Lidocaine induces apoptosis in head and neck squamous cell carcinoma through activation of bitter taste receptor T2R14.

Head and neck squamous cell carcinomas (HNSCCs) have high mortality and significant treatment-related morbidity. It is vital to discover effective, minimally invasive therapies that improve survival and quality of life. Bitter taste receptors (T2Rs) are expressed in HNSCCs, and T2R activation can induce apoptosis. Lidocaine is a local anesthetic that also activates bitter taste receptor 14 (T2R14). Lidocaine has some anti-cancer effects, but the mechanisms are unclear. Here, we find that lidocaine causes intracellular Ca2+ mobilization through activation of T2R14 in HNSCC cells. T2R14 activation with lidocaine depolarizes mitochondria, inhibits proliferation, and induces apoptosis. Concomitant with mitochondrial Ca2+ influx, ROS production causes T2R14-dependent accumulation of poly-ubiquitinated proteins, suggesting that proteasome inhibition contributes to T2R14-induced apoptosis. Lidocaine may have therapeutic potential in HNSCCs as a topical gel or intratumor injection. In addition, we find that HPV-associated (HPV+) HNSCCs are associated with increased TAS2R14 expression. Lidocaine treatment may benefit these patients, warranting future clinical studies.

Savory Signaling: T1R Umami Receptor Modulates Endoplasmic Reticulum Calcium Store Content and Release Dynamics in Airway Epithelial Cells.

T1Rs are expressed in solitary chemosensory cells of the upper airway where they detect apical glucose levels and repress bitter taste receptor Ca2+ signaling pathways. Microbial growth leads to a decrease in apical glucose levels. T1Rs detect this change and liberate bitter taste receptor signaling, initiating an innate immune response to both kill and expel pathogens through releasing antimicrobial peptides and increasing nitric oxide production and ciliary beat frequency. However, chronic inflammation due to disease, smoking, or viral infections causes a remodeling of the epithelial airway. The resulting squamous metaplasia causes a loss of multi-ciliated cells and solitary chemosensory cells, replaced by basal epithelial cells. To understand how T1R function is altered during disease, we used basal epