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Abstract—Recent data demonstrated that chemokine CXCL13 mediates neuroinflammation and con- tributes to the maintenance of neuropathic pain after nerve ...
Inflammation ( # 2017) DOI: 10.1007/s10753-017-0520-x

ORIGINAL ARTICLE

Chemokine CXCL13 activates p38 MAPK in the trigeminal ganglion after infraorbital nerve injury Qian Zhang,1 Ming-Di Zhu,2 De-Li Cao,1 Xue-Qiang Bai,1 Yong-Jing Gao,1 and Xiao-Bo Wu

1,3

Abstract—Recent data demonstrated that chemokine CXCL13 mediates neuroinflammation and contributes to the maintenance of neuropathic pain after nerve injury in the spinal cord. Pro-nociceptive chemokines activate mitogen-activated protein kinases (MAPKs) which are potential signaling pathways contributing to the nociceptive behavior in inflammatory or neuropathic pain. However, whether activation of p38 and JNK MAPK signaling pathway in the trigeminal ganglion (TG) are involved in CXCL13 and its receptor CXCR5-mediated orofacial pain has not yet been clarified. Here, we show that the unilateral partial infraorbital nerve ligation (pIONL) induced a profound orofacial pain in wild-type (WT) mice. Western blot results showed that pIONL induced p38 but not JNK activation in the TG of WT mice. However, the orofacial pain induced by pIONL was alleviated in Cxcr5−/− mice, and the activation of p38 was also abrogated in Cxcr5−/− mice. Furthermore, intra-TG injection of CXCL13 evoked mechanical hypersensitivity and increased p-p38 expression in WT mice. But CXCL13 had no effect on pain behavior or p-p38 expression in Cxcr5−/− mice. Finally, pretreatment with p38 inhibitor, SB203580, attenuated the pIONL-induced mechanical allodynia and decreased the mRNA expression of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the TG. Taken together, our data suggest that CXCL13 acts on CXCR5 to increase p38 activation and further contributes to the pathogenesis of orofacial neuropathic pain. KEY WORDS: CXCL13; CXCR5; p38 MAPK; trigeminal ganglion; orofacial pain.

INTRODUCTION Orofacial pain is a common symptom in clinic, potentially resulting from surgery, trauma, or infection in craniofacial tissues including temporomandibular joint, periodontal tissue, masticatory muscles, and trigeminal nerve [1]. Trigeminal neuralgia is the most common orofacial neuropathic pain. Recent Qian Zhang and Ming-Di Zhu contributed equally to this work. 1

Pain Research Laboratory, Institute of Nautical Medicine, Jiangsu Key Laboratory of Inflammation and Molecular Drug Target, Nantong University, 9 Seyuan Road, Nantong, Jiangsu 226019, China 2 Department of Orthopedics, The Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China 3 To whom correspondence should be addressed at Pain Research Laboratory, Institute of Nautical Medicine, Jiangsu Key Laboratory of Inflammation and Molecular Drug Target, Nantong University, 9 Seyuan Road, Nantong, Jiangsu 226019, China. E-mail: [email protected]

studies have demonstrated that inflammatory mediators play an important role in the pathogenesis of neuropathic pain [2, 3]. The pro-inflammatory cytokines, such as IL-1β, TNF-α, and chemokines, such as CX3CL1 and CCL2, were increased in trigeminal ganglion after injury of infraorbital nerve or inflammation of temporalis muscle [4–6]. Blocking the function of chemokines including CCL2, CXCL1, and CXCL12 in the spinal cord or dorsal root ganglion markedly prevented hyperalgesia behavior induced by hind paw inflammation and incision [7–9]. The mitogen-activated protein kinases (MAPKs) are a family of intracellular signaling, which include extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). All the MAPKs are activated by phosphorylation and contribute to pathological conditions through distinct molecular and cellular mechanism [10]. Previous studies have

0360-3997/17/0000-0001/0 # 2017 Springer Science+Business Media New York

Zhang, Zhu, Cao, Bai, Gao, and Wu demonstrated that p38, JNK, and ERK are downstream signaling in responding to several pronociceptive chemokines activation and contribute to inflammation- or nerve injury-induced hyperalgesia [3, 7, 11, 12]. Dysregulation of p38 MAPK activation was associated with nociceptive behavior following injury of the trigeminal nerve root-induced neuropathic pain [13]. CXCL13 and its receptor CXCR5 have been detected in neural tissues including neurons and glia and are involved in neuroinflammation [14]. Our recent study demonstrated that the CXCL13/CXCR5 in the spinal cord participated in the development and maintenance of neuropathic pain through neuronal/astrocytic interaction [15] and also contributed to the trigeminal neuralgia through ERK signaling pathway in trigeminal ganglion [16]. Taking the above evidence together, we hypothesize that the other members of MAPKs’ family p38 and JNK may also function as downstream signal pathways for CXCL13/CXCR5 and contribute to the pathogenesis of orofacial pain. Therefore, we tested the orofacial pain behavior in WT and Cxcr5 knockout (KO) mice using the well-established partial infraorbital nerve ligation (pIONL) model. We also examined the protein expression for phosphorylated p38 (p-p38) and JNK (pJNK) in the trigeminal ganglion after pIONL or CXCL13 intra-TG injection. The results showed that p-p38 expression was inhibited in Cxcr5 KO mice and pIONL-induced mechanical allodynia was alleviated after inhibition of p38 activation.

MATERIALS AND METHOD Animals Adult ICR and C57/BL6 male mice (25–35 g) used for experiments were purchased from animal experiment central of Nantong University. Cxcr5−/− (KO) mice (B6.129S2 (Cg)-Cxcr5tm1Lipp/J) were purchased from the Jackson Laboratory. Animals were housed in standard clear plastic cages under a regular light-dark cycle with free access to food and water. All animal procedures performed in this study were reviewed and approved by the Animal Care and Use Committee of Nantong University and were performed in accordance with the guidelines of the International Association for the Study of Pain.

Drugs and Administration CXCL13 was purchased from Peprotech (USA). SB203580, an inhibitor of p38 activation, was purchased from Sigma (USA). The trigeminal ganglion injection was made with a 30-G needle via the infraorbital foramen along infraorbital canal to the middle of ganglia. Surgery As previously described [6], mice were anesthetized with sodium pentobarbital, laid on the back, and a modified partial infraorbital nerve ligation (pIONL) was carefully performed. In brief, the oral cavity was exposed, and a longitudinal incision (1 mm) on the buccal mucosa and at the level of the maxillary first molar was made to expose the left ION. The ION was carefully isolated using fine forceps without damaging nearby facial nerve branches. Approximately 1/2 the diameter of the nerve was tightly ligated with 8-0 silk suture and cut, and then the incision was closed. For the sham-operated mice, the ION was exposed on the left side using the same procedure without damaging ION. DNA Extraction and Genotyping About 3 mm of the mouse tails were cut from C57BL/ 6 wild-type and Cxcr5−/− mice and then used to extract DNA with the phenol-chloroform method. PCR was performed using the primer sets and genotyping protocol described below from the Jackson Laboratory. Primers included common forward primer oIMR7120: 5′-CGG AGA TTC CCC TAC AGG AC-3′, wild-type reverse primer oIMR7121: 5′-GAT CTT GTG CAG AGC GAT CA-3′, mutant reverse primer oIMR8963: 5′-AAT TCG CCA ATG ACA AGA CG-3′. For PCR amplification, approximately 500 ng DNA was used in a 50-μl reaction volume containing 25 μl 2× Taq PCR MasterMix (Tiangen Biotech) and 1 μM primers of oIMR7120, oIMR7121, or oIMR8963. Reactions initially were denatured at 94 °C for 3 min followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 2 min. Amplicons were separated using 1.5% agarose gel, stained with DuRed (Biotium) and photographed with the GelDoc-It Imaging System (UVP). Behavioral Testing Oral-Facial Stimulation Pain Behavior. After 12 h fasting of the previous night, mice were placed individually in cages and habituated for 10 min. Then the orofacial stimulation test (31300-002) by Ugo Basile was used to

