Bao-Chun Jiang

CXCL13 drives spinal astrocyte activation and neuropathic pain via CXCR5

Recent studies have implicated chemokines in microglial activation and pathogenesis of neuropathic pain. C-X-C motif chemokine 13 (CXCL13) is a B lymphocyte chemoattractant that activates CXCR5. Using the spinal nerve ligation (SNL) model of neuropathic pain, we found that CXCL13 was persistently upregulated in spinal cord neurons after SNL, resulting in spinal astrocyte activation via CXCR5 in mice. shRNA-mediated inhibition of CXCL13 in the spinal cord persistently attenuated SNL-induced neuropathic pain. Interestingly, CXCL13 expression was suppressed by miR-186-5p, a microRNA that colocalized with CXCL13 and was downregulated after SNL. Spinal overexpression of miR-186-5p decreased CXCL13 expression, alleviating neuropathic pain. Furthermore, SNL induced CXCR5 expression in spinal astrocytes, and neuropathic pain was abrogated in Cxcr5-/- mice. CXCR5 expression induced by SNL was required for the SNL-induced activation of spinal astrocytes and microglia. Intrathecal injection of CXCL13 was sufficient to induce pain hypersensitivity and astrocyte activation via CXCR5 and ERK. Finally, intrathecal injection of CXCL13-activated astrocytes induced mechanical allodynia in naive mice. Collectively, our findings reveal a neuronal/astrocytic interaction in the spinal cord by which neuronally produced CXCL13 activates astrocytes via CXCR5 to facilitate neuropathic pain. Thus, miR-186-5p and CXCL13/CXCR5-mediated astrocyte signaling may be suitable therapeutic targets for neuropathic pain.

Chemokine CXCL1 enhances inflammatory pain and increases NMDA receptor activity and COX-2 expression in spinal cord neurons via activation of CXCR2.

Recent studies have shown that CXCL1 upregulation in spinal astrocytes is involved in the maintenance of neuropathic pain. However, whether and how CXCL1 regulates inflammatory pain remains unknown. Here we show that intraplantar injection of CFA increased mRNA and protein expressions of CXCL1 and its major receptor CXCR2 in the spinal cord at 6h and 3days after the injection. Immunofluorescence double staining showed that CXCL1 and CXCR2 were expressed in spinal astrocytes and neurons, respectively. Intrathecal injection of CXCL1 neutralizing antibody or CXCR2 antagonist SB225002 attenuated CFA-induced mechanical and heat hypersensitivity on post-CFA day 3. Patch-clamp recordings showed that CXCL1 potentiated NMDA-induced currents in lamina II neurons via CXCR2, and this potentiation was further increased in CFA-treated mice. Furthermore, intrathecal injection of CXCL1 increased COX-2 expression in dorsal horn neurons, which was blocked by pretreatment with SB225002 or MEK (ERK kinase) inhibitor PD98059. Finally, pretreatment with SB225002 or PD98059 decreased CFA-induced heat hyperalgesia and COX-2 mRNA/protein expression and ERK activation in the spinal cord. Taken together, our data suggest that CXCL1, upregulated and released by spinal astrocytes after inflammation, acts on CXCR2-expressing spinal neurons to increase ERK activation, synaptic transmission and COX-2 expression in dorsal horn neurons and contributes to the pathogenesis of inflammatory pain.

TRAF6 upregulation in spinal astrocytes maintains neuropathic pain by integrating TNF-α and IL-1β signaling.

Summary TRAF6, upregulated in spinal cord astrocytes in the late phase after nerve injury, maintains neuropathic pain by integrating tumor necrosis factor &agr; and interleukin 1&bgr; signaling and activating the JNK/CCL2 pathway in astrocytes. ABSTRACT The proinflammatory cytokines tumor necrosis factor (TNF) &agr; and interleukin (IL) 1&bgr; have been strongly implicated in the pathogenesis of neuropathic pain, but the intracellular signaling of these cytokines in glial cells is not fully understood. TNF receptor‐associated factor 6 (TRAF6) plays a key role in signal transduction in the TNF receptor superfamily and the IL‐1 receptor superfamily. In this study, we investigated the role of TRAF6 in neuropathic pain in mice after spinal nerve ligation (SNL). SNL induced persistent TRAF6 upregulation in the spinal cord. Interestingly, TRAF6 was mainly colocalized with the astrocytic marker glial fibrillary acidic protein on SNL day 10 and partially expressed in microglia on SNL day 3. In cultured astrocytes, TRAF6 was upregulated after exposure to TNF‐&agr; or IL‐1&bgr;. TNF‐&agr; or IL‐1&bgr; also increased CCL2 expression, which was suppressed by both siRNA and shRNA targeting TRAF6. TRAF6 siRNA treatment also inhibited the phosphorylation of c‐Jun N‐terminal kinase (JNK) in astrocytes induced by TNF‐&agr; or IL‐1&bgr;. JNK inhibitor D‐NKI‐1 dose‐dependently decreased IL‐1&bgr;–induced CCL2 expression. Moreover, spinal injection of TRAF6 siRNA decreased intrathecal TNF‐&agr;– or IL‐1&bgr;–induced allodynia and hyperalgesia. Spinal TRAF6 inhibition via TRAF6 siRNA, shRNA lentivirus, or antisense oligodeoxynucleotides partially reversed SNL‐induced neuropathic pain and spinal CCL2 expression. Finally, intrathecal injection of TNF‐&agr;–activated astrocytes induced mechanical allodynia, which was attenuated by pretreatment of astrocytes with TRAF6 siRNA. Taken together, the results suggest that TRAF6, upregulated in spinal cord astrocytes in the late phase after nerve injury, maintains neuropathic pain by integrating TNF‐&agr; and IL‐1&bgr; signaling and activating the JNK/CCL2 pathway in astrocytes.

Ligustilide attenuates inflammatory pain via inhibition of NFκB-mediated chemokines production in spinal astrocytes

Ligustilide (LIG) is a major component of Radix Angelica Sinensis, and reportedly has neuroprotective and anti‐inflammatory effects. Recent studies have demonstrated that spinal astrocyte‐mediated neuroinflammation plays an important role in the pathogenesis of chronic pain. Here we investigated the anti‐nociceptive effect of systemic treatment with LIG on chronic inflammatory pain and explored possible mechanisms. Unilateral hindpaw injection of complete Freunds adjuvant (CFA) induced persistent pain hypersensitivity. Repeated daily intravenous treatment with LIG, either before or after CFA injection, attenuated CFA‐induced thermal hyperalgesia and mechanical allodynia. The same treatment also inhibited CFA‐induced keratinocyte‐derived chemokine (KC) and monocyte chemoattractant protein‐1 (MCP‐1) mRNA and protein increases in astrocytes of the spinal cord. In vitro study showed LIG dose‐dependently reduced lipopolysaccharide (LPS)‐induced upregulation of KC and MCP‐1 mRNA in astrocyte cultures. Interestingly, LIG treatment did not affect CFA‐ or LPS‐induced glial fibrillary acidic protein upregulation, but did inhibit CFA‐induced phosphorylated nuclear factor‐κB (p‐NFκB) upregulation in spinal astrocytes. Furthermore, intrathecal injection of NFκB inhibitor attenuated CFA‐induced pain hypersensitivity and upregulation of KC and MCP‐1 in the spinal cord. Finally, single intravenous injection of LIG attenuated intrathecal injection of LPS‐induced mechanical allodynia. The same treatment also decreased LPS‐induced NFκB activation and KC and MCP‐1 upregulation in the spinal cord. These data indicate that LIG attenuates chronic inflammatory pain potentially via inhibiting NFκB‐mediated chemokines production in spinal astrocytes. These results provide direct evidence of the anti‐nociceptive and anti‐inflammatory effects of LIG, suggesting a new application of LIG for the treatment of chronic inflammatory pain.

MicroRNA-146a-5p attenuates neuropathic pain via suppressing TRAF6 signaling in the spinal cord

Glia-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. Our recent study demonstrated that TNF receptor associated factor-6 (TRAF6) is expressed in spinal astrocytes and contributes to the maintenance of spinal nerve ligation (SNL)-induced neuropathic pain. MicroRNA (miR)-146a is a key regulator of the innate immune response and was shown to target TRAF6 and reduce inflammation. In this study, we found that in cultured astrocytes, TNF-α, IL-1β, or lipopolysaccharide (LPS) induced rapid TRAF6 upregulation and delayed miR-146a-5p upregulation. In addition, miR-146a-5p mimic blocked LPS-induced TRAF6 upregulation, as well as LPS-induced c-Jun N-terminal kinase (JNK) activation and chemokine CCL2 expression in astrocytes. Notably, LPS incubation with astrocytes enhanced the DNA binding activity of AP-1 to the promoters of mir-146a and ccl2. TRAF6 siRNA or JNK inhibitor SP600125 significantly reduced LPS-induced miR-146a-5p increase in astrocytes. In vivo, intrathecal injection of TNF-α or LPS increased spinal TRAF6 expression. Pretreatment with miR-146a-5p mimic alleviated TNF-α- or LPS-induced mechanical allodynia and reduced TRAF6 expression. Finally, SNL induced miR-146a-5p upregulation in the spinal cord at 10 and 21days. Intrathecal injection of miR-146a-5p mimic attenuated SNL-induced mechanical allodynia and decreased spinal TRAF6 expression. Taken together, the results suggest that (1) miR-146a-5p attenuates neuropathic pain partly through inhibition of TRAF6 and its downstream JNK/CCL2 signaling, (2) miR-146a-5p is increased by the activation of TRAF6/JNK pathway. Hence, miR-146a-5p may be a novel treatment for chronic neuropathic 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.

Exogenous induction of HO-1 alleviates vincristine-induced neuropathic pain by reducing spinal glial activation in mice.

Chemotherapy drugs such as vincristine can produce painful peripheral neuropathy for which is still lack of effective treatment. Recent studies have demonstrated that neuroinflammation plays an important role in the pathogenesis of neuropathic pain. Heme oxygenase 1 (HO-1) was shown to mediate the resolution of inflammation. In this study, we investigated the contribution of HO-1 in the modulation of vincristine-induced pain and the mechanisms implicated. Injection of vincristine induced persistent mechanical allodynia and thermal hyperalgesia in mice. The expression of HO-1 mRNA and protein was increased in 2 weeks in the spinal cord. Immunostaining showed that HO-1 was mainly expressed in neurons of spinal cord dorsal horn in naïve animals, but induced in astrocytes and microglia after vincristine injection. Intraperitoneal injection of HO-1 inducer increased HO-1 expression in the spinal cord and attenuated vincristine-induced pain. Persistent induction of HO-1 by intraspinal injection of HO-1-expressing lentivirus alleviated vincristine-induced pain for more than 2 weeks. Furthermore, vincristine induced activation of glial cells (astrocytes and microglia), phosphorylation of MAPKs (JNK, ERK, and p38), and production of TNF-α and monocyte chemoattractant protein-1 in the spinal cord, which were all reduced by intrathecal injection of HO-1 inducer. Taken together, our data provide the first evidence that induction of HO-1 attenuates vincristine-induced neuropathic pain via inhibition of glia-mediated neuroinflammation in the spinal cord. This suggests that exogenously induced HO-1 may have potential as therapy in chemotherapy-induced neuropathic pain.

Chemokine CXCL13 mediates orofacial neuropathic pain via CXCR5/ERK pathway in the trigeminal ganglion of mice

BackgroundTrigeminal nerve damage-induced neuropathic pain is a severely debilitating chronic orofacial pain syndrome. Spinal chemokine CXCL13 and its receptor CXCR5 were recently demonstrated to play a pivotal role in the pathogenesis of spinal nerve ligation-induced neuropathic pain. Whether and how CXCL13/CXCR5 in the trigeminal ganglion (TG) mediates orofacial pain are unknown.MethodsThe partial infraorbital nerve ligation (pIONL) was used to induce trigeminal neuropathic pain in mice. The expression of ATF3, CXCL13, CXCR5, and phosphorylated extracellular signal-regulated kinase (pERK) in the TG was detected by immunofluorescence staining and western blot. The effect of shRNA targeting on CXCL13 or CXCR5 on pain hypersensitivity was checked by behavioral testing.ResultspIONL induced persistent mechanical allodynia and increased the expression of ATF3, CXCL13, and CXCR5 in the TG. Inhibition of CXCL13 or CXCR5 by shRNA lentivirus attenuated pIONL-induced mechanical allodynia. Additionally, pIONL-induced neuropathic pain and the activation of ERK in the TG were reduced in Cxcr5−/− mice. Furthermore, MEK inhibitor (PD98059) attenuated mechanical allodynia and reduced TNF-α and IL-1β upregulation induced by pIONL. TNF-α inhibitor (Etanercept) and IL-1β inhibitor (Diacerein) attenuated pIONL-induced orofacial pain. Finally, intra-TG injection of CXCL13 induced mechanical allodynia, increased the activation of ERK and the production of TNF-α and IL-1β in the TG of WT mice, but not in Cxcr5−/− mice. Pretreatment with PD98059, Etanercept, or Diacerein partially blocked CXCL13-induced mechanical allodynia, and PD98059 also reduced CXCL13-induced TNF-α and IL-1β upregulation.ConclusionsCXCL13 and CXCR5 contribute to orofacial pain via ERK-mediated proinflammatory cytokines production. Targeting CXCL13/CXCR5/ERK/TNF-α and IL-1β pathway in the trigeminal ganglion may offer effective treatment for orofacial 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.

Altered T-UCRs expression profile in the spinal cord of mice with neuropathic pain

Spinal cord plays an important role in the transmission and modulation of nociceptive information. Global changes in gene expression in the spinal cord contribute to the induction and maintenance of neuropathic pain. Transcribed Ultraconserved Regions (T-UCRs), a novel class of long noncoding RNAs, can regulate gene expression at both transcriptional and post-transcriptional levels and are related to many human diseases such as cancer, Alzheimers disease, and heart diseases. In this study, we screened abnormal T-UCRs expression in the spinal cord under spinal nerve ligation (SNL)-induced neuropathic pain condition. Microarray data showed the alternation of T-UCRs at the transcriptional level in the spinal cord 10 days after SNL. Among 78 altered T-UCRs, 23 T-UCRs were upregulated by more than 1.5-fold and 55 ones downregulated by less than 0.5-fold after SNL. Hierarchical cluster analysis of T-UCRs expression profiles showed the opposite expression pattern between SNL and sham-operated mice. The quantitative real-time reverse transcription polymerase chain reaction analysis further confirmed the expression patterns of uc.305, uc.189, uc.46, and uc.217 after SNL. The gene ontology annotation and signaling pathway analysis for the T-UCRs host genes indicated that differentially expressed T-UCRs were involved in several intracellular activities and signaling pathways, including Ephrin receptor activity, soluble NSF attachment protein receptor (SNARE) interactions in vesicular transport pathway, and WNT signaling pathway. Collectively, the current data suggest the possible role of T-UCR in the pathogenesis of neuropathic pain. T-UCRs may serve as a new kind of target for the treatment of neuropathic pain.