Effects of methanandamide on human periodontal ligament cells CURRENT STATUS:

Background Endocannabinoid system is Synthetic cannabinoid methanandamide (Meth-AEA) has an improved stability and affinity to cannabinoid receptors compared to its endogenous analogue anandamide. In the present study, we investigated the effect of methanandamide on the production of pro-inflammatory mediators in primary human periodontal ligament cells (hPdLCs). Methods hPdLCs were treated with Meth-AEA for 24 h and resulting production of interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1 was measured under normal condition as well as under inflammatory conditions mimicked by the presence of Porphyromonas gingivalis lipopolysaccharide (LPS). Additionally, the effect of Meth-AEA on the proliferation/viability of hPdLCs was measured by MTT method.

2 Abstract Background Endocannabinoid system is involved in the regulation of periodontal tissue homeostasis. Synthetic cannabinoid methanandamide (Meth-AEA) has an improved stability and affinity to cannabinoid receptors compared to its endogenous analogue anandamide. In the present study, we investigated the effect of methanandamide on the production of pro-inflammatory mediators in primary human periodontal ligament cells (hPdLCs).

Conclusion
Our study supports the influence of cannabinoid system on the inflammatory processes in periodontal tissue and its potential involvement in the progression of periodontal disease.

Background
Periodontitis is a plaque biofilm-induced chronic inflammatory disease, which affects teeth supporting structures including the gingival tissue, periodontal ligament and alveolar bone [1]. Clinical signs of periodontitis are gingival inflammation, periodontal pocket formation, periodontal tissue destruction and in advanced cases periodontitis might lead to the alveolar bone resorption and tooth loss [2].
Gram negative anaerobic bacteria Porphyromonas gingivalis (P. gingivalis) is thought to be one of the primary etiological agents of periodontitis [3,4]. P. gingivalis possess multiple virulence factors that could either induce periodontal tissue inflammation or subvert host immune system [4,5].
Lipopolysaccharide (LPS) is one of the most important virulence factors of P. gingivalis [6,7] The endocannabinoid (EC) system consists of endocannabinoids and cannabinoid receptor proteins.
Endocannabinoids are a family of endogeneous lipid neurotransmitter which activates cannabinoid receptors. Several endocannabinoids have been discovered, the most characterized ones are the anandamide (AEA) and 2-arachidonoylglycerol (2-AG) [8], which might be produced by various cells like osteoblasts, osteoclasts, and endothelial cells [9,10]. Cannabinoid receptors a transmembrane Gprotein coupled receptor family. The major endocannabinoid receptors cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) are expressed in various cells and tissues and particularly in dental tissues [11]. EC system is thought to regulate several brain processes; however, actual studies suggest its involvement in the regulation of bone physiology and immune response [12,13].
Since endocannabinoid system is involved in the regulation of bone formation and immune response, several studies of last years investigated a mutual role of this system in the homeostasis of periodontal tissue under healthy and inflammatory conditions. Both AEA and 2-AG are detectable in gingival crevicular fluid and their level seem to be increased in periodontally diseased individuals [14,15]. There are some controversies about the changes in the expression of CB1 and CB2 receptors in periodontitis. One study suggests that the expression of CB1 and CB2 is upregulated under pathological conditions [14]. In contrast, other study shows that bacterial inflammation results in the decrease of CB1 expression and the increase of CB2 expression [16]. Activation of EC system promotes survival and neuronal differentiation of periodontal ligament stem cells [17]. However, the exact role of EC system into progression of periodontal disease still remains unknown.
A mutual role of EC system in homeostasis of periodontal tissue is investigated by several experimental studies. In vitro studies show that anandamide stimulates proliferation of human gingival fibroblasts [15] and diminish cytokine production by these cells in response to stimulation 4 with P. gingivalis LPS [14]. Our recent study shows that AEA and 2-AG have different effect on P.
gingivalis LPS induced production of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 [18]. Particularly, P. gingivalis LPS induced response was inhibited by AEA and enhanced by 2-AG [18]. In vivo study using ligature periodontitis model in rat shows that the local application of AEA decreases the content of tumor necrosis factor alpha and IL-1β in gingival tissue [19]. The effect of AEA was abolished by simultaneous application of CB1 and CB2 inhibitors [19].
One of the major problems of application of EC, and particularly AEA, in research is their low aqueous stability, which might doubt the quality of obtained results [20]. This problem can be solved by development of synthetic analogues of ECs [21]. Methanandamide (Meth-AEA), a synthetic analogue of AEA, has a four-fold higher affinity to receptor than AEA itself and additionally exists a high resistance to enzymatic hydrolysis [22]. In comparison with AEA, Meth-AEA is suggested to be more selective for CB1 receptor and less selective for CB2 receptor [23]. Compared to ECs, the information about the effect of Meth-AEA on periodontal tissue is very limited. Only one report investigated the effect of topical application of Meth-AEA in LPS induced periodontitis model in rats to date [24]. This study shows that Meth-AEA significantly diminishes the alveolar bone loss in this periodontitis model. However, the ability of Meth-AEA to influence the inflammatory response in human cells of periodontium is still unknown. Therefore, in the present study we investigated the effect of Meth-AEA on the basal and P. gingivalis LPS induced production of some pro-inflammaytory mediators by primary human periodontal ligament cells (hPdLCs).