Chemokine CXCL13 activates p38 MAPK in the trigeminal ganglion measure the hypersensitivity to mechanical stimulation of the trigeminal area within 10 min. The duration of feeding milk and the number of feeding attempts for animals were measured by interruption of an infrared barrier traversing the opening to the reward. The feeding time was indicative the pain threshold for animals. The ORO software (Ugo Basile, Italy) was used to behavior recording and analysis. Mechanical Allodynia. All behavior experiments were carried out in a quiet room. Mice were habituated to handling and testing equipment 30 min before experiments. A modified Dixon’s up-and-down method was used in behavior tested [17]. Briefly, a graded series of von Frey filaments (Semmes-Weinstein monofilaments, Stoelting, Wood Dale, IL) was used for mechanical stimulation of the left cheek. The brisk withdrawal of the head behavior was recorded as pain of response threshold. Testing six times of responds for each mouse were used. The stimulation always began with the lowest force filament. The lower the force that causes a response means, the lower the pain threshold for mice. Rota-rod Test. Mice were trained on the rota-rod for 3 min at a speed of 10 rpm, till the mice no longer fell off it. For testing, the speed was set at 10 rpm for 60 s and subsequently accelerated to 80 rpm in 5 min. The time taken for mice to fall after the beginning of the acceleration was recorded.

The Comparative CT Method (2_△△CT) was used to analysis the changes. Western Blot Animals were transcardially perfused with 0.01 M cold PBS. The ganglia were dissected and homogenized in a lysis buffer containing protease and phosphatase inhibitors (Roche, USA). Protein concentrations were determined by BCA Protein Assay (Thermo Scientific, USA). The protein (30 μg) was loaded for each lane and separated on SDS-PAGE gel. The separate the protein was transferred to the PVDF film via wet transfer method and blocked with 5% skim milk for 2 h. Then, the blots were incubated overnight at 4 °C with primary antibody. Primary antibody concentrations were as follows: rabbit against p-38 (1:1000, Cell Signaling) and rabbit against pp38 (1:1000, Cell Signaling); rabbit against JNK (1:1000, Cell Signaling) and rabbit against pJNK (1:1000, Cell Signaling). For loading control, the blots were probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (mouse, 1:20,000, Millipore). These blots were further incubated with secondary antibody and exposed on Hyperfilm (Odyssey, USA). Specific bands were evaluated by apparent molecular size. The intensity of the selected bands was analyzed using the ImageJ software (NIH, Bethesda, MD, USA). Statistical Analysis

Quantitative Real-Time RT-PCR Assay According to our previous study [6], Trizol reagent (Invitrogen, USA) was used to prepare total RNA from acutely isolated left trigeminal ganglia. The PCR reactions were run on a Rotor-Gene 3000 RT-PCR machine (Corbett Research, Qiagen Inc., USA). Total RNA (1 μg) was reversely transcripted to cDNA. The melting curves were performed to validate the specificity of each PCR product. The following primers were used for real-time PCR reactions: TNF-α forward, 5′-GTT CTATGG CCC AGA CCC TCA C-3′; TNF-α reverse, 5′-GGC ACC ACT AGT TGG TTG TCT TTG-3′; IL-1β forward, 5′-TCC AGG ATG AGG ACA TGA GCA C-3′; IL-1β reverse 5′-GAA CGT CAC ACA CCA GCA GGT TA-3′; GAPDH forward, 5′GCT TGA AGG TGT TGC CCT CAG-3′; GAPDH reverse, 5′-AGA AGC CAG CGT TCA CCA GAC-3′. PCR amplification was performed for 30 s at 95 °C and was followed by 40 cycles (5 s at 95 °C following 45 s at 60 °C) and extended 30 s at 72 °C. The PCR amplification of GAPDH mRNA was used as the normalization control.

