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Exp Neurobiol 2021; 30(5): 329-340
Published online October 31, 2021
© The Korean Society for Brain and Neural Sciences
Pa Reum Lee†, Jin-Hee Lee†, Ji Min Park and Seog Bae Oh*
Department of Neurobiology and Physiology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 03080, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-740-8656, FAX: 82-2-762-5107
†These authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pulpitis (toothache) is a painful inflammation of the dental pulp and is a prevalent problem throughout the world. This pulpal inflammation occurs in the cells inside the dental pulp, which have host defense mechanisms to combat oral microorganisms invading the pulp space of exposed teeth. This innate immunity has been well studied, with a focus on Toll-like receptors (TLRs). The function of TLR4, activated by Gram-negative bacteria, has been demonstrated in trigeminal ganglion (TG) neurons for dental pain. Although Gram-positive bacteria predominate in the teeth of patients with caries and pulpitis, the role of TLR2, which is activated by Gram-positive bacteria, is poorly understood in dental primary afferent (DPA) neurons that densely innervate the dental pulp. Using Fura-2 based Ca2+ imaging, we observed reproducible intracellular Ca2+ responses induced by Pam3CSK4 and Pam2CSK4 (TLR2-specific agonists) in TG neurons of adult wild-type (WT) mice. The response was completely abolished in TLR2 knock-out (KO) mice. Single-cell RT-PCR detected
Keywords: Neurobiology, Dental pulp exposure, Pulpitis, Toll-like receptors, Trigeminal nerve
Pulpitis (toothache) is a painful inflammation of the dental pulp and is a problem of high prevalence throughout the world. It is caused primarily by oral bacteria that infect the pulp, where blood vessels and dental primary afferent (DPA) neurons are richly supplied . The most common entry points of these oral bacteria are dental caries, followed by dental trauma or cracked teeth . The major organisms in dental caries are Gram-positive bacteria, such as
Pulpitis commonly accompanies the innate dental pulp immunity that is mediated by the initial sensing of the bacterial pathogens, intracellular signal transduction, and the release of by-products, such as cytokines and inflammatory mediators [1, 2]. A well-known mechanism underlying this innate immunity is the activation of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) in odontoblasts [1, 10], pulp fibroblasts , and sentinel cells [1, 11]. A highly relevant class of PRRs is the family of Toll-like receptors (TLRs), of which 12 TLRs have been found in mammals ; therefore, TLRs play an essential role in the first-line defense of the dental pulp.
As the known cell surface TLRs in rodents are TLR1, TLR2, TLR4, TLR5, and TLR6, they can bind and respond to invading bacteria or their specific structural motifs [13, 14]. For example, TLR4 is activated by lipopolysaccharides present in the cell wall of Gram-negative bacteria, whereas TLR2, which forms heterodimers with either TLR1 or TLR6, is activated by peptidoglycan, lipoteichoic acid (LTA), or lipoproteins derived from Gram-positive bacteria . Gram-positive bacteria predominate over Gram-negative bacteria on the teeth of patients diagnosed with caries and/or pulpitis [3, 4], and upregulation of TLR2 is greater than that of TLR4 in the dental pulp exposed to the oral environment . Nevertheless, the contribution of TLR2 expressed in DPA neurons is not yet fully understood, while the role of TLR4 expressed in trigeminal ganglion (TG) neurons has been studied on dental pulp inflammatory pain [16-19].
Thus, in this study, we focused on the functional and molecular expression of TLR2 in DPA neurons and its change in pulpal inflammation from adult mice. We examined whether TLR2 expressed in TG neurons responds to the synthetic bacterial lipopeptides, Pam3CSK4 and Pam2CSK4. Next, we examined
A total of 39 male C57BL/6J WT mice (5 to 8-week-old) (Doo Yeol Biotech, South Korea), two male transient receptor potential vanilloid 1 (TRPV1) KO mice, and four male TRPV1-ZsGreen mice (crossed the Trpv1-Cre mice  with Ai6 Rosa26ZsGreen1 mice) of C57BL/6J background (5 to 7-week-old) (Jackson Laboratory, USA) were used for this study. A total of eight male and female TLR2 KO mice (5 to 7-week-old) were kindly provided by Dr. Seung Hyun Han (Seoul National University, South Korea). The mice were maintained on ad libitum standard lab chow (pellets) and water under standard conditions (23±1℃, 12 h light-dark cycle). All the surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (protocol code: SNU-190313-6). This study also conformed to the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines for preclinical animal studies.
