TAS2R16 Activation Suppresses LPS-Induced Cytokine Expression in Human Gingival Fibroblasts

Sustained and non-resolved inflammation is a characteristic of periodontitis. Upon acute inflammation, gingival fibroblasts release cytokines to recruit immune cells to counter environmental stimuli. The intricate regulation of pro-inflammatory signaling pathways, such as NF-κB, is necessary to maintain periodontal homeostasis. Nonetheless, how inflammation is resolved has not yet been elucidated. In this study, 22 subtypes of taste receptor family 2 (TAS2Rs), as well as the downstream machineries of Gα-gustducin and phospholipase C-β2 (PLCβ2), were identified in human gingival fibroblasts (HGFs). Various bitter agonists could induce an intensive cytosolic Ca2+ response in HGFs. More importantly, TAS2R16 was expressed at a relatively high level, and its agonist, salicin, showed robust Ca2+ evocative effects in HGFs. Activation of TAS2R16 signaling by salicin inhibited the release of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, at least in part, by repressing LPS-induced intracellular cAMP elevation and NF-κB p65 nuclear translocation in HGFs. These findings indicate that TAS2Rs activation in HGFs may mediate endogenous pro-inflammation resolution by antagonizing NF-κB signaling, providing a novel paradigm and treatment target for the better management of periodontitis.


INTRODUCTION
Periodontitis is a chronic inflammatory disease that is triggered by the accumulation of dental plaques. It involves a severe chronic inflammation that destroys tooth-supporting tissues, and if left untreated, it leads to tooth loss (1). Periodontitis is the primary cause of tooth loss in adults. It not only affects oral health but also has a close relationship with systemic diseases, such as diabetes mellitus, cardiovascular diseases, rheumatoid arthritis, etc (2). Gingival fibroblasts are the predominant cells in gingival connective tissues, contributing to sustained inflammation in periodontal diseases (3,4). Under inflammatory conditions, human gingival fibroblasts (HGFs) function as accessory immune cells, and together with other cells such as gingival epithelial cells, secreting pro-inflammatory cytokines and promoting neutrophils homing (5,6). Nonetheless, sustained release of cytokines and immune cell recruitment will cause non-resolved inflammation, thus contributing to inflammatory tissue breakdown and the development of periodontitis. The regulation of cytokine release by fibroblasts during inflammation is mainly mediated by NF-kB signaling (7), while the mechanisms of periodontal inflammation resolution remain poorly understood.
Taste receptor family 2 (TAS2Rs), which belong to the Gprotein-coupled receptor family, are initially identified in taste buds and function as peripheral taste receptors for bitter stimuli (8,9). Recent studies have identified the expression of TAS2Rs in a variety of extra-gustatory tissues, including the respiratory tract, gastrointestinal mucosa, urethra, heart, and gingiva (10)(11)(12)(13)(14). The TAS2Rs-expressing cells that reside in these tissues also express several downstream machineries that are essential for the taste signaling cascade in type II taste cells, such as G protein subunit Ga-gustducin, phospholipase C-b2 (PLCb2), and transient receptor potential cation channel melanostatin (TRPM) 5 (15)(16)(17). Most studies have demonstrated that extragustatory TAS2Rs function as immune sentinels in mammalian innate immune responses to environmental stimuli, including metabolites derived from bacteria or parasites (18)(19)(20)(21). In addition, intracellular Ca 2+ elevation is involved in TAS2Rsmediated downstream effects, such as antimicrobial peptide (AMP) secretion, pathogen clearance, and respiratory reflexes (18,22). Of note, TAS2Rs activation can antagonize lipopolysaccharide (LPS)-induced inflammatory mediator production both in human whole blood and lung macrophages (23,24), suggesting the potential role of TAS2Rs in the tight control of inflammation. However, the mechanisms and biological significance of TAS2Rs-induced anti-inflammatory effects have not been well documented. Our recent study revealed that bitter tastant denatonium benzoate could alleviate periodontitis in a TAS2Rs-dependent manner in mice and confirmed that gingival solitary chemosensory cells (gSCCs) contributed to such an effect by mediating AMP expression (25).
Given the wide expression of TAS2Rs in various types of cells, we hypothesize that gingival fibroblasts express TAS2Rs, which are involved in the attenuation of excessive inflammatory responses and contribute to the tight regulation of periodontal inflammation. In this study, we confirmed the expression of TAS2Rs and downstream signaling components in HGFs. Activation of TAS2R16 by salicin antagonized LPS-induced cytokine expression by downregulating intracellular cAMP and inhibiting the NF-kB signaling cascade in HGFs.

