and astrocytes hyperactivities and pain hypersensitivities in both the development and maintenance phases. Conversely, mimicking the upregulation of CBX2 in the spinal cord facilitated the activities of neurons and astrocytes and produced evoked nociceptive hypersensitivity and spontaneous pain. Our results also revealed that activating the ERK pathway, upregulating CXCL13 in neurons, and CXCL13 further inducing astrocyte activation were possible downstream signaling mechanisms of CBX2 in pain processing. In conclusion, upregulation of CBX2 after nerve injury leads to nociceptive hyperalgesia by promoting neuronal and astrocyte hyperactivities through the ERK pathway. Inhibiting CBX2 upregulation may be therapeutically beneficial.

Nerve Injury-Induced γH2AX Reduction in Primary Sensory Neurons Is Involved in Neuropathic Pain Processing.

Phosphorylation of the serine 139 of the histone variant H2AX (γH2AX) is a DNA damage marker that regulates DNA damage response and various diseases. However, whether γH2AX is involved in neuropathic pain is still unclear. We found the expression of γH2AX and H2AX decreased in mice dorsal root ganglion (DRG) after spared nerve injury (SNI). Ataxia telangiectasia mutated (ATM), which promotes γH2AX, was also down-regulated in DRG after peripheral nerve injury. ATM inhibitor KU55933 decreased the level of γH2AX in ND7/23 cells. The intrathecal injection of KU55933 down-regulated DRG γH2AX expression and significantly induced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. The inhibition of ATM by siRNA could also decrease the pain threshold. The inhibition of dephosphorylation of γH2AX by protein phosphatase 2A (PP2A) siRNA partially suppressed the down-regulation of γH2AX after SNI and relieved pain behavior. Further exploration of the mechanism revealed that inhibiting ATM by KU55933 up-regulated extracellular-signal regulated kinase (ERK) phosphorylation and down-regulated potassium ion channel genes, such as potassium voltage-gated channel subfamily Q member 2 (Kcnq2) and potassium voltage-gated channel subfamily D member 2 (Kcnd2) in vivo, and KU559333 enhanced sensory neuron excitability in vitro. These preliminary findings imply that the down-regulation of γH2AX may contribute to neuropathic pain.

Involvement of Histone Lysine Crotonylation in the Regulation of Nerve-Injury-Induced Neuropathic Pain.

Histone lysine crotonylation (KCR), a novel epigenetic modification, is important in regulating a broad spectrum of biological processes and various diseases. However, whether KCR is involved in neuropathic pain remains to be elucidated. We found KCR occurs in macrophages, sensory neurons, and satellite glial cells of trigeminal ganglia (TG), neurons, astrocytes, and microglia of the medulla oblongata. KCR in TG was detected mainly in small and medium sensory neurons, to a lesser extent in large neurons. Peripheral nerve injury elevated KCR levels in macrophages in the trigeminal and dorsal root ganglia and microglia in the medulla oblongata but reduced KCR levels in sensory neurons. Inhibition of histone crotonyltransferases (p300) by intra-TG or intrathecal administration of C646 significantly alleviated partial infraorbital nerve transection (pIONT)- or spinal nerve ligation (SNL)-induced mechanical allodynia and thermal hyperalgesia. Intra-TG or intrathecal administration of Crotonyl coenzyme A trilithium salt to upregulate KCR dose-dependently induced mechanical allodynia and thermal hyperalgesia in mice. Mechanismly, inhibition of p300 alleviated pIONT-induced macrophage activation and reduced the expression of pain-related inflammatory cytokines Tnfα, Il1ß and chemokines Ccl2 and Cxcl10. Correspondingly, exogenous crotonyl-CoA induced macrophage activation and the expression of Tnfα, Il1ß, Il6, Ccl2 and Ccl7 in TG, which C646 can repress. These findings suggest that histone crotonylation might be functionally involved in neuropathic pain and neuroinflammation regulation.

Decoding gene expression signatures in mice trigeminal ganglion across trigeminal neuropathic pain stages via high-throughput sequencing.

Trigeminal neuropathic pain (TNP) arises due to peripheral nerve injury, the mechanisms underlying which are little known. The altered gene expression profile in sensory ganglia is critical for neuropathic pain generation and maintenance. We, therefore, assessed the transcriptome of the trigeminal ganglion (TG) from mice at different periods of pain progression. Trigeminal neuropathic pain was established by partial infraorbital nerve transection (pIONT). High-throughput RNA sequencing was applied to detect the mRNA profiles of TG collected at 3 and 10 days after modeling. Injured TG displayed dramatically altered mRNA expression profiles compared to Sham. Different gene expression profiles were obtained at 3 and 10 days after pIONT. Moreover, 314 genes were significantly upregulated, and 81 were significantly downregulated at both 3 and 10 days post-pIONT. Meanwhile, enrichment analysis of these persistent differentially expressed genes (DEGs) showed that the MAPK pathway was the most significantly enriched pathway for upregulated DEGs, validated by immunostaining. In addition, TG cell populations defined by single-nuclei RNA sequencing displayed cellular localization of DEGs at a single-cell resolution. Protein-protein interaction (PPI) and sub-PPI network analyses constructed networks and identified the top 10 hub genes for DEGs at different time points. The present data provide novel information on the gene expression signatures of TG during the development and maintenance phases of TNP, and the identified hub genes and pathways may serve as potential targets for treatment.

NFAT1 Orchestrates Spinal Microglial Transcription and Promotes Microglial Proliferation via c-MYC Contributing to Nerve Injury-Induced Neuropathic Pain.

Peripheral nerve injury-induced spinal microglial proliferation plays a pivotal role in neuropathic pain. So far, key intracellular druggable molecules involved in this process are not identified. The nuclear factor of activated T-cells (NFAT1) is a master regulator of immune cell proliferation. Whether and how NFAT1 modulates spinal microglial proliferation during neuropathic pain remain unknown. Here it is reported that NFAT1 is persistently upregulated in microglia after spinal nerve ligation (SNL), which is regulated by TET2-mediated DNA demethylation. Global or microglia-specific deletion of Nfat1 attenuates SNL-induced pain and decreases excitatory synaptic transmission of lamina II neurons. Furthermore, deletion of Nfat1 decreases microglial proliferation and the expression of multiple microglia-related genes, such as cytokines, transmembrane signaling receptors, and transcription factors. Particularly, SNL increases the binding of NFAT1 with the promoter of Itgam, Tnf, Il-1b, and c-Myc in the spinal cord. Microglia-specific overexpression of c-MYC induces pain hypersensitivity and microglial proliferation. Finally, inhibiting NFAT1 and c-MYC by intrathecal injection of inhibitor or siRNA alleviates SNL-induced neuropathic pain. Collectively, NFAT1 is a hub transcription factor that regulates microglial proliferation via c-MYC and guides the expression of the activated microglia genome. Thus, NFAT1 may be an effective target for treating neuropathic pain.

C/EBPß Participates in Nerve Trauma-Induced TLR7 Upregulation in Primary Sensory Neurons.

Nerve trauma-induced toll-like receptor 7 (TLR7) expression level increases in primary sensory neurons in injured dorsal root ganglion (DRG) avails to neuropathic pain, but the reason is still unknown. In the current study, we showed that unilateral lumbar 4 (L4) spinal nerve ligation (SNL) upregulated CCAAT/enhancer-binding protein-ß (C/EBPß) expression in ipsilateral L4 DRG. Preventing this elevation attenuated the SNL-induced upregulation of TLR7 in the ipsilateral L4 DRG and inhibited cold/thermal hyperalgesia and mechanical allodynia. In injected DRG, mimicking nerve trauma-induced C/EBPß upregulation increased TLR7 levels, augmented responses to cold/thermal/mechanical stimuli, and caused ipsilateral spontaneous pain with no SNL. Mechanistically, SNL upregulated binding of increased C/EBPß to Tlr7 promoter in ipsilateral L4 DRG. Accorded that C/EBPß could trigger the activation of Tlr7 promoter and co-expressed with Tlr7 mRNA in individual DRG neurons, our findings strongly suggest the role of C/EBPß in nerve trauma-mediated TLR7 upregulation in injured primary sensory neurons.