Results are expressed as the mean ± SEM. The behavioral data was analyzed by two-way repeated measures ANOVA followed by Bonferroni test as the multiple comparison analysis. The qPCR data was analyzed by one-way ANOVA followed by Bonferroni test. An unpaired Student’s t test was used to determine the significant difference between two groups of data. P < 0.05 was considered significant.

RESULTS pIONL Induces the Activation of p38 but Not JNK in the TG After 3 days habituation, the basal feeding time was tested from sham-operated and pIONL mice and they showed similar total feeding times. The feeding time of sham group did not significantly changed 10 days after surgery. However, pIONL induced a markedly decrease in

Zhang, Zhu, Cao, Bai, Gao, and Wu

Fig. 1. pIONL induces p38 activation in the TG. a pIONL induced orofacial pain behavior in wild-type mice 3 and 10 days after surgery (***P < 0.001, twoway RM ANOVA followed with Bonferroni test). b Western blot shows that the p-JNK/JNK expression relative to naive was not changed in the trigeminal ganglion 10 days after pIONL (Student’s t test, naïve vs. pIONL, P > 0.05, n = 3 mice/group). c Western blot shows that the p-p38/p38 expression relative to naive was increased in the trigeminal ganglion 10 days after pIONL (one-way ANOVA, naïve vs. pIONL, ***P < 0.001; sham vs. pIONL, *P < 0.05; n = 3 mice per group).

feeding time compared with the sham-operated group (Fig. 1a, P < 0.001, two-way RM ANOVA). At postoperative day 10, we examined the expression of p-JNK, which has been demonstrated to be involved in the pathogenesis of neuropathic pain in the dorsal root ganglia (DRGs) and spinal cord [7, 18]. However, Western blot showed that the p-JNK expression was not changed in pIONL group compared with non-operated mice (Fig. 1b, P > 0.05, Student’s t test). We then checked p38 activation in the TG after pIONL. As shown in Fig. 1c, the p-p38 expression in the trigeminal ganglion was significantly increased in the ipsilateral side after pIONL, compared with naive or sham mice (Fig. 1c, P < 0.001, naive vs. pIONL; P < 0.05, sham vs. pIONT, one-way ANOVA). pIONL Induces CXCR5-Dependent p38 Activation in the TG To confirm the role of CXCR5 in orofacial pain, we measured the behavior in Cxcr5−/− mice before and after pIONL surgery. The genotype of WT and Cxcr5−/− was confirmed by RT-PCR (Fig. 2a). The feeding time in

Cxcr5−/− mice was significantly increased compared with WT group (Fig. 2b, P < 0.01, two-way RM ANOVA). We also examined the p38 activation in trigeminal ganglion in Cxcr5−/− mice. The expression of p-p38 was not changed in wild-type and Cxcr5−/− mice in sham-operated group (Fig. 2c, P > 0.05, Student’s t test). However, compared with the WT mice, the protein of p-p38 in TG was markedly decreased in Cxcr5−/− mice after 10 days of pIONL (Fig. 2c, P < 0.001, Student’s t test). CXCL13 Induces CXCR5-Dependent Mechanical Allodynia and p38 Activation Single intra-TG injection of CXCL13 (100 ng in 5 μl) in naïve WT mice resulted in decreased threshold of head withdraw as compared to those recorded in vehicle-administrated group (Fig. 3a, P < 0.001, two-way RM ANOVA). By contrast, behavior data showed that no significant change was observed in Cxcr5 −/− mice (Fig. 3b, P > 0.05, two-way RM ANOVA). Western-blot showed that p-p38 protein expression in the TG was significantly increased after 1 h of CXCL13 injection in WT mice (Fig. 3c,

Chemokine CXCL13 activates p38 MAPK in the trigeminal ganglion

Fig. 2. pIONL induces CXCR5-dependent p38 activation in the TG. a Genotyping of WT and Cxcr5−/− mice. b pIONL-induced orofacial pain was markedly attenuated in Cxcr5−/− animals (**P < 0.01, ***P < 0.001, two-way RM ANOVA followed with Bonferroni test). c Western blot shows that the p-p38/p38 expression relative to WT was significantly inhibited in Cxcr5−/− mice, compared with WT group (***P < 0.001, Student’s t test).