Primary Human Gingival Fibroblasts Culture
The current study was approved and supported by the Institution Review Board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-OT-2020-049). The experiment was in accordance with the Declaration of Helsinki principles. Gingival tissues were collected from healthy volunteers (n=4, aged between 18~30 years old) who experienced the third molar extraction with no clinical symptoms of either oral or systematic diseases. Tissues were immersed into dulbecco's modified eagle medium (DMEM; Gibco, New York, NY, USA) with 10% fetal bovine serum (FBS; Gibco, New York, NY, USA) and 1% penicillin-streptomycin solution (Hyclone, Logan, UT, USA) on ice and quickly transferred to the lab. The pooled tissues were rinsed by Hank's Balanced Salt Solution (HBSS, Hyclone, Logan, UT, USA) with 1% penicillin-streptomycin, torn off epithelium, cut into small pieces, and then incubated with 2 mM type I collagenase (Sigma-Aldrich, Steinheim, Germany) at 37°C for 60 min. After centrifugation (200 g, 3 min), cells were resuspended in DMEM and then seeded on a T25 culture flask (Corning, New York, NY, USA), and cultured in the humidified atmosphere at 37°C with 5% CO 2 . P3-P8 of the cells were used for further experiments.

RNA Extraction and Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from fresh gingival tissues (n=8) and cultured HGFs (n=4) with TRIzol regent (Life Technologies, Carlsbad, CA, USA) and MiniBest Universal RNA Extraction Kit (TAKARA, Tokyo, Japan), respectively, according to the manufacturer's instructions. The precipitate was air-dried and resuspended in RNase-free water. The purity and concentration of isolated RNA were determined by a nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was generated from a total of 1 µg RNA using RT reagent Kit with gDNA Eraser (TAKARA, Tokyo, Japan). Aliquot without reverse transcriptase was prepared as the negative control. PCR was performed using PrimeSTAR ® Max DNA Polymerase (TAKARA, Tokyo, Japan) to detect the existence of TAS2Rs and downstream molecules (primers listed in Supplementary Table 1). Besides, quantitative real-time PCR was performed to determine the relative expression level of TAS2Rs in HGFs (primers listed in Supplementary Table 1). The expression level of target genes normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated by 2 -DDCT method.

Immunofluorescence Staining
Cultured HGFs were fixed in 4% formaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 for 15 min at room temperature. After blocked with 5% goat or rabbit serum for 60 min at room temperature, cells were incubated at 4°C overnight with the desired primary antibodies for vimentin (

Calcium Mobilization Assay
Calcium mobilization traces were recorded using Flexstation III (Molecular Devices, Sunnyvale, CA, USA). Relative fluorescence units (excitation at 488 nm, emission at 525 nm, and cutoff at 515 nm) were read every 2 s for a duration of 200 s (bitter compounds were added at 30 s). For calcium imaging, cells were examined under an Olympus IX-83 P2ZF microscope with a standard GFP filter. Images were acquired every 1 s for 140 s (bitter compounds were injected into the wells at~10 s). 10 µM ATP (Solarbio, Beijing, China) and DPBS were used as positive and negative controls, respectively. Curves of fluorescence intensity normalized by baseline (F/F 0 ; F 0 was determined as the average of the first 5 readings) to time were illustrated with GraphPad 8.0 (GraphPad Software, San Diego, CA, USA).

