Role of macrophage-mediated Toll-like receptor 4-interleukin-1R signaling in ectopic tongue pain associated with tooth pulp inflammation.

BACKGROUND: The existence of referred pain and ectopic paresthesia caused by tooth pulp inflammation may make definitive diagnosis difficult and cause misdiagnosis or mistreatment; thus, elucidation of that molecular mechanism is urgent. In the present study, we investigated the mechanisms underlying ectopic pain, especially tongue hyperalgesia, after tooth pulp inflammation. METHODS: A rat model with mandibular first molar tooth pulp exposure was employed. Tooth pulp exposure-induced heat and mechanical-evoked tongue hypersensitivity was measured, and immunohistochemical staining for Iba1, a marker of active macrophages, IL-1ß, IL-1 type I receptor (IL-1RΙ), and toll-like receptor 4 in the trigeminal ganglion was performed. In addition, we investigated the effects of injections of liposomal clodronate Clophosome-A (LCCA), a selective macrophage depletion agent, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS, a toll-like receptor 4 antagonist), IL-1ß, or heat shock protein 70 (Hsp70, a selective agonist of toll-like receptor 4), to examine changes in tongue hypersensitivity and in the regulation of IL-1RΙ, toll-like receptor 4, and transient receptor potential vanilloid 1 (TRPV1) biosynthesis. RESULTS: At day 1 after tooth pulp exposure, obvious tooth pulp inflammation was observed. Tooth pulp exposure-induced heat and mechanical tongue hypersensitivity was observed from days 1 to 3 after tooth pulp exposure. The production of IL-1ß in activated macrophages and toll-like receptor 4 and IL-1RΙ expression were significantly increased in trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of LCCA significantly suppressed tongue hypersensitivity; however, toll-like receptor 4 and IL-1RΙ expression in trigeminal ganglion neurons innervating the tongue was not significantly altered. Intra-trigeminal ganglion injection of LPS-RS significantly suppressed tongue hypersensitivity and reduced IL-1RΙ expression in the trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of recombinant Hsp70 significantly promoted tongue hypersensitivity and increased IL-1RI expression in trigeminal ganglion neurons innervating the tongue in naive rats. Furthermore, intra-trigeminal ganglion injection of recombinant IL-1ß led to tongue hypersensitivity and enhanced TRPV1 expression in trigeminal ganglion neurons innervating the tongue in naive rats. CONCLUSIONS: The present findings suggest that the neuron-macrophage interaction mediated by toll-like receptor 4 and IL-1RI activation in trigeminal ganglion neurons affects the pathogenesis of abnormal tongue pain following tooth pulp inflammation via IL-1RI and TRPV1 signaling in the trigeminal ganglion. Further research may contribute to the establishment of new therapeutic and diagnostic methods.

Involvement of TNFα in the enhancement of hypersensitivity in the adulthood-injured face associated with facial injury in infancy.

To evaluate the mechanisms underlying acceleration of hypersensitivity in the adulthood-injured face following facial injury in infants, we developed the rats model with facial skin injury in infants and adulthoods (incision + incision), and facial skin suture in infants and facial skin injury in adulthoods (sham + incision), and analyzed the mechanical head-withdrawal threshold (MHWT) of the facial skin, immunohistochemical analysis of trigeminal ganglion (TG) and the effects of intra-ganglionic administration of neutralizing ant-TNFα antibody and recombinant TNFα on nocifensive behavior. The MHWT became considerably lower in incision + incision rats than in sham + incision rats at 10-14 days after the surgery. We observed many TG neurons encircled by glial fibrillary acidic protein-immunoreactive (GFAP-IR) cells and those exhibited TNFα immunoreactivity. TNFα was also expressed in GFAP-IR cells in incision + inicision TG. TNFα protein levels and the relative number of TNFα-IR cells were significantly higher in incision + incision rats than in sham + incision rats. The MHWT was significantly recovered during the intra-ganglionic administration of neutralizing anti-TNFα antibody 4-14 days after the incision. Furthermore, the MHWT was significantly decreased in sham + incision rats following the intra-ganglionic administration of recombinant TNFα. The present findings suggest that the neuron-satellite glial cell communication via TNFα is a critical mechanism in the enhancement of mechanical hypersensitivity in the adulthood-injured face following facial injury in infants.

Connexin 43 expression in satellite glial cells contributes to ectopic tooth-pulp pain.