Methods
Cell culture and reagents hPdLCs were isolated from periodontal ligament tissue obtained from wisdom molars extracted for orthodontic reason in healthy individuals similarly to method described earlier [25]. All donors were systematically healthy, aged from 18 to 22 y.o. Periodontal ligament tissue was scraped from the teeth root surface with a scalpel, cut into small pieces and placed into Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), streptomycin (50 µg/ml) and penicillin (100 U/ml) under humidified air atmosphere of 5% CO 2 at 37°C. Outgrowing cells were 5 collected and further grown in DMEM medium. hPdLCs between the third and sixth passages were Cell proliferation/viability assay Cell proliferation/viability was measured by MTT method as described in our previous study [18]. As shown by our recent study, sCD14 enhances the response of periodontal ligament cells to bacterial 6 LPS [26] After 24 h stimulation, the expression of IL-6, IL-8, and MCP-1 in hPdLCs was measured by real time PCR and the content of corresponding protein in conditioned media was assayed by ELISA similarly to the methods described previously [18,27].
Isolation of mRNA from hPdLCs , subsequent transcription to cDNA, and amplification was performed using commercially available TaqMan Gene Expression Cells-to-CT kit (Ambion/Applied Biosystems, Foster City, CA, USA), which provides good accuracy and superior sensitivity of gene-expression analysis [28]. qPCR was performed on an ABI StepOnePlus device (Applied Biosystems, Foster City,