In Vitro LPS-Induced Inflammation Model
HGFs were grown into~50% confluency and then transfected with 50 nM TAS2R16-siRNA or non-target-siRNA (listed in Supplementary Table 1; synthesized by Hippo biotechnology) using lipofectamine 2000 according to the manufacturer's instruction. TAS2R16 silencing efficiency was confirmed by RT-qPCR. The transfected cells were cultured till~90% confluency (for 48 h) and treated with LPS (5 µg/mL; Cat. No. L2880, Sigma, Steinheim, Germany) and (or) D (-)-salicin (2 mM; Cat. No. S0625, Sigma, Steinheim, Germany) for 24 h. In other experiments, HGFs were grown into~80% confluency. LPS (5 µg/mL for all assays), salicin (2 mM for all assays except for the cell proliferation assay and the calcium mobilization assay), U73122 (1 µM for RT-qPCR, and 0.5 µM for Western Blot), and forskolin (5 µM for RT-qPCR and Western Blot) were alone or combinatorically applicated to HGFs for 24 h. Total RNA was extracted using MiniBest Universal RNA Extraction Kit. Inflammatory cytokines were quantified by real-time quantitative RT-PCR (primers listed in Supplementary Table 1). The phosphorylation of NF-kB was evaluated by western blotting and CLSM. Additionally, culture supernatant or cell lysate was collected for subsequent experiments as described below.

Cell Proliferation
HGFs were seeded at a density of 1×10 4 onto a 96-well plate and incubated for 24 h. The cells were treated with an increasing concentration of salicin (range from 0.1 to 10 mM). After 24 h, the medium was changed and the mixture of Cell Counting Kit-8 (CCK-8) solution (DMEM: CCK-8 = 9:1; ApexBio, Houston, Texas, USA) was supplemented to every well for 1.5 h at 37°C when the optical density (OD) at 450 nm of non-treated HGFs is about 1.

Neutrophil Isolation and Cell Migration Assay
Human peripheral blood was collected from consented healthy volunteers. Neutrophils were isolated from EDTAanticoagulated whole blood by Percoll density centrifugation using 2 discontinuous gradients (1.075 and 1.090) followed by erythrocyte lysis as previously described (29). In brief, 2 mL 60% Percoll solution (Sigma, Steinheim, Germany) was layered over 2 mL 75% Percoll solution in the FBS-pretreated tube. 2 mL peripheral blood was centrifugated and suctioned for leukocytic cream, which was carefully layered over 60% Percoll. The suspended substance between the two layers was extracted, and residual red cells were lysed by Red Cell Lysis Buffer (Biosharp, Hefei, China). Cells were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, New York, NY, USA) at a density of 1×10 6 cells/mL. Cell viability and purity (typically >98%) were determined by Trypan blue exclusion (Solarbio, Beijing, China) and Wright's-Giemsa staining solution (Solarbio, Beijing, China), respectively. Chemotaxis assay was conducted as previously reported with minor modification using the transwell system (Cat# 3422, Corning, New York, NY, USA) (30). Culture supernatant of HGFs treated with LPS and (or) D (-)-salicin was collected and filtered. 100 µL cells were seeded into upper chambers, and 600 µl supernatant was added to lower chambers. After 2 h incubation (37°C, 5% CO 2 ), 500 µl aliquot in the lower chambers was harvested to a 96-well plate. Randomly selected fields in each well were recorded by an Olympus invert microscope. The migrated neutrophils were counted by ImageJ (National Institutes of Health, Bethesda, MD, USA).