All the procedures were performed using the method described previously . Briefly, the pulp of the maxillary first molar was exposed with a low-speed dental drill after induction of anesthesia with pentobarbital (60 to 70 mg/kg, intraperitoneal). The cavity was then filled with crystals of DiI (Molecular Probes, USA) and was occluded with a glass ionomer sealant (GC Fuji II, GC Corporation, Japan). The TG neurons containing DiI-labeled DPA neurons were cultured after two weeks post-labeling.
The DPA neurons were visible with DiI labeling, and they were collected by micromanipulation using a glass pipette. Each neuron was photographed for measuring neuron size (µm2) that could represent neuronal types (e.g., small to medium size for nociceptive neurons and large size for non-nociceptive neurons) before cell collection. A single neuron was placed into each tube containing reverse transcription agents (Superscript III, Invitrogen, USA). Complementary DNA (cDNA) synthesis and nested PCR amplification using nested primer pairs (listed in Table 1) were sequentially performed as previously described .
Following previously described methods , the enamel surfaces of the maxillary first molars on both sides were peeled using a low-speed dental drill under pentobarbital anesthesia (60 to 70 mg/kg, intraperitoneal). Then, we performed an etching with a 32% phosphoric acid etchant (Denkist, South Korea) for approximately 10 s, followed by irrigation using a sterilized saline solution. The cavity was filled with crystals of FG (Fluorochrome, USA) and was occluded with a glass ionomer sealant.
One week post-labeling with FG, the pulp of the right maxillary first molar in each mouse was exposed using a low-speed dental drill, as previously described . Dentinal debris was removed from the dental pulp surface using dental k-files (#06, MANI, INC., Japan), followed by irrigation with a sterilized saline solution. After recovering from the anesthetic, the mice were returned to their cages and were supplied with regular food and water.
The mice were sacrificed at 24 h post-injury and were perfused with diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, USA)-treated phosphate-buffered saline (PBS) solution (pH 7.4) to prevent blood contaminants. The TG tissues were then harvested immediately and were used for RNA isolation. Total RNA (250 ng), which was purified using an RNeasy Mini Plus Kit (Qiagen, Germany), was synthesized to cDNA using a QuantiTect Reverse Transcription kit (Qiagen, Germany) according to the manufacturer’s instructions. All the PCR amplification procedures were performed as previously described , and the relative expressions of the genes were calculated using the ΔΔCT method with
At 24 h post-injury, the mice were perfused and fixed with 4% paraformaldehyde (PFA) solution, and the harvested TG tissues were transversely sectioned (thickness: 14 µm). The TG sections were incubated with a blocking solution containing 10% normal rabbit serum (NRS) in PBS containing 0.3% Triton X-100 (PBST) and then were stained with rat anti-TLR2 antibody (1:100 dilution, Abcam, Cat. #ab1186, USA) in 0.3% PBST containing 1% NRS. As a secondary antibody, rabbit anti-rat Cy3 (1:200 dilution, Jackson ImmunoResearch, USA) was used with the same diluents of the primary antibody, and NeuroTrace 640/660 Deep-Red Fluorescent Nissl Stain (1:100 dilution, Molecular Probes, USA) was followed to identify the cell bodies and their sizes (data not shown). For double immunostaining for TLR2 and CGRP, we sequentially incubated a second blocking solution containing 10% normal donkey serum (NDS) in 0.3% PBST after TLR2 immunostaining, followed by goat anti-CGRP (1:1,000 dilution, Abcam, Cat. #ab36001, USA) in 0.3% PBST containing 1% NDS and donkey anti-goat Alexa Fluor 647 (1:200 dilution, Jackson ImmunoResearch, USA) in the same diluents of the primary antibody. All images were examined using a confocal microscope (LSM700, Zeiss, Germany).
All the subsequent immunohistochemical analyses, including cross-sectional area (µm2) and fluorescence intensity of FG-labeled DPA neurons within each TG section, were performed using ImageJ (National Institutes of Health, USA). A total of 3 to 6 TG sections per mouse were randomly selected. The image of the selected sections was converted to 8-bit greyscale, and the brightness/contrast and color threshold were adjusted equally for all the images. Each neuron was determined to be either positive or negative based on its mean intensity value.
TG neurons were used for Ca2+ imaging within 24 h of the primary culture. Fura-2-based ratiometric Ca2+ imaging was performed as previously described . Fura-2 AM (2 μM; Invitrogen, USA) was loaded for one hour at 37℃. A bath solution (containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose) and a zero Ca2+ bath solution (containing 140 mM NaCl, 5 mM KCl, 2 mM EGTA, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose) were adjusted to pH 7.3±0.1 with NaOH and were prepared fresh before each experiment. The 340/380 ratiometric images and [Ca2+]i changes were acquired using MetaFluor software (v 126.96.36.199; Molecular Devices, USA). In all the Ca2+ imaging experiments, TLR2 agonist-positive neurons, displayed as Ca2+ transients with ≥7% of the maximal amplitude (in 50 mM KCl) and reproducibility of at least two out of three, were used for the analysis.
Pam3CSK4 (Invivogen, Cat. #tlrl-pms, USA) and Pam2CSK4 (Invivogen, Cat. #tlrl-pm2s-1, USA) were dissolved in the supplied endotoxin-free water (1 mg/ml). Capsaicin (Sigma-Aldrich, USA) was prepared in ethanol (10 mM) and stored at -20℃. The drugs were diluted to their final concentration with the bath solution for Ca2+ imaging.
The statistical data are presented as the mean±SEM. The differences between three groups were compared using a one-way ANOVA with a Bonferroni post hoc test, and the differences between two groups were compared using a two-tailed Student’s
To determine the functional expression patterns of TLR2, we chose to use in our study the synthetic bacterial lipopeptide Pam3CSK4 (a TLR2/TLR1-specific agonist) typically used for TLR2 activation [13, 24]. Activation of TLR2 by three sequential applications of Pam3CSK4 (1 μg/ml; 100 s/application) elicited reproducible [Ca2+]i responses in a subset of TG neurons (Fig. 1A, 1B). When we further confirmed the source of the Ca2+, the Pam3CSK4-induced [Ca2+]i responses were increased even by depletion of the [Ca2+]i store with a 1 μM thapsigargin pretreatment, but the Ca2+ response remained unchanged by the following Pam3CSK4 application after washout with bath solution (Fig. 1C, 1D). Conversely, the [Ca2+]i responses were completely abolished by a zero Ca2+ bath solution (Fig. 1E, 1F) and in TLR2 KO mice (Fig. 1I). Among the 50 mM KCl-responsive TG neurons, a subset of TG neurons responded to Pam3CSK4 (approximately 5.7%,
Similar to Pam3CSK4, three sequential applications of Pam2CSK4 (a TLR2/TLR6-specific agonist) (1 μg/ml; 100 s/application) elicited reproducible [Ca2+]i responses in a subset of TG neurons (Fig. 2A, 2B). The Pam2CSK4-responding neurons were also observed in TRPV1-positive neurons as well as TRPV1-negative neurons (Fig. 2C), and the CAP (1 µM)-induced Ca2+ transients were elicited in half of the Pam2CSK4-responding neurons (50%,
We investigated the TLR2 expression patterns in individual DPA neurons that were retrogradely labeled with DiI using scRT-PCR (Fig. 3A). Approximately 17.1% of the DPA neurons expressed
Since significant morphological changes and pain-like behaviors can occur within 24 h of the experimental dental pulp injury [17, 23], we assessed the molecular changes to
Since non-neuronal cells like infiltrated macrophages can be contained in the injured or inflamed TG tissues , we examined the changes that occur in TLR2 protein expression by pulpal inflammation in the cell bodies of DPA neurons that were labeled with the retrograde neuronal tracer FG. Immunohistochemical analysis showed a significant increase of the TLR2-positive DPA neurons in the injured side, compared to the non-injured side, at 24 h post-injury (Fig. 5A, 5B), and the increase in small- to medium-sized neurons (up to 700 µm2) was greater than in large-sized neurons, suggesting that TLR2 has functional roles in nociceptive neurons associated with pain (Fig. 5C). Next, we found that both TLR2 and CGRP (pain-related neuropeptides) expression levels were upregulated in the injured DPA neurons, compared to the non-injured DPA neurons, at 24 h post-injury (Fig. 5D, 5F), and the increase was also observed in TRPV1-lineage DPA neurons, suggesting that upregulation of TLR2 in DPA neurons can contribute to pain during pulpitis (Fig. 5D, 5G). The anti-TLR2 antibody specificity used in our work was verified by a significant decrease in TLR2 immunoreactivity in TLR2 KO mice (Fig. 5E, 5F).
In this study, we present a possible role of neuronal TLR2 in pulpitis with the following findings: (1) Both Pam3CSK4 and Pam2CSK4 (TLR2-specific agonists) increased the [Ca2+]i levels in a subset of TG neurons, which were responsive to capsaicin (a TRPV1 agonist), but this response was abolished entirely in TLR2 KO mice. (2)
TLR2 activation triggers downstream signal transduction pathways, usually in non-neuronal cells such as macrophages and epithelial cells, by inducing the release of Ca2+ from intracellular stores . However, the mechanisms for this action are different in sensory neurons such as DRG neurons . Wang et al.  has demonstrated that Pam3CSK4 dramatically increases the [Ca2+]i concentrations, depending on the activation of downstream TRPV1 and TRPA1 channels, by confirming a significant inhibition of this response in TRPV1 KO and TRPA1 KO mice. Our Ca2+ imaging results confirmed that Pam3CSK4-induced Ca2+ influxes were obtained from the extracellular source (Fig. 1E, 1F), but not from the intracellular store (Fig. 1C, 1D). Moreover, TRPV1-expressing TG neurons responded to either Pam3CSK4 (approximately 82.6%,
Although we primarily used Pam3CSK4 to verify the function of TLR2 in sensory neurons, the heterodimeric TLR2 receptors (TLR2/TLR1 or TLR2/TLR6) are distinctly activated by different agonists . The Pam3CSK4 is the triacylated lipopeptide commonly derived from Gram-negative bacteria, while Pam2CSK4 is the diacylated lipopeptide derived from Gram-positive bacteria . Compared to Pam3CSK4 actions (approximately 5.7%,
The experimental dental pulp injury model used in this study is well established and reviewed for studying neuroinflammatory interactions by allowing the molecular changes in the pulp tissue and somatic TG induced by dental injury [17, 23, 37, 38]. Using this model, we confirmed that 24 h following dental pulp injury is a sufficient amount of time to address the upregulation of (1) TLR2 expression in both the injured TG (Fig. 4A) and in (2) individual DPA neurons on the injured side (Fig. 5B) and especially (3) inflammatory marker genes, such as TNF-α and IL-1β (Fig. 4E, 4F), and MYD88, which is associated with the TLR downstream signaling pathway (Fig. 4G), in injured TG from WT mice, but not from TLR2 KO mice. Although this model cannot completely exclude the damage itself following pulpal injury, it sufficiently allows for bacterial accumulation in the exposed dental cavity [37, 39]. Accordingly, our study has limitations in demonstrating that molecular changes are caused by PAMPs but not by damage-associated molecular patterns (DAMPs). However, we showed a significant increase of both TLR2 and CGRP expression levels in TRPV1-lineage DPA neurons, which have been reported to be upregulated by bacterial infection , suggesting that upregulation of TLR2 can contribute to pain transmission during pulpitis (Fig. 5F, 5G).
Mammals host a complex community of commensal bacteria, with an estimated 200 to 700 species residing in the oral cavity [5, 41]. Dental caries is commonly caused by Gram-positive bacteria, such as
In conclusion, our results provide a better molecular understanding of pulpitis by revealing that TLR2 expression is upregulated in DPA neurons, and this study uncovers potential neural functions of TLR2 in pulpal inflammation.
Authors thank Dr. Seung Hyun Han (School of Dentistry, Seoul National University) for providing TLR2 KO mice. This research was supported by National Research Foundation (NRF) of Korea (grant number: 2017M3C7A1025602, 2018R1A5A2024418, and 2021R1A2C3003334) funded by the Korean government (Ministry of Education, Science and Technology). All authors gave their final approval and agree to be accountable for all aspects of the work.
All authors declare no conflict of interest.
List of primers used
|Target gene||Outer primer pairs||Inner primer pairs||GenBank no.|
|(F) 5’-GTTCCTACCCCCAATGTGTCC-3’||(F) 5’-TTGTGATGGGTGTGAACCAC-3’||NM_001289726.1, NM_008084.3|
|(R) 5’-ACCTGTTGCTGTAGCCGTATC-3’||(R) 5’-CCTTCCACAATGCCAAAGTTG-3’|
|(F) 5’-TGCGTATAGACAGGAGGCAGA-3’||(F) 5’-TGGAGGAGGAGATCCAGTTCTT-3’||NM_001131020.1, NM_010277.3|
|(R) 5’-GGCGATAGTCGTTAGCTTCGT-3’||(R) 5’-GCCACTGCCTCGTATTGAGT-3’|
|(F) 5’-GCAGCCAAAGTGAACACAGA-3’||(F) 5’-AGAGTTGGAGGCCCTGAAAA-3’||NM_010904.3|
|(R) 5’-AATGTCCAGGGCCATCTTGA-3’||(R) 5’-GTGCAGCCATCTCCCACTT-3’|
|(F) 5’-TCTCTGGGCAGTCTTGAACA-3’||(F) 5’-AGACACTGGGGGTAACATCG-3’||NM_011905.3|
|(R) 5’-TCGCGGATCGACTTTAGACT-3’||(R) 5’-AGAGAAGTCAGCCCAGCAAA-3’|
|Target gene||Primer pairs||GenBank no.|
|(F) 5’- AGTGGCCCTACCAAGTCTCA-3’|
(R) 5’- GTGTCTCAGGCTGTTTGTTCC-3’
|NM_001384171.1, NM_011604.5, NM_001359180.2|