ZNF382 controls mouse neuropathic pain via silencer-based epigenetic inhibition of Cxcl13 in DRG neurons.

Nerve injury-induced changes of gene expression in dorsal root ganglion (DRG) are critical for neuropathic pain genesis. However, how these changes occur remains elusive. Here we report the down-regulation of zinc finger protein 382 (ZNF382) in injured DRG neurons after nerve injury. Rescuing this down-regulation attenuates nociceptive hypersensitivity. Conversely, mimicking this down-regulation produces neuropathic pain symptoms, which are alleviated by C-X-C motif chemokine 13 (CXCL13) knockdown or its receptor CXCR5 knockout. Mechanistically, an identified cis-acting silencer at distal upstream of the Cxcl13 promoter suppresses Cxcl13 transcription via binding to ZNF382. Blocking this binding or genetically deleting this silencer abolishes the ZNF382 suppression on Cxcl13 transcription and impairs ZNF382-induced antinociception. Moreover, ZNF382 down-regulation disrupts the repressive epigenetic complex containing histone deacetylase 1 and SET domain bifurcated 1 at the silencer-promoter loop, resulting in Cxcl13 transcriptional activation. Thus, ZNF382 down-regulation is required for neuropathic pain likely through silencer-based epigenetic disinhibition of CXCL13, a key neuropathic pain player, in DRG neurons.

MMP24 Contributes to Neuropathic Pain in an FTO-Dependent Manner in the Spinal Cord Neurons.

Nerve injury-induced gene expression change in the spinal cord is critical for neuropathic pain genesis. RNA N6-methyladenosine (m6A) modification represents an additional layer of gene regulation. We showed that spinal nerve ligation (SNL) upregulated the expression of matrix metallopeptidase 24 (MMP24) protein, but not Mmp24 mRNA, in the spinal cord neurons. Blocking the SNL-induced upregulation of spinal MMP24 attenuated local neuron sensitization, neuropathic pain development and maintenance. Conversely, mimicking MMP24 increase promoted the spinal ERK activation and produced evoked nociceptive hypersensitivity. Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and RNA Immunoprecipitation (RIP) assay indicated the decreased m6A enrichment in the Mmp24 mRNA under neuropathic pain condition. Moreover, fat-mass and obesity-associated protein (FTO) was colocalized with MMP24 in spinal neurons and shown increased binding to the Mmp24 mRNA in the spinal cord after SNL. Overexpression or suppression of FTO correlates with promotion or inhibition of MMP24 expression in cultured spinal cord neurons. In conclusion, SNL promoted the m6A eraser FTO binding to the Mmp24 mRNA, which subsequently facilitated the translation of MMP24 in the spinal cord, and ultimately contributed to neuropathic pain genesis.

G protein-coupled receptor GPR151 is involved in trigeminal neuropathic pain through the induction of Gßγ/extracellular signal-regulated kinase-mediated neuroinflammation in the trigeminal ganglion.