Western Blot
HGFs treated with LPS, and (or) D (-)-salicin were scraped from wells and washed with 4°C PBS. Total protein was extracted using cell lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 1% protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instruction. The equivalent amount of protein was loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gels. The gels were transferred to polyvinylidene difluoride (PVDF) membrane for 1 h. The membranes were blocked with 5% non-fat powdered milk (Sangon Biotech, Shanghai, China) for 1.5 h at room temperature and then incubated with desired primary antibodies for p65 (1:

IL-8 and Intracellular cAMP Quantification
HGFs treated with LPS and (or) D (-)-salicin were centrifugated for 10 min at 4°C. The supernatant was collected for IL-8 quantification (Zci Bio, Shanghai, China), and the cell lysate was prepared by Ultrasonic Cell Disruption System (QIAGEN, Germantown, Germany) to perform cAMP quantification (Zci Bio, Shanghai, China) according to the manufacturer's instructions, respectively. The results were collected by Flexstation III (OD560), analyzed using the generated standard curves, and illustrated as nmol/L.

Statistical Analysis
Each in vitro experiment was repeated separately three times. Statistical analysis was performed using GraphPad 8.0. Data were presented as the mean ± standard deviation. Comparisons between different groups were performed by one-way ANOVA followed by Tukey's multiple comparisons test or unpaired t-test in different contexts. Differences were considered significant when the two-tailed P value was <0.05.

Primary HGFs Express Multiple TAS2Rs and Downstream Machineries
Fresh gingival tissues were collected to determine the expression of TAS2Rs. RT-PCR revealed the expression of 22 subtypes of TAS2Rs ( Figure 1A and Supplementary Figure 1). As fibroblasts are predominant in gingival connective tissue and are closely involved in the inflammatory responses to environmental stimuli (5,31,32), we investigated the expression of TAS2Rs in HGFs. In the cultured primary HGFs (pooled from four gingival samples), TAS2R16, TAS2R31, TAS2R38, TAS2R39, and TAS2R43 were the most expressed among the 19 TAS2Rs detected in HGFs ( Figure 1B). Meanwhile, the expression of TAS2R4, TAS2R7, TAS2R8, TAS2R9, TAS2R10, and TAS2R60 was not detected ( Figure 1B) in the samples. In addition to taste receptors, taste signaling elements Ga-gustducin (GNAT3) and PLCb2 (PLCB2) were identified by real-time quantitative RT-PCR ( Figure 1C) and immunofluorescence staining ( Figure 1D) in cultured HGFs. Moreover, TRPM4, instead of TRPM5, was detected in HGFs ( Figure 1C). To further confirm the expression of TAS2Rs and downstream machinery in HGFs, we analyzed several datasets containing HGF expression profiles from the Gene Expression Omnibus. TRPM4, TRPM5, PLCB2, and a variety of TAS2Rs, including TAS2R16, TAS2R31, TAS2R38, TAS2R39, and TAS2R43, were identified in HGFs in most of the selected datasets ( Figure 1E and Supplementary Table 3). Of note, the expression levels of TRPM5 in these datasets were significantly lower relative to TRPM4, particularly in datasets GSE140523, in which only one out of the five samples expressed TRPM5 (Supplementary Table 4). The expression of GNAT3 was reported in three datasets ( Figure 1E

Salicin Activates TAS2R16 in Primary HGFs
To further confirm the function of TAS2Rs expressed in HGFs, we screened candidate bitter tastants that could induce intracellular calcium accumulation in HGFs. Several agonists evoked increased intracellular Ca 2+ levels in primary HGFs, including salicin, quinine, phenylthiocarbamide (PTC), aristolochic acid, camphor, and thiamine. In addition, low-level but sustained Ca 2+ increment was observed when stimulating HGFs with sodium cyclamate, chloroquine, chlorphenamine, and chloramphenicol (Figure 2A). Salicin induced the most robust Ca 2+ response among the tested agents, and TAS2R16, the specific receptor for salicin (Supplementary Figure 2) (26,33), showed a relatively high expression level in HGFs. We focused on the physiological function of TAS2R16 activation in the following experiments.

