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

Abstract

Background: Trigeminal 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.

Methods: The 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.

Results: pIONL 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.

Conclusions: CXCL13 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.

Keywords: CXCL13; CXCR5; ERK; Orofacial pain; Proinflammatory cytokines; Trigeminal ganglion.

Fig. 1

pIONL induces mechanical allodynia assessed by orofacial operant test and increases ATF3 expression in the maxillary division of TG neurons. a Total contact number of orofacial operant test with mechanical stimulation was comparable between sham and pIONL mice. P > 0.05. Two-way repeated measures ANOVA. b Total contact time was significantly decreased from 1 to 21 days after pIONL, compared to sham group. **P < 0.01, ***P < 0.001, pIONL vs. sham. Two-way repeated measures ANOVA followed by Bonferroni test. c The percentage of ATF3-positive cells were dramatically increased in the maxillary division (V2) of TG 10 days after pIONL, whereas ATF3-positive cells were not significantly changed in the ophthalmic (V1) division or mandibular (V3) division. ***P < 0.001, pIONL vs. sham. Student’s t test. d–g Representative images show the expression of ATF3 in the TG of sham-treated (d, e) or pIONL (f, g) animals. e, g High-magnification images of d and f, indicated in the white boxes

Fig. 2

pIONL induces persistent CXCL13 expression in the TG. a The time course of Cxcl13 mRNA expression in the ipsilateral TG from naïve, sham, and pIONL-operated mice. pIONL increased Cxcl13 expression at 3, 10, and 21 days, compared to sham. *P < 0.05, ***P < 0.001. Two-way ANOVA followed by Bonferroni test. b Western blot shows increased CXCL13 protein level 10 days after pIONL, compared to sham. *P < 0.05. Student’s t test. c Representative images of CXCL13 immunofluorescence in the TG. CXCL13-IR was low in naïve mice (c) and sham mice (d), but increased in the TG of pIONL mice (e). f–h Double staining of CXCL13 (f) and neuronal marker β-III tubulin (g) shows the neuronal expression of CXCL13

Fig. 3

Inhibition of CXCL13 by shRNA lentivirus attenuated pIONL-induced mechanical allodynia. a Pretreatment with LV-Cxcl13 shRNA increased the contact time between 3 and 21 days after the operation, compared to LV-NC-injected mice. Intra-TG injection was performed 7 days before pIONL (arrow). *P < 0.05, **P < 0.01, ***P < 0.001. Two-way repeated measures ANOVA followed by Bonferroni test. b Posttreatment with LV-Cxcl13 shRNA increased the contact time between 7 and 14 days after the operation, compared to LV-NC treatment. Intra-TG injection was performed 3 days after pIONL (arrow). *P < 0.05, **P < 0.01. Two-way repeated measures ANOVA followed by Bonferroni test. c Representative fluorescence photomicrograph shows GFP expression in the TG 7 days after intra-TG infusion of lentivirus vector. d Real-time PCR assay of Cxcl13 shows that pretreatment with LV-Cxcl13 shRNA inhibited pIONL-induced Cxcl13 upregulation. **P < 0.01, ***P < 0.001. Student’s t test

Fig. 4

pIONL induces persistent CXCR5 expression in the TG. a The time course of Cxcr5 mRNA expression in the ipsilateral TG from naïve, sham, and pIONL-operated mice. pIONL increased Cxcr5 expression at 3, 10, and 21 days, compared to sham. *P < 0.05, **P < 0.01. Two-way ANOVA followed by Bonferroni test. b Western blot shows increased CXCR5 protein level 10 days after pIONL, compared to sham. *P < 0.05. One-way ANOVA followed by Bonferroni test. c Representative images of CXCR5 immunofluorescence in the TG. CXCR5-IR was low in naïve mice (c) and sham mice (d), but increased in the TG of pIONL mice (e). f–h Double staining of CXCR5 (f) and neuronal marker β-III tubulin (g) shows the neuronal expression of CXCR5

Fig. 5

CXCR5 is essential for pIONL-induced mechanical allodynia. a Mechanical sensitivity assessed by Von Frey test was indistinguishable in WT and KO mice. b pIONL-induced mechanical allodynia was significantly reduced in Cxcr5 KO mice, compared to WT mice. *P < 0.05, **P < 0.01. Two-way repeated measures ANOVA followed by Bonferroni test. c Pretreatment with LV-Cxcr5 shRNA increased the contact time between 3 and 21 days after the operation, compared to LV-NC-injected mice. *P < 0.05, ***P < 0.001. Two-way repeated measures ANOVA followed by Bonferroni test. d Posttreatment with LV-Cxcr5 shRNA increased the contact time between 7 and 21 days after the operation, compared to LV-NC treatment. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way repeated measures ANOVA followed by Bonferroni test. e Real-time PCR assay of Cxcr5 shows that pretreatment with LV-Cxcr5 shRNA inhibited pIONL-induced Cxcr5 upregulation. *P < 0.05, **P < 0.01, ***P < 0.001. Student’s t test

Fig. 6

pIONL induces the activation of ERK in the TG. a Western blot shows the expression of pERK in naive, sham, and pIONL mice. pIONL increased the expression of pERK expression in WT mice, and the increase was reduced in Cxcr5 KO mice. *P < 0.05. One-way ANOVA followed by Bonferroni test (left histogram) or Student’s t test (right histogram). b Representative images of pERK immunofluorescence in the TG of WT and KO mice 10 days after the operation. pERK-IR was increased after pIONL in WT mice, but not in KO mice

Fig. 7

pIONL-induced mechanical allodynia is mediated by ERK-dependent proinflammatory cytokines production in the TG. a pIONL increased TNF-α and IL-1β expression 10 days after pIONL in WT mice, but not in Cxcr5 KO mice. *P < 0.05, ***P < 0.001. One-way ANOVA followed by Bonferroni test. b Intra-TG injection of MEK inhibitor, PD98059 10 days after pIONL attenuated pIONL-induced mechanical allodynia at 3 h. **P < 0.01. Two-way repeated measures ANOVA followed by Bonferroni test. The same treatment reduced the expression of TNF-α (c) and IL-1β (d). **P < 0.01, Student’s t test. e Intra-TG injection of TNF-α inhibitor, Etanercept 10 days after pIONL alleviated pIONL-induced mechanical allodynia. The effect was shown 1 h after injection and maintained for more than 6 h. *P < 0.05, ***P < 0.001. Two-way repeated measures ANOVA followed by Bonferroni test. f Intra-TG injection of IL-1β inhibitor, Diacerein 10 days after pIONL alleviated pIONL-induced mechanical allodynia from 1 to 6 h. *P < 0.05, ***P < 0.001. Two-way repeated measures ANOVA followed by Bonferroni test

Fig. 8

Intra-TG injection of CXCL13 induces CXCR5/ERK-dependent mechanical allodynia and proinflammatory cytokines production. a, b Intra-TG injection of CXCL13 (100 ng) induced mechanical allodynia in WT mice (a), but not in Cxcr5 KO mice (b). ***P < 0.001, WT-vehicle vs. WT-CXL13. Two-way repeated measures ANOVA followed by Bonferroni test. c Western blot showing that intra-TG CXCL13 significantly increased pERK expression in the TG in WT mice, but not in Cxcr5 KO mice. *P < 0.05. Student’s t test. d Intra-TG injection of CXCL13 increases TNF-α and IL-1β expression at 1 and 3 h in WT mice, but not in Cxcr5 KO mice. *P < 0.05, **P < 0.01. One-way ANOVA followed by Bonferroni test. e Intra-TG injection of PD98059, Etanercept, or Diacerein partially blocked intra-TG CXCL13-induced mechanical allodynia. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way repeated measures ANOVA followed by Bonferroni test