ABSTRACT: Trigeminal nerve injury-induced neuropathic pain is a debilitating chronic orofacial pain syndrome but lacks effective treatment. G protein-coupled receptors (GPCRs), especially orphan GPCRs (oGPCRs) are important therapeutic targets in pain medicine. Here, we screened upregulated oGPCRs in the trigeminal ganglion (TG) after partial infraorbital nerve transection (pIONT) and found that Gpr151 was the most significantly upregulated oGPCRs. Gpr151 mRNA was increased from pIONT day 3 and maintained for more than 21 days. Furthermore, GPR151 was expressed in the neurons of the TG after pIONT. Global mutation or knockdown of Gpr151 in the TG attenuated pIONT-induced mechanical allodynia. In addition, the excitability of TG neurons was increased after pIONT in wild-type (WT) mice, but not in Gpr151-/- mice. Notably, GPR151 bound to Gαi protein, but not Gαq, Gα12, or Gα13, and activated the extracellular signal-regulated kinase (ERK) through Gßγ. Extracellular signal-regulated kinase was also activated by pIONT in the TG of WT mice, but not in Gpr151-/- mice. Gene microarray showed that Gpr151 mutation reduced the expression of a large number of neuroinflammation-related genes that were upregulated in WT mice after pIONT, including chemokines CCL5, CCL7, CXCL9, and CXCL10. The mitogen-activated protein kinase inhibitor (PD98059) attenuated mechanical allodynia and reduced the upregulation of these chemokines after pIONT. Collectively, this study not only revealed the involvement of GPR151 in the maintenance of trigeminal neuropathic pain but also identified GPR151 as a Gαi-coupled receptor to induce ERK-dependent neuroinflammation. Thus, GPR151 may be a potential drug target for the treatment of trigeminal neuropathic pain.

Intravenous Administration of Triptonide Attenuates CFA-Induced Pain Hypersensitivity by Inhibiting DRG AKT Signaling Pathway in Mice.

BACKGROUND: Currently, medical treatment of inflammatory pain is limited by a lack of safe and effective therapies. Triptonide (TPN), a major component of Tripterygium wilfordii Hook.f. with low toxicity, has been shown to have good anti-inflammatory and neuroprotective effects. The present study aims to investigate the effects of TPN on chronic inflammatory pain. MATERIALS AND METHODS: Inflammatory pain was induced by intraplantar injection of complete Freund's adjuvant (CFA). TPN's three different doses were intravenously administered to compare the analgesic efficacy: 0.1 mg/kg, 0.5 mg/kg, and 2.0 mg/kg. The foot swelling was quantitated by measuring paw volume. Mechanical allodynia and thermal hyperalgesia were assessed with von Frey filament testing and Hargreaves' test, respectively. Western blots, qRT-PCR and immunofluorescence tests were used to analyze the expression of pAKT, tumor necrosis factor-α (TNF-α), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6). Two AKT inhibitors, AKT inhibitor Ⅳ and MK-2206, were used to examine AKT's effects on pain behavior and cytokines expression. RESULTS: Intravenous treatment with TPN attenuated CFA-induced paw edema, mechanical allodynia, and thermal hyperalgesia. Western blotting and immunofluorescence results showed that CFA induced AKT activation in the dorsal root ganglion (DRG) neurons. However, these effects were suppressed by treatment with TPN. Furthermore, TPN treatment inhibited CFA-induced increase of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6. Consistent with the in vivo data, TPN inhibited LPS-induced Akt phosphorylation and inflammatory mediator production in ND7/23 cells. Finally, intrathecal treatment with AKT inhibitor Ⅳ or MK-2206, attenuated CFA-induced mechanical allodynia and thermal hyperalgesia, and simultaneously decreased the mRNA expression of TNF-α, IL-1ß, and IL-6 in DRG. CONCLUSION: These data indicate that TPN attenuates CFA-induced pain potentially via inhibiting AKT-mediated pro-inflammatory cytokines production in DRG. TPN may be used for the treatment of chronic inflammatory pain.

Chemokines in chronic pain: cellular and molecular mechanisms and therapeutic potential.

Chronic pain resulting from nerve injury, tissue inflammation, and tumor invasion or treatment, is a major health problem impacting the quality of life and producing a significant economic and social burden. However, the current analgesic drugs including non-steroidal anti-inflammatory drugs and opioids are inadequate to relieve chronic pain due to the lack of efficacy or severe side-effects. Chemokines are a family of small secreted proteins that bind to G protein-coupled receptors to trigger intracellular signaling pathways and direct cell migration, proliferation, survival, and inflammation under homeostatic and pathological conditions. Accumulating evidence supports the important role of chemokines and chemokine receptors in the peripheral and central nervous system in mediating chronic pain via enhancing neuroinflammation. In this review, we focus on recent progress in understanding the comprehensive roles of chemokines and chemokine receptors in the generation and maintenance of different types of chronic pain, including neuropathic pain, inflammatory pain, cancer pain, and visceral pain. The current review also summarizes the upstream signaling of transcriptional and epigenetic regulation on the expression of chemokines and chemokine receptors as well as the downstream signaling of chemokine receptors underlying chronic pain. As chronic itch and chronic pain share some common mechanisms, we also discuss the emerging roles of chemokines and chemokine receptors in chronic itch. Targeting specific chemokines or chemokine receptors by siRNAs, blocking antibodies, or small-molecule antagonists may offer new therapeutic potential for the management of chronic pain.

Increased CXCL13 and CXCR5 in Anterior Cingulate Cortex Contributes to Neuropathic Pain-Related Conditioned Place Aversion.

Pain consists of sensory-discriminative and emotional-affective components. The anterior cingulate cortex (ACC) is a critical brain area in mediating the affective pain. However, the molecular mechanisms involved remain largely unknown. Our recent study indicated that C-X-C motif chemokine 13 (CXCL13) and its sole receptor CXCR5 are involved in sensory sensitization in the spinal cord after spinal nerve ligation (SNL). Whether CXCL13/CXCR5 signaling in the ACC contributes to the pathogenesis of pain-related aversion remains unknown. Here, we showed that SNL increased the CXCL13 level and CXCR5 expression in the ACC after SNL. Knockdown of CXCR5 by microinjection of Cxcr5 shRNA into the ACC did not affect SNL-induced mechanical allodynia but effectively alleviated neuropathic pain-related place avoidance behavior. Furthermore, electrophysiological recording from layer II-III neurons in the ACC showed that SNL increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), decreased the EPSC paired-pulse ratio, and increased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor/N-methyl-D-aspartate receptor ratio, indicating enhanced glutamatergic synaptic transmission. Finally, superfusion of CXCL13 onto ACC slices increased the frequency and amplitude of spontaneous EPSCs. Pre-injection of Cxcr5 shRNA into the ACC reduced the increase in glutamatergic synaptic transmission induced by SNL. Collectively, these results suggest that CXCL13/CXCR5 signaling in the ACC is involved in neuropathic pain-related aversion via synaptic potentiation.

TLR8 and its endogenous ligand miR-21 contribute to neuropathic pain in murine DRG.

Toll-like receptors (TLRs) are nucleic acid-sensing receptors and have been implicated in mediating pain and itch. Here we report that Tlr8 -/- mice show normal itch behaviors, but have defects in neuropathic pain induced by spinal nerve ligation (SNL) in mice. SNL increased TLR8 expression in small-diameter IB4+ DRG neurons. Inhibition of TLR8 in the DRG attenuated SNL-induced pain hypersensitivity. Conversely, intrathecal or intradermal injection of TLR8 agonist, VTX-2337, induced TLR8-dependent pain hypersensitivity. Mechanistically, TLR8, localizing in the endosomes and lysosomes, mediated ERK activation, inflammatory mediators' production, and neuronal hyperexcitability after SNL. Notably, miR-21 was increased in DRG neurons after SNL. Intrathecal injection of miR-21 showed the similar effects as VTX-2337 and inhibition of miR-21 in the DRG attenuated neuropathic pain. The present study reveals a previously unknown role of TLR8 in the maintenance of neuropathic pain, suggesting that miR-21-TLR8 signaling may be potential new targets for drug development against this type of chronic pain.

Demethylation of G-Protein-Coupled Receptor 151 Promoter Facilitates the Binding of Krüppel-Like Factor 5 and Enhances Neuropathic Pain after Nerve Injury in Mice.

G-protein-coupled receptors are considered to be cell-surface sensors of extracellular signals, thereby having a crucial role in signal transduction and being the most fruitful targets for drug discovery. G-protein-coupled receptor 151 (GPR151) was reported to be expressed specifically in the habenular area. Here we report the expression and the epigenetic regulation of GRP151 in the spinal cord after spinal nerve ligation (SNL) and the contribution of GPR151 to neuropathic pain in male mice. SNL dramatically increased GPR151 expression in spinal neurons. GPR151 mutation or spinal inhibition by shRNA alleviated SNL-induced mechanical allodynia and heat hyperalgesia. Interestingly, the CpG island in the GPR151 gene promoter region was demethylated, the expression of DNA methyltransferase 3b (DNMT3b) was decreased, and the binding of DNMT3b with GPR151 promoter was reduced after SNL. Overexpression of DNMT3b in the spinal cord decreased GPR151 expression and attenuated SNL-induced neuropathic pain. Furthermore, Krüppel-like factor 5 (KLF5), a transcriptional factor of the KLF family, was upregulated in spinal neurons, and the binding affinity of KLF5 with GPR151 promoter was increased after SNL. Inhibition of KLF5 reduced GPR151 expression and attenuated SNL-induced pain hypersensitivity. Further mRNA microarray analysis revealed that mutation of GPR151 reduced the expression of a variety of pain-related genes in response to SNL, especially mitogen-activated protein kinase (MAPK) signaling pathway-associated genes. This study reveals that GPR151, increased by DNA demethylation and the enhanced interaction with KLF5, contributes to the maintenance of neuropathic pain via increasing MAPK pathway-related gene expression.SIGNIFICANCE STATEMENT G-protein-coupled receptors (GPCRs) are targets of various clinically approved drugs. Here we report that SNL increased GPR151 expression in the spinal cord, and mutation or inhibition of GPR151 alleviated SNL-induced neuropathic pain. In addition, SNL downregulated the expression of DNMT3b, which caused demethylation of GPR151 gene promoter, facilitated the binding of transcriptional factor KLF5 with the GPR151 promoter, and further increased GPR151 expression in spinal neurons. The increased GPR151 may contribute to the pathogenesis of neuropathic pain via activating MAPK signaling and increasing pain-related gene expression. Our study reveals an epigenetic mechanism underlying GPR151 expression and suggests that targeting GPR151 may offer a new strategy for the treatment of neuropathic pain.

Chemokine receptor CCR2 contributes to neuropathic pain and the associated depression via increasing NR2B-mediated currents in both D1 and D2 dopamine receptor-containing medium spiny neurons in the nucleus accumbens shell.

Patients with neuropathic pain are usually accompanied by depression. Chemokine-mediated neuroinflammation is involved in a variety of diseases, including neurodegenerative diseases, depression, and chronic pain. The nucleus accumbens (NAc) is an important area in mediating pain sensation and depression. Here we report that spinal nerve ligation (SNL) induced upregulation of chemokine CCL2 and its major receptor CCR2 in both dopamine D1 and D2 receptor (D1R and D2R)-containing neurons in the NAc. Inhibition of CCR2 by shRNA lentivirus in the NAc shell attenuated SNL-induced pain hypersensitivity and depressive behaviors. Conversely, intra-NAc injection of CCL2-expressing lentivirus-induced nociceptive and depressive behaviors in naïve mice. Whole-cell patch clamp recording of D1R-positive or D2R-positive medium spiny neurons (MSNs) showed that SNL increased NMDA receptor (NMDAR)-mediated currents that are induced by stimulation of prefrontal cortical afferents to MSNs, which was inhibited by a CCR2 antagonist. Furthermore, Ccr2 shRNA also reduced NMDAR-mediated currents, and this reduction was mainly mediated via NR2B subunit. Consistently, NR2B, colocalized with CCR2 in the NAc, was phosphorylated after SNL and was inhibited by intra-NAc injection of Ccr2 shRNA. Furthermore, SNL or CCL2 induced ERK activation in the NAc. Inhibition of ERK by a MEK inhibitor reduced NR2B phosphorylation induced by SNL or CCL2. Finally, intra-NAc injection of NR2B antagonist or MEK inhibitor attenuated SNL-induced pain hypersensitivity and depressive behaviors. Collectively, these results suggest that CCL2/CCR2 signaling in the NAc shell is important in mediating neuropathic pain and depression via regulating NR2B-mediated NMDAR function in D1R- and D2R-containing neurons following peripheral nerve injury.

Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury.

Chemokines-mediated neuroinflammation in the spinal cord plays a critical role in the pathogenesis of neuropathic pain. Chemokine CXCL9, CXCL10, and CXCL11 have been identified as a same subfamily chemokine which bind to CXC chemokine receptor 3 to exert functions. Our recent work found that CXCL10 is upregulated in spinal astrocytes after spinal nerve ligation (SNL) and acts on chemokine receptor CXCR3 on neurons to contribute to central sensitization and neuropathic pain, but less is known about CXCL9 and CXCL11 in the maintenance of neuropathic pain. Here, we report that CXCL9 and CXCL11, same as CXCL10, were increased in spinal astrocytes after SNL. Surprisingly, inhibition of CXCL9 or CXCL11 by spinal injection of shRNA lentivirus did not attenuate SNL-induced neuropathic pain. In addition, intrathecal injection of CXCL9 and CXCL11 did not produce hyperalgesia or allodynia behaviors, and neither of them induced ERK activation, a marker of central sensitization. Whole-cell patch clamp recording on spinal neurons showed that CXCL9 and CXCL11 enhanced both excitatory synaptic transmission and inhibitory synaptic transmission, whereas CXCL10 only produced an increase in excitatory synaptic transmission. These results suggest that, although the expression of CXCL9 and CXCL11 are increased after SNL, they may not contribute to the maintenance of neuropathic pain.

Chemokine CCL8 and its receptor CCR5 in the spinal cord are involved in visceral pain induced by experimental colitis in mice.

Visceral hypersensitivity induced by inflammatory bowel disease (IBD) is a clinical challenge since the underlying mechanisms remain elusive. Chemokines and their receptors have been suggested to modulate inflammatory pain and neuropathic pain. However, the exact chemokines involved in visceral pain remain to be determined. Here, we investigated the effects of spinal chemokine CCL8 and its major receptor CCR5 on the development of visceral hyperalgesia. We showed that intracolonic injection of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice produced significant colonic inflammation and visceral hypersensitivity to colorectal distension. Moreover, the mRNA and protein expression of CCL8 and CCR5 in the lumbosacral spinal cord were significantly upregulated. Both of CCL8 and CCR5 were expressed in spinal neurons. Furthermore, TNBS induced the activation of extracellular signal-regulated kinase (ERK) in the spinal cord. The induction of visceral pain by TNBS was attenuated by injection of ERK upstream kinase (MEK) inhibitor PD98059. Finally, intrathecal CCL8 neutralizing antibody or CCR5 antagonist DAPTA dose-dependently suppressed TNBS-evoked visceral hyperalgesia and spinal ERK activation. Taken together, these data demonstrated that CCL8 and CCR5, expressed and upregulated in spinal neurons after colonic inflammation, are involved in the maintenance of visceral hyperalgesia via the activation of spinal ERK. Targeting CCL8/CCR5/ERK pathway in the spinal cord might provide a novel treatment for the relief of visceral pain.

Chemokines in neuron-glial cell interaction and pathogenesis of neuropathic pain.