TAS2R16 Activation Inhibits LPS-Induced Cytokine Expression in HGFs
HGFs secrete multiple cytokines in the gingival tissues of patients with periodontitis (32). Upon acute inflammation, pathogenassociated molecular patterns, such as LPS, induce cytokine expression in gingival fibroblasts (34). We investigated whether TAS2R16 activation impacted cytokine expression in HGFs in the context of LPS-induced inflammation. Salicin (0-2 mM) did not significantly repress the cell viability of HGFs (Supplementary Figure 3). Under the normal condition, cytokine expression of HGFs was maintained at a relatively low level and was unchanged after salicin treatment (Supplementary Figure 4). In addition, TAS2R16 silencing also did not affect the expression level of selected cytokines (Supplementary Figure 5), implying that TAS2R16 function and our siRNA known-down manipulation had no effects on cytokine expression in HGFs at baseline. Twenty-two candidate cytokines or chemokines were screened based on their expression levels in HGFs or their possible relationship with periodontal inflammation. As expected, LPS treatment significantly induced cytokine expression in HGFs ( Figure 3A). The top five most expressed cytokines after LPS treatment including IL-6, IL-8, CXCL1, CXCL3, and CCL2 (Supplementary Table 5) were selected for further investigation. Simultaneous treatment with salicin significantly inhibited the LPS-induced expression of IL-6, IL-8, and CXCL3, rather than CXCL1 and CCL2 ( Figure 3B). Consistently, TAS2R16-silencing diminished the inhibitory effect of salicin on the expression of IL-6, IL-8, and CXCL3 in HGFs ( Figure 3C), and promoted IL-8 secretion that was suppressed by salicin in the supernatant of HGF-cultures ( Figure 3D), further suggesting that salicin can inhibit LPS-induced cytokine expression in HGFs in a TAS2R16-dependent manner.
IL-8, also known as CXCL8, is closely related to neutrophil recruitment during periodontal inflammation (35,36). Transwell assay revealed that the conditioned medium from LPS-treated HGFs significantly enhanced neutrophil migration, while simultaneous treatment with salicin significantly repressed this effect ( Figure 3E). Furthermore, treatment with salicin did not alter the ability of the conditioned medium to attract neutrophils when TAS2R16 was silenced in HGFs ( Figure 3E).

TAS2R16 Activation Represses LPS-Induced Intracellular cAMP Accumulation and NF-kB Nuclear Translocation
The expression of IL-8 is regulated by multiple transcriptional factors, of which NF-kB plays a key role (31). In cultured primary HGFs, salicin treatment significantly repressed the LPS-induced phosphorylation of p65 at ser 276 , as well as the level of p65 in nuclear extracts, in a TAS2R16-dependent manner ( Figures 4A-D), implying that TAS2R16 activation countered LPS-induced cytokine expression via negatively regulating NF-kB signaling activation in HGFs. It has been reported that cAMP is related to the phosphorylation of NF-kB p65 (37,38), which functions as a central downstream effector in LPS-induced inflammation (7,39). We confirmed the increased intracellular cAMP level in HGFs induced by LPS ( Figure 5A). cAMP is also one of the well-documented downstream effectors of taste receptor signaling. TAS2R activation can decrease the cAMP level. We, therefore, speculated that TAS2R16 activation might inhibit the LPS-induced p65 phosphorylation via downregulation of intracellular cAMP. Although salicin treatment alone had no significant effect on the intracellular level of cAMP in HGFs ( Figure 5B), it decreased the LPS-induced cAMP accumulation by~20%, an effect that could be abolished by siRNA knock-down of TAS2R16 ( Figure 5C). Of note, stimulation of cAMP synthesis in HGFs by treatment of adenylate cyclase (AC) activator forskolin, weakened the inhibitory effects of salicin on p65 ser 276 phosphorylation ( Figures 5D, E), and rescued LPSinduced IL-8 expression that was repressed by salicin (Figures 5F), further demonstrating that cAMP was involved in the inhibitory effects of TAS2R16 signaling against LPSinduced inflammation.
Another well-known downstream effect of TAS2Rs activation is the elevation of intracellular Ca 2+ in a PLCb2-dependent way, which was confirmed in our cultured HGFs (Figures 2A, B). Several studies reported that LPS-induced cytokine expression was also partly dependent on intracellular calcium elevation mediated by PLC (40,41). Consistently, we also found largely abrogated expression of IL-8, IL-6, and CXCL3 in the treatment of U73122 under the LPS-induced inflammatory condition (Supplementary Figure 6A). In addition, U73122 treatment did not mitigate the inhibitory effect of salicin on LPS-induced expression of IL-8 and CXCL3, as well as p65 nuclear translocation (Supplementary Figures 6B, C), suggesting that salicin is less likely to antagonize LPS-induced cytokine expression and NF-kB activation via Ca 2+ .

