Melatonin antagonizes lipopolysaccharide-induced pulpal fibroblast responses

Pulpal inflammation is an outcome of complex host-bacteria interactions that involves multiple intracellular machineries in tooth pulp. Early stage of pulpal injury caused by bacterial stimuli is mediated via innate immune sensors i.e. Toll-like receptor (TLR), particularly TLR4 which recognizes lipopolysaccharide (LPS), a potent immunostimulatory component derived from gram-negative bacterial cell walls [1, 2]. Recent study has shown that LPS-mediated inflammation of the pulp leads to an upregulation of an array of inflammatory cytokines and mediators including interleukin-1 β (IL-1 β), tumor necrosis factor- α (TNF- α) and antimicrobial peptides such as human β-defensins, chemokine CCL20, [1], thereby ensuring rapid innate immune defense in response to microbial challenge in dental pulp.

In deep dental caries, the chance of chronic exposure of pulp cells to polymicrobial infections is dramatically increased due to the abundance of cariogenic bacterial species found in carious dentin [3]. Such microorganisms may cause potential dangers to the dental pulp upon their direct engagement with innate immune receptors. An unavoidable microbial burden that reaches the dental pulp via dentinal tubules triggers not only an increased expression profile of inflammatory mediators, but also an elevated amount of host protective protein such as transforming growth factor (TGF)- β 1 [4]. Strikingly, the interaction of TGF- β 1 with odontoblasts results in dampened pulpal immune response via suppression of TLR4 [5]. Interestingly, LPS stimulation of human pulpal cells leads to the synthesis of cyclooxygenase (COX) enzymes [6], underscoring the implication of TLR4 in modulating pulpal inflammation. COX is known for its inherent property to promote prostaglandin synthesis, resulting in inflammation and pain [7‐9]. Therefore, homeostasis of the dental pulp is crucially dependent on the intricate interaction between invading microorganisms and host cells that comprise pulpal soft tissues. The resulting induction of host inflammatory products play a significant role in not only overcoming microbial insults, but also in initiating tooth pain as an early clinical symptom of irreversible pulpitis.

Previous studies have elucidated the contribution of melatonin on the modulation of host tissue homeostasis in a number of tissues. Melatonin is produced from the mammal pineal gland by melanocytes and is known for its distinct versatility on the variety of tissues such as salivary glands and the oral epithelium [10‐12]. In bony tissues, administration of melatonin increases mineral components and bone mass, possibly due to an inhibition of receptor activator of nuclear factor kappa-B ligand (RANKL), and induction of osteoprotegerin (OPG), as well as with osteogenic gene expression i.e. bone sialoprotein, osteocalcin, and alkaline phosphatase [13]. It is thus suggested that melatonin is responsible for the maintenance of hard tissue homeostasis.

It is important to note that immunomodulatory properties of melatonin have been extensively investigated. Melatonin potentiates an inflammatory immune response in human myeloid cells involved in both innate and adaptive immunity [14]. Remarkably, previous studies have shown that melatonin is able to regulate glucocorticoid receptor and promote apoptotic resistance in the thymus [15, 16]. In addition, melatonin suppresses LPS-mediated nuclear factor-kappa B (NF- κ B) signaling in human stem cells derived from dental pulp [17], suggesting its antagonistic activity at human TLR4. Collectively, current evidence suggest that melatonin is an essential component required for normal cellular functioning. However, whether melatonin plays a role in LPS antagonism in pulpal fibroblasts during pulpal inflammation still remains poorly understood. Therefore, the objective of this study was to determine an in situ expression of melatonin in human dental pulpal tissues as well as melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2). In addition, a potential melatonin-mediated immunomodulatory mechanisms utilized by pulpal fibroblasts in response to LPS, a major virulent factor of gram-negative microbes, was investigated. Altogether, our in vitro investigations indicate a potential role of pulpal melatonin in fine tuning inflammatory responses to LPS infection.

Immune response of dental pulp to microbial stimuli is triggered via a repertoire of innate immune sensors, such as TLRs including LPS sensor TLR4. Concurrently, host proteins released in response to microbial recognition loop back to autoregulate and fine tune host defense response. For one example, TGF- β 1 released after TLR4 engagement, autoregulates TLR4 signaling in odontoblasts by suppressing TLR4 at both mRNA and protein levels, dampening the inflammatory response [5]. Similarly, melatonin is able to modulate immune responses in pulpal stem cells [17]. Worth noting is that melatonin is globally expressed in different tissues and possibly affects their phenotypes. Notably, expression of melatonin was found to be significantly upregulated in oral lichen planus lesions. Morphometric analysis of oral mucosa in oral lichen planus lesions showed an abundance of MT1 in the spinous cell layer and connective tissue layer of the epithelium when compared to normal epithelium [18]. This expression pattern suggests the association of melatonin and its receptor with the pathogenesis of oral mucosal disease. Remarkably, melatonin and its derivatives exert anti-oxidative effects against human gingival fibroblast cell death induced by hydrogen peroxide possibly via several mechanisms including the inhibition of nitric oxide synthesis and reduced lipid peroxidation [19]. Melatonin was also detected in saliva and gingival crevicular fluids, and the amount was surprisingly higher in subjects with healthy periodontium compared to individuals with periodontitis [20, 21]. It is thus possible that an increased expression of melatonin in the oral epithelium provides an instant cytoprotective barrier against immune-mediated cellular damage of oral epithelial cells [22].

In healthy pulpal tissues, the strongest melatonin expression was in odontoblastic layers consisting of the cell-rich zone. In addition, abundant melatonin was found in pulpal stromal cells and in endothelial cells. This expression profile is analogous to the transepithelial gradient of IL-8, where IL-8 was found most abundantly on the superficial epithelial cell layer in juxtaposition to subgingival plaque [23]. Even in caries-free teeth, melatonin-expressing cell layers in dental pulp may thus act as instant front-line defenders in response to an approaching microbial burden. There it is postulated that melatonin in dental pulp may be inducible by a cariogenic microbiome and requires further investigation.

This study also showed that odontoblasts and pulp cells express melatonin receptors MT1 and MT2. Notably, an elevated expression level of MT1 but not MT2 was correlated with diseased pulp. In the developing human tooth, MT1 was found to be expressed in odontoblasts and dental papilla cells [24]. Our immunohistochemistry data have led to the hypothesis that MT1 plays a pivotal role in the synthesis of reactionary dentin in response to polymicrobial infection in the pulp due to an increased MT1 expression during pulpal inflammation. Contrasting expression profiles of melatonin receptors MT1 and MT2 during pulpal disease suggest that coordination between these two receptor isoforms may play an essential role in the modulation of innate immunity within the pulp, and that these two receptors might have an opposite function in the pulp. MT1 may presumably act as a selective sensor to recognize melatonin synthesized in response to microbial infection. In agreement with this notion, increased MT1 expression was found in osteosarcoma cells, relative to normal bone cells [25], thus indicating the responsiveness of MT1 in bony pathogenesis. However in contrast to MT1, there is limited knowledge about the function of MT2. It has been shown that stimulation of melatonin receptors with their specific agonist Ramelteon increased survival in rats with polymicrobial sepsis [26]. In line with this finding, administration of short-term melatonin in septic mice reduced rates of septic shock compared to mice treated with a vehicle control [27]. Topical application of melatonin on oral mucosal surfaces has been shown to reduce RANKL, but increase OPG levels in saliva, suggesting the efficacy of melatonin on arresting the destruction of periodontal tissue [28]. Altogether, these studies suggest that melatonin receptors may play protective roles when activated by melatonin. Therefore, it is possible that during microbial infection-induced pulpal inflammation, increased melatonin inhibits pulpal inflammation via local engagement with melatonin receptors abundantly expressed in inflamed pulps.

Similar to other tissue sites, pulpal inflammation is associated with an intricate interaction between host inflammatory mediators and host cells under a hostile microenvironment of various inflammatory mediators. Induction of COX enzymes is a prerequisite for prostaglandin synthesis, particularly COX-2 that is known for its inducible profile in response to bacterial LPS and inflammatory cytokines [29‐31]. In inflamed dental pulps, COX-2 expression was found predominantly in the area of infiltrating leukocytes, but was rarely detectable in normal healthy pulps [32]. Importantly, the presence of pleiotropic inflammatory cytokines such as IL-1 α and TNF- α in addition to microbial stimuli in the microenvironment induced a robust COX-2 response in cultured pulp cells [33]. These findings highlight a significant role of COX-2 signaling in pulpal innate immune defense, particularly by pulpal fibroblasts. Black-pigmented pathogenic bacteria including Porphyromonas endodontalis also conferred the ability to induce COX-2 expression in human pulp cells [34]. Our in vitro investigation in cultured human pulpal fibroblasts harvested from dental pulp tissue explants showed that COX-2, but not COX-1, was induced by E. coli LPS after stimulation for 3 h. Importantly, COX-1 was constitutively expressed in dental pulp cells harvested from 3 donors which had a negligible increase of COX-1 transcript when stimulated with LPS.

Surprisingly, we found that the addition of exogenous melatonin into the culture medium attenuated basal expression of COX-2. This finding underscores the requirement of a select signaling pathway for pulpal homeostasis via COX-2 induction in dental pulp. Co-culture of LPS-stimulated pulpal fibroblasts with exogenous melatonin reduced COX-2, but not COX-1, during the early-phase response to LPS infection. This antagonistic phenotype was lost during late phase of LPS stimulation. Loss of LPS antagonism mediated by melatonin after 24-h may be the result of autocrine activity of inflammatory cytokines such as IL-1 β and TNF- α that automatically induce COX-2 expression. It is possible that COX-2 signaling is necessary for fine tuning an adaptive immune response within the pulp providing that prostaglandin E2 (PGE2) is able to promote vascular permeability [35], and induces immunoreactive calcitonin gene-related peptide (iCGRP) via bradykinin, a mechanism believed to be responsible for pulpal pain [36]. Interestingly, COX-2 deletion resulted in a decreased level of IL-1 β and interferon- γ in response to LPS infection in vivo [37]. Thus, in the context of the pulpal environment, inhibition of inflammatory cascades via LPS-induced COX-2 upregulation may result in a modified adaptive inflammatory response through the modulation of PGE2 synthesis and suppression of COX-2, while simultaneously reducing peripheral sensitization. Whether LPS-stimulated pulpal fibroblasts utilize this mechanism to maintain tissue homeostasis requires additional examinations.

A recent study has shown that systemic administration of melatonin during LPS-induced pulpal inflammation dampened TLR4-mediated inflammatory response in dental pulp cells [17]. Our results were consistent with their findings as a 3-h co-culture of E. coli LPS-stimulated human pulpal fibroblasts with melatonin reduced COX-2 and IL-1 β transcripts. Antagonism mediated by melatonin is thought to occur in part at TLR4 because LPS derived from E. coli exerts host hyperresponsiveness specifically at human TLR4 [38, 39]. Although not examined in this study, previous investigations have demonstrated the abundance of TLR4 in human pulpal fibroblasts [2, 40] and LPS infection increased PGE2 and COX-2 expression [40], indicating the participation of TLR4 in pulpal innate immune response during an infection with cariogenic microbes. Remarkably, LPS-induced human dental pulp stem cells (DPSCs) upregulated the expression of functional TLR4 and increased cell migration, but resulted in a reduced proliferation of DPSCs [41]. This suggests there is a distinct local immune response to bacterial stimuli by the recruitment of dental pulp cells in deep dental caries. Interestingly, concomitant activation of eukaryotic cells with bacterial LPS and melatonin seemingly modulates an expression of inflammatory mediators. An earlier study has demonstrated that melatonin suppressed LPS-induced acetylation of the p52 isoform of NF- κ B, an important activation step for COX-2 induction in macrophages [42]. In addition, melatonin blocked the activation of NLRP3 inflammasome required for IL-1 β biosynthesis [43]. In agreement with these notions, we clearly elucidated the antagonistic activity of exogenous melatonin against LPS infection of pulpal fibroblasts via the suppression of COX-2 and IL-1 β. It is striking that melatonin similarly suppressed TLR4 and NF- κ B in mice infected with LPS and provided protection against mastitis [44]. However, whether pulpal fibroblasts utilize blocking mechanisms at NLRP3 and the p52 NF- κ B subunit to regulate chronic pulpal stimulation remains to be elucidated. Collectively, our results might shed light on a functional interplay between melatonin receptors and TLR4 in pulpal fibroblasts. Activation of innate immune receptors in dental pulp by invading bacteria along with pulpal hormone signaling may coordinate host receptors with distinct functions to enhance dental pulp defense. However, the influence of physiological conditions of the body on pulpal melatonin level is still unclear. Whether pulpal melatonin is induced in response to polymicrobial infection found in dental caries also warrants additional investigation. Considering the inhibition of cellular activities by melatonin, multiple intracellular signaling cascades could be targeted by exogenous melatonin deposited in extra-pineal tissues and may be a suitable candidate hormone that could be used in targeted therapy of inflammatory diseases.

In summary, we have reported the ubiquity of melatonin and its receptors i.e. MT1 and MT2 in human dental pulp and a possible implication of melatonin in pulpal tissue homeostasis via the antagonism of LPS-induced COX-2 and IL-1 β signaling. Abundance of extra-pineal melatonin and its hormone receptors MT1 and MT2 in pulpal tissue suggests that melatonin might license dental pulp an immune regulation both via a direct melatonin-receptor engagement and its cross interactions with pulpal microbial sensors in the vicinity, which has yet to be elucidated. Interesting to note is the increased MT1, but not MT2 expression in inflamed pulps, indicating a distinct utilization of melatonin receptors during pulpal pathogenesis. Melatonin in orchestration with its receptors might thus provide a first line defense against microbial insult in dental pulp. Perhaps, melatonin may prove beneficial for the treatment of inflamed dental pulpal tissues. Nonetheless, induction of melatonin expression by polymicrobial infection especially in pulpal disease still needs further elucidation.