Neuropathic pain resulting from damage or dysfunction of the nervous system is a highly debilitating chronic pain state and is often resistant to currently available treatments. It has become clear that neuroinflammation, mainly mediated by proinflammatory cytokines and chemokines, plays an important role in the establishment and maintenance of neuropathic pain. Chemokines were originally identified as regulators of peripheral immune cell trafficking and were also expressed in neurons and glial cells in the central nervous system. In recent years, accumulating studies have revealed the expression, distribution and function of chemokines in the spinal cord under chronic pain conditions. In this review, we provide evidence showing that several chemokines are upregulated after peripheral nerve injury and contribute to the pathogenesis of neuropathic pain via different forms of neuron-glia interaction in the spinal cord. First, chemokine CX3CL1 is expressed in primary afferents and spinal neurons and induces microglial activation via its microglial receptor CX3CR1 (neuron-to-microglia signaling). Second, CCL2 and CXCL1 are expressed in spinal astrocytes and act on CCR2 and CXCR2 in spinal neurons to increase excitatory synaptic transmission (astrocyte-to-neuron signaling). Third, we recently identified that CXCL13 is highly upregulated in spinal neurons after spinal nerve ligation and induces spinal astrocyte activation via receptor CXCR5 (neuron-to-astrocyte signaling). Strategies that target chemokine-mediated neuron-glia interactions may lead to novel therapies for the treatment of neuropathic pain.

Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice.

DNA methylation has been implicated in the pathogenesis of chronic pain. However, the specific genes regulated by DNA methylation under neuropathic pain condition remain largely unknown. Here we investigated how chemokine receptor CXCR3 is regulated by DNA methylation and how it contributes to neuropathic pain induced by spinal nerve ligation (SNL) in mice. SNL increased Cxcr3 mRNA and protein expression in the neurons of the spinal cord. Meanwhile, the CpG (5'-cytosine-phosphate-guanine-3') island in the Cxcr3 gene promoter region was demethylated, and the expression of DNA methyltransferase 3b (DNMT3b) was decreased. SNL also increased the binding of CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine)/enhancer binding protein α (C/EBPα) with Cxcr3 promoter and decreased the binding of DNMT3b with Cxcr3 promoter in the spinal cord. C/EBPα expression was increased in spinal neurons after SNL, and inhibition of C/EBPα by intrathecal small interfering RNA attenuated SNL-induced pain hypersensitivity and reduced Cxcr3 expression. Furthermore, SNL-induced mechanical allodynia and heat hyperalgesia were markedly reduced in Cxcr3-/- mice. Spinal inhibition of Cxcr3 by shRNA or CXCR3 antagonist also attenuated established neuropathic pain. Moreover, CXCL10, the ligand of CXCR3, was increased in spinal neurons and astrocytes after SNL. Superfusing spinal cord slices with CXCL10 enhanced spontaneous EPSCs and potentiated NMDA-induced and AMPA-induced currents of lamina II neurons. Finally, intrathecal injection of CXCL10 induced CXCR3-dependent pain hypersensitivity in naive mice. Collectively, our results demonstrated that CXCR3, increased by DNA demethylation and the enhanced interaction with C/EBPα, can be activated by CXCL10 to facilitate excitatory synaptic transmission and contribute to the maintenance of neuropathic pain. SIGNIFICANCE STATEMENT: Peripheral nerve injury induces changes of gene expression in the spinal cord that may contribute to the pathogenesis of neuropathic pain. CXCR3 is a chemokine receptor. Whether it is involved in neuropathic pain and how it is regulated after nerve injury remain largely unknown. Our study demonstrates that spinal nerve ligation downregulates the expression of DNMT3b, which may cause demethylation of Cxcr3 gene promoter and facilitate the binding of CCAAT/enhancer binding protein α with Cxcr3 promoter and further increase CXCR3 expression in spinal neurons. The upregulated CXCR3 may contribute to neuropathic pain by facilitating central sensitization. Our study reveals an epigenetic mechanism underlying CXCR3 expression and also suggests that targeting the expression or activation of CXCR3 signaling may offer new therapeutics for neuropathic pain.