DISCUSSION
TAS2Rs and their downstream signaling molecules have been identified in multiple extra-gustatory tissues throughout the body, including the respiratory tract, gastrointestinal mucosa, and heart. Our recent study also identified a specific group of TAS2Rexpressing cells in the gingiva of mice, defined as gingival solitary chemosensory cells (25). HGFs, the predominant cell type in gingival connective tissues, together with other cells like gingival epithelial cells, play an essential role in the maintenance of periodontal homeostasis. However, HGF-originated TAS2Rs have barely been investigated. Here, we found that HGFs expressed various TAS2Rs and downstream machineries, including TAS2R16, TAS2R38, TAS2R31, TAS2R39, TAS2R43, and TRPM4, and responded to a diverse range of bitter tastants, particularly salicin. TRPM4, like TRPM5, is a voltage-sensitive and monovalent-selective channel activated by oscillatory changes in intracellular Ca 2+ (42). A recent study revealed the critical role of TRPM4 in response to bitter, sweet, and umami stimuli (43). TRPM4 activation can increase initial cell depolarization, activate the continuous release of ATP, and thus mediate taste responses (43). The current study identified TRPM4 instead of TRPM5 in HGF samples. In the selected datasets, the expression levels of TRPM5 in HGFs were also significantly lower in comparison with that of TRPM4, especially in GSE140523, in which only one out of the five samples expressed TRPM5 (44). Nonetheless, the discrepancy between our RT-qPCR data and GEO RNA-seq data regarding the TRPM5 expression in HGFs may be caused by different sampling and detection methods. Additionally, we detected higher expression levels of TAS2R16, TAS2R38, TAS2R31, TAS2R39, and TAS2R43 in primary HGFs. TAS2R43 is responsive to a diverse constellation of structurally different compounds and has been identified in human airway epithelia (45,46). Denatonium benzoate, a bitter agonist of TAS2R43 and other TAS2Rs, can stimulate airway epithelial ciliary motility via taste signaling (45). TAS2R39 is highly expressed in human airway smooth muscle cells and human monocyte-derived macrophages (6,22). Increased phagocytosis can be induced by the TAS2R14/TAS2R39 agonists apigenin and chrysin (6). TAS2R38 encodes the bitter taste receptor in humans, which preferentially recognizes PTC. Clinical studies have revealed that gene polymorphisms of TAS2R38 are correlated with protective effects against caries (47,48). In addition, TAS2R38 appears to be an essential mediator of sinonasal epithelial defense and is responsible for respiratory bacterial infections (18). TAS2R16 has been identified in human neuronal tissue. Salicin, a specific agonist of TAS2R16, may modulate neurite outgrowth via TAS2R16 activation (49). Given the relatively higher expression of TAS2R16, TAS2R38, TAS2R31, TAS2R39, and TAS2R43 in HGFs and the evident Ca 2+ evocative effects of their agonists, the potential physiological functions of these TAS2Rs in the periodontium can be expected. Among the five most expressed TAS2Rs, TAS2R16 mediated the most robust Ca 2+ accumulative effects in response to salicin and thus may exert regulatory effects on the physiology of HGFs and periodontal health. Instead of taste perception, TAS2Rs-expressing extra-gustatory cells are immune sentinels in the mammalian innate immune response. TAS2Rs are broadly tuned for bacterial compounds and play a role in nitric oxide production, cilia beating, type 2 immunity initiation, or direct bactericidal effects in the airway and gut (18,20,50,51). In addition, studies have documented the role of TAS2Rs activation in anti-inflammatory effects by suppressing cytokine expression in different cells or tissues (23,24,52,53). Consistently, our