Discussion
Meth-AEA is a highly stable synthetic analogue of AEA and therefore is widely used in the research on cannabinoid system [21,22]. In the present study, we investigated for the first time the effect of Meth-AEA on proliferation viability and inflammatory response in primary hPdLCs in order to further clarify a potential role of EC system in periodontitis. Inflammatory response was assessed by measuring production of IL-6, IL-8, and MCP-1, which are involved in progression of periodontal disease [30][31][32].
We have focused on these pro-inflammatory mediators, because their production in hPdLCs is strongly increased by bacterial LPS [26,33], whereas production of other cytokines like IL-1β or tumor necrosis factor α by hPdLCs in response to LPS stimulation is rather low [34].
Proliferation/viability of hPdLCs was not significantly affected by Meth-AEA in concentrations up to 10 µM and was inhibited in concentration of 30 µM. The effect of Meth-AEA on hPdLCs proliferation/viability differs from that observed for AEA. In our previous study we found that AEA slightly stimulates proliferation/viability of hPdLCs [18]. The exact reason for this discrepancy is not There are several signaling pathways, which could be potentially involved in the biological effects of Meth-AEA. A biochemical study shows that Meth-AEA exhibits high affinity for CB1 receptor, whereas its affinity for CB2 receptor is rather low [23]. Particularly, this study reports that affinity of Meth-AEA is about 20 nM for CB1 and about 900 nM for CB2 receptor. In our study, the effects of Met-AEA were observed only for concentrations of 10 µM, whereas no significant effect was observed for Meth-AEA concentrations up to 1 µM. Thus, it seems that the effects of Meth-AEA could also be contributed to the activation of CB2 receptor. Interestingly, one study shows that Meth-AEA induces IL-6 secretion by prostate cancer cells and this effect is inhibited by CB2 receptor antagonist SR144528 and not by CB1 receptor antagonist rimonabant [35]. An anti-inflammatory role of CB2 receptor activation in periodontitis is recently confirmed by a study of LPS-induced periodontitis in rats [36]. Here, topical application of CB2 receptor agonist HU-308 significantly attenuates the bone loss and inflammatory parameters in this rat model. A recent study shows that different CB-2 receptors agonist have antiinflammatory effect in human periodontal fibroblasts [37].
A possibility of CB1-independent mechanisms in the biological effects of Meth-AEA cannot be totally excluded. A study using CB1 receptor knockout mice shows that Meth-AEA exerts physiological effect even in this mice model, which suggests existence of CB1-independent effects of Meth-AEA [38]. The most investigated CB1-independent effect of Meth-AEA is its ability to activate the transient receptor potential vanilloid type-1 receptor (TRPV1). A contribution of this mechanism into anti-inflammatory effect of Meth-AEA cannot be neglected because an ablation of TRPV1 in mice results in exacerbated immune response [39]. However, there are also some CB1-independent and TRPV1 independent effects of Meth-AEA [40]. Particularly, Meth-AEA is shown to interact with the muscarinic acetylcholine receptors [41]. Nevertheless, the role of CB1-independent mechanisms in the effect of Meth-AEA in hPdLCs is not known and must be further clarified.
Our results on Meth-AEA provide further evidences of the involvement of EC system into progression of periodontal disease. Our data are in line with a study on rats showing that Meth-AEA reduces the clinical parameters in LPS-induced periodontitis [24]. Similarly, a local injection of AEA reduced ligature induced periodontitis in rats [19]. An involvement of EC system in periodontitis is also suggested by studies on cannabinoid receptor expression in periodontal tissue. In vivo study on human periodontal biopsies shows that periodontitis is associated with a decreased expression of CB1 receptor and an increased expression of CB2 receptor [11]. Another study shows an increased expression of CB1 and CB2 receptors in inflamed gingival tissue. All these data suggest that EC system plays an important role in periodontitis.
EC system can be speculated to mediate a link between stress and periodontitis. Stress is an important risk factor influencing immune system and potentially contributing to the progression of periodontitis [42,43]. EC system plays an important role in the stress response [44]. Recently, EC system is suggested as an important part of neuroimmunoendocrine response in periodontal disease [45]. Our data as well as data of previous studies [11,14,15,18,19] suggest that EC system regulates host response in periodontitis. Emotional stress is associated with the increased salivary levels of IL-6 and IL-8 [46]. Acute psychological stress is associated with the decreased tissue content of AEA in brain, which is presumably due to an increased AEA hydrolysis [47]. It can be hypothesized that stress can also influence the levels of endocannabinoids in periodontal tissues and thus modulate their effect on inflammatory response. However, the exact role of EC system in the association between stress and periodontal disease should be further investigated.

Ethics approval and consent to participate
The study protocol was approved by the Ethics Committee of the Medical University of Vienna (ethical approval number: 1694/2015). The methods were carried out in accordance with the relevant guidelines and regulations; all patients got informed before the surgical procedure and gave their written consent.

Consent for publication
Not applicable

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request