Fig. 3. CXCL13 induces CXCR5-dependent mechanical allodynia and p38 activation. a CXCL13 (100 ng in 5 μl) produced a significant mechanical allodynia after 1 h of intra-ganglionic injection (***P < 0.001, two-way RM ANOVA followed with Bonferroni test). b CXCL13 administration-induced mechanical allodynia was blocked in Cxcr5−/− animals (P > 0.05, two-way RM ANOVA followed with Bonferroni test). c Western blot shows that the level of p-p38 protein expression was increased in the trigeminal ganglion after 1 h of CXCL13 injection (*P < 0.05, Student’s t test), which was not significantly changed in Cxcr5−/− mice.

Zhang, Zhu, Cao, Bai, Gao, and Wu

Fig. 4. pION-induced pain is attenuated by p38 inhibitor. a The intra-ganglionic injection of SB203580, a potent and selective p-p38 antagonist, attenuated pIONL-induced mechanical allodynia 10 days after pIONL (***P < 0.001, two-way RM ANOVA followed with Bonferroni test). b No changes in the animals’ motor function following 3 h after intra-TG administration of the SB203580 in pIONL mice (P > 0.05, two-way RM ANOVA). c Real-time PCR results show the marked decrease of TNF-α and IL-1β mRNA level relative to control in the trigeminal ganglion intra-ganglionic injection of SB203580 in pIONL animals (**P < 0.01, Student’s t test).

P < 0.05, Student’s t test), but not significantly changed in Cxcr5 − / − mice (Fig. 3c, P > 0.05, Student’s t test). Inhibition of P38 Attenuated pIONL-Induced Pain and the Expression of TNF-α and IL-1β in the TG We then checked whether p-p38 MAPK is involved in pIONL-induced orofacial mechanical allodynia behavior. Intra-TG administration of SB203580 (1 μg in 5 μl), a p38 MAPK selective inhibitor, attenuated pIONL-induced mechanical allodynia, compared to the vehicle-treated group (Fig. 4a, P < 0.05, two-way RM ANOVA). The same treatment did not affect the animals’ motor function (Fig. 4b, P > 0.05, two-way RM ANOVA). Previous studies have demonstrated that pro-inflammatory cytokines are vital for chronic pain [19] and also regulated by p38 MAPK activation in rodent spinal cord and human gestational tissue [20, 21]. To confirm this probability, we assessed the mRNA expression of TNF-α and IL-1β in the TG 3 h after SB203580 administration in pIONL mice. We found that the mRNA expression for TNF-α and IL-1β were both significantly decreased after SB203580 treatment, compared to the vehicle-treated ones (Fig. 4c, P < 0.01, Student’s t test).

DISCUSSION The present study showed that pIONL induced a profound orofacial pain in WT mice, but the symptom of hyperalgesia was significantly alleviated in Cxcr5−/− mice.