Pulpitis often causes referred pain in opposing teeth. However, the precise mechanism underlying ectopic pain associated with tooth-pulp inflammation remains unclear. We performed the present study to test the hypothesis that functional interactions between satellite glial cells (SGCs) and trigeminal ganglion (TG) neurons are involved in ectopic orofacial pain associated with tooth-pulp inflammation. Digastric muscle electromyograph (D-EMG) activity elicited by administration of capsaicin into the upper second molar pulp (U2) was analyzed to evaluate noxious reflex responses. D-EMG activity was significantly increased in rats with lower first molar (L1) inflammation relative to saline-treated rats. Significantly increased expression of glial fibrillary acid protein (GFAP), a marker of activated glial cells, and connexin 43 (Cx43), a gap-junction protein, was observed in activated SGCs surrounding U2-innervating TG-neurons after L1-pulp inflammation. Daily administration of Gap26, a Cx43-inhibiting mimetic peptide, into the TG significantly suppressed capsaicin-induced D-EMG activity enhancement and reduced the percentage of fluorogold-labeled (U2-innervated) cells that were surrounded by GFAP-immunoreactive (IR) and Cx43-IR cells after L1-pulp inflammation. These findings indicate that tooth-pulp inflammation induces SGC activation and subsequent spread of SGC activation in the TG via Cx43-containing gap junctions. Thus, remote neuron excitability becomes enhanced in the TG following tooth-pulp inflammation, resulting in ectopic tooth-pulp pain in the contralateral tooth.

Interaction between calcitonin gene-related peptide-immunoreactive neurons and satellite cells via P2Y12 R in the trigeminal ganglion is involved in neuropathic tongue pain in rats.

The P2Y12 receptor expressed in satellite cells of the trigeminal ganglion is thought to contribute to neuropathic pain. The functional interaction between neurons and satellite cells via P2Y12 receptors and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) underlying neuropathic pain in the tongue was evaluated in this study. Expression of P2Y12 receptor was enhanced in pERK1/2-immunoreactive cells encircling trigeminal ganglion neurons after lingual nerve crush. The administration to lingual nerve crush rats of a selective P2Y12 receptor antagonist, MRS2395, attenuated tongue hypersensitivity to mechanical and heat stimulation and suppressed the increase in the relative numbers of calcitonin gene-related peptide (CGRP)-immunoreactive neurons and neurons encircled by pERK1/2-immunoreactive cells. Administration of the P2Y1,12,13 receptor agonist, 2-(methylthio)adenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), to naïve rats induced neuropathic pain in the tongue, as in lingual nerve crush rats. Co-administration of 2-MeSADP + MRS2395 to naïve rats did not result in hypersensitivity of the tongue. The relative number of CGRP-immunoreactive neurons increased following this co-administration, but to a lesser degree than observed in 2-MeSADP-administrated naïve rats, and the relative number of neurons encircled by pERK1/2-immunoreactive cells did not change. These results suggest that the interaction between activated satellite cells and CGRP-immunoreactive neurons via P2Y12 receptors contributes to neuropathic pain in the tongue associated with lingual nerve injury.

PHD1 interacts with ATF4 and negatively regulates its transcriptional activity without prolyl hydroxylation.

Cellular response to hypoxia plays an important role in both circulatory and pulmonary diseases and cancer. Hypoxia-inducible factors (HIFs) are major transcription factors regulating the response to hypoxia. The α-subunits of HIFs are hydroxylated by members of the prolyl-4-hydroxylase domain (PHD) family, PHD1, PHD2, and PHD3, in an oxygen-dependent manner. Here, we report on the identification of ATF4 as a protein interacting with PHD1 as well as PHD3, but not with PHD2. The central region of ATF4 including the Zipper II domain, ODD domain and ß-TrCP recognition motif were involved in the interaction with PHD1. Coexistence of PHD1 stabilized ATF4, as opposed to the destabilization of ATF4 by PHD3. Moreover, coexpression of ATF4 destabilized PHD3, whereas PHD1 stability was not affected by the presence of ATF4. Mutations to alanine of proline residues in ATF4 that satisfied hydroxylation consensus by PHDs did not affect binding activity of ATF4 to PHD1 and PHD3. Furthermore, in vitro prolyl hydroxylation assay clearly indicated that ATF4 did not serve as a substrate of both PHD1 and PHD3. Coexpression of PHD1 or PHD3 with ATF4 repressed the transcriptional activity of ATF4. These results suggest that PHD1 and PHD3 control the transactivation activity of ATF4.