study also demonstrated that salicin suppressed the LPS-induced expression of pro-inflammatory cytokines, including IL-6 and IL-8 via TAS2R16 activation. Neutrophils constitute the majority of leukocytes recruited to the periodontium. Neutrophils are necessary for maintaining gingival health; nonetheless, they are recently recognized as major players in periodontitis by contributing to substantial tissue destruction (54). The hyperactivation and excessive neutrophil-mediated tissue injury have been detected in aggressive periodontitis and all stages of chronic periodontitis (55)(56)(57). HGFs, as the major components of the gingival connective tissue, are transcriptionally active in expressing chemokines and specifically wire toward neutrophils recruitment when encountering infections and damages (5). Among the chemokines that regulate neutrophil migration, IL-8 is the most significant and well-characterized chemotactic and activating factor for neutrophils (58,59). Here, we also found that salicin weakened LPS-induced neutrophil recruitment in a TAS2R16-dependent manner, likely due to the downregulated release of proinflammatory cytokines, particularly IL-8. The suppressed overactivation of cytokine release and over-migration of neutrophils may protect periodontal tissue injury and thus benefit inflammation control.  The NF-kB signaling pathway plays a central role in the LPSinduced expression of cytokines in various cell types (7,39,60), and is a major contributor to numerous chronic inflammatory diseases, including periodontitis (61,62). However, the potential relationship between the taste signaling cascade and the NF-kB pathway has not yet been well documented. Here, we demonstrated that TAS2R16 activation by salicin inhibited the LPS-induced NF-kB cascade by repressing the phosphorylation and nuclear translocation of p65 in HGFs. Activation of TAS2Rs can elevate intracellular calcium levels via the PLCb2-inositol 1,4,5-trisphosphate (IP 3 )-Ca 2+ axis (9,22). PLC signaling positively correlates with inflammatory responses in various cell types (63)(64)(65), and Plcb2-deficiency alleviates LPS-induced inflammation and tissue injury (66). In addition, inhibition of PLC signaling and intracellular calcium release can suppress NF-kB translocation (67,68). Consistently, our study also demonstrated that blocking of PLC and its subsequent Ca 2+ accumulation by U73122 effectively inhibited pro-inflammatory cytokine expression and NF-kB p65 nuclear translocation induced by LPS. More importantly, we demonstrated that salicin treatment significantly elevated the intracellular level of calcium, and reduced the inflammatory cytokine expression in LPS-induced HGFs. Although LPS-induced cytokine expression is partly dependent on intracellular calcium elevation mediated by PLC were performed by one-way ANOVA followed by Tukey's multiple comparisons test. In (A, F), and the right panal in (C), the comparisons were performed by unpaired t-test. *p < 0.05; **p < 0.01; n.s., not significant. (40,41), LPS per se can reduce PLCb2 expression in a timedependent manner (69,70). In addition, our data showed that inhibition of intracellular calcium by U73122 failed to counter the inhibitory effect of salicin on LPS-induced IL-8 expression and p65 nuclear translocation, further suggesting that salicin is less likely to antagonize LPS-induced cytokine expression and NF-kB activation via PLCb2-IP 3 -Ca 2+ under this context. Other pathway(s) that can be elicited by TAS2Rs activation may contribute to the inhibitory effect of salicin on the inflammatory cytokine expression and NF-kB activation in the LPSinduced HGFs. Activation of TAS2Rs can cause the separation of the heterotrimeric G-protein subunits into Ga-gustducin and the bg-gustducin