This change is correlated with the activation of p38, not JNK activation in pIONL group. We also found that intraTG injection of CXCL13 induced mechanical allodynia and upregulation of p-p38 protein expression in WT, not in Cxcr5−/− mice. Inhibition the activation of p38 in TG significantly relieved pIONL-induced mechanical allodynia and downregulated the mRNA expression for cytokines including TNF-α and IL-1β. Previous studies have shown that CXCL13 plays an essential role in the development of lymph nodes and Peyer’s patches [22] and involves in neuroinflammation in multiple sclerosis [14]. In present study, we found that the symptom of hyperalgesia induced by pIONL was significantly attenuated in Cxcr5−/− mice. Previous studies demonstrated that the changes in the production of chemokines and pro-cytokines in the TG contribute to orofacial pain pathological mechanisms. For example, application of CX3CL1 or CCL2 to trigeminal ganglion resulted in orofacial pain behavior in mice [4, 23]. In the present study, we further confirmed that injection of CXCL13 into the trigeminal ganglion did result in a mechanical allodynia. Conversely, the mechanical allodynia was abrogated in Cxcr5−/− mice. These results fit with those observations in Cxcr5−/− mice with spinal nerve injury [15] and infraorbital nerve damage-induced chronic pain [16]. The MAPK family usually provides a link between the extracellular stimuli and the intracellular responses [24]. The p38 MAPK signaling has been shown to play a pivotal role in nociceptive processing in the spinal cord and is involved in the generation of hyperalgesia and allodynia after nerve injury [25]. Consistent with this, we found an

Chemokine CXCL13 activates p38 MAPK in the trigeminal ganglion increase of p-p38 protein expression level in the TG from pIONL WT mice. Similar to those observed in pIONL animals, the p-p38 MAPK protein level was also increased after CXCL13 intra-TG injection in WT mice. The increase of p-p38 protein induced by pIONL or CXCL13 administration in TG was abrogated in Cxcr5−/− mice. Researches on the role of p38 MAPK in sensory neurons or immune cells suggest that p38 may be a potential clinical target in pain control [10, 26]. For example, direct inhibition of p38 MAPK in spinal microglia results in the relief of inflammatory pain, neuropathic pain, as well as postoperative pain [27, 28]. In the present study, we found that intraganglionic injection of a p38 selective inhibitor SB203580 attenuated mechanical sensitization induced by pIONL. This is consistent with previous researches which demonstrated that the activation of p38 was involved in ectopic orofacial inflammatory pain and trigeminal neuropathic pain [29, 30]. Inhibition of the activation of p38 also exhibits an efficacy in relief from acute postsurgical dental pain [31]. In the previous work, we have demonstrated that CXCL13/CXCR5 drives neuropathic pain via neuronal-astrocytic interaction in the spinal cord. In addition, ERK is a downstream kinase of CXCL13/CXCR5 signaling in the spinal cord [15] and trigeminal ganglion [16]. Certainly, we also considered the role of JNK activation in pIONL-induced pain model. However, the protein of p-JNK was not significantly changed in the TG after pIONL. Previous studies have shown that glial cells and neurons can release numerous pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which might increase pain hypersensitivity via MAPK pathway [32]. Both TNF-α and IL-1β are vital cytokines and might directly regulate the function of ion channels in primary nociceptors, such as the transient receptor potential cation channel subfamily V member 1 (TRPV1) [33] and persistent TTX-resistant sodium channel [34]. Inhibition of TNF-α or IL-1β in TG by selective inhibitors attenuated trigeminal nerve injury induced mechanical allodynia [16]. Here, we found that blockade of p38 activity in trigeminal ganglion effectively decreased the mRNA expression of TNF-α and IL-1β. These findings consistent with previous investigation demonstrating that activation of p38 in microglia resulted in an upregulation of multiple cytokines including TNF-α, IL-1β, and IL-6 in the spinal cord and dorsal root ganglion [25]. Taken together, our results suggest that CXCL13/ CXCR5 activation may mediate pIONL-induced orofacial pain through at least partially the activation of p38 MAPK and further upregulate the expression of pro-inflammatory cytokines including TNF-α and IL-1β in trigeminal

ganglion. Targeting CXCL13/CXCR5 signaling may be a treatment for chronic orofacial pain. ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (NSFC 31371121 and 81300954), the Natural Science Research Program of Jiangsu Province (13KJB180017), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

COMPLIANCE WITH ETHICAL STANDARDS Conflict of Interest. The authors declare that they have no conflict of interest. REFERENCES

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