dimer. The bg-gustducin increases intracellular calcium via the PLCb2-IP 3 -Ca 2+ axis, while the a-gustducin stimulates phosphodiesterase (PDE) that hydrolyzes cAMP, thereby decreasing intracellular cAMP levels (71,72). Recent studies have indicated that cAMP positively affects NF-kB activity by targeting the p65/RelA ser 276 residue through its main effector protein kinase A (PKA) in different cells (37,38,(73)(74)(75)(76)(77)(78)(79). Moreover, cAMP/PKA/cAMP-response element binding protein (CREB) signaling pathway may promote LPS-induced pro-inflammatory cytokine release, including IL-6, IL-33, and TNF-a (80)(81)(82). On the other hand, LPS can cause a significant dose and time-dependent increase in forskolin-stimulated adenylate cyclase (AC) activity (83), which in turn elevates intracellular cAMP levels. In the current study, the activation of TAS2R16 by salicin did not decrease the intracellular level of cAMP in the absence of LPS induction, consistent with that reported in other cells (84,85). Nonetheless, LPS-induction significantly increased cAMP levels in the HGFs, and salicin treatment decreased the LPS-induced cAMP accumulation in a TAS2R16-dependent manner and inhibited the inflammatory cytokine expression and NF-kB activation in this context. In addition, the inhibitory effects of salicin on the LPS-induced NF-kB cascade were countered by elevation of cAMP using forskolin, an AC activator, further suggesting that salicin can activate TAS2R signaling and exert anti-inflammatory effects on the LPS-induced HGFs via repressing intracellular cAMP.
Given the wide existence of TAS2Rs and their physiological functions, the application of bitter compounds to the treatment of diseases is possible. TAS2Rs may play a critical role in the pharmaceutical activities of herbal medicines (86). Berberine, a potential bitter agonist for TAS2R38 and TAS2R46 (86), exerts a protective effect in inhibiting inflammatory responses and has a long history in the treatment of inflammatory bowel diseases (87,88). Scutellaria baicalensis Georgi, one of the most widely used herbal medicines, shows anti-inflammatory effects in treating respiratory tract and gut diseases (89). Baicalin, baicalein, and wogonin, the most abundant bioactive components extracted from Scutellaria baicalensis Georgi, can activate TAS2R14 as bitter agonists (86,89,90). Moreover, our previous study showed that in mice with periodontitis, treatment with the bitter component denatonium benzoate activated gSCCs to produce more antimicrobial peptides and inhibit bacterial colonization, thus alleviating alveolar bone loss in periodontitis (25). Given that TAS2R16 activation inhibited the inflammatory response in HGFs, TAS2Rs may be exploited as a potential target for the treatment of periodontitis. Further studies are warranted to investigate whether TAS2R16 activation can alleviate alveolar bone loss in animal models as well as in clinical cohorts.
Taken together, the current study identified and profiled the expression of TAS2Rs in HGFs and discovered calcium signaling in response to various bitter tastants. Furthermore, salicin, a specific agonist of TAS2R16, exerted anti-inflammatory effects in a TAS2R16-dependent manner by inhibiting the cAMP and NF-kB cascade. Thus, our data suggest the possibility of using TAS2Rs as a drug target for the treatment of periodontitis.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institution Review Board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-OT-2020-049). The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
ZZ, RX, JXL, and XP carried out the experiments. XX, JYL, XDZ, and XZ conceived the study and provided advice. ZZ, LZ, and RX drafted the manuscript. All authors edited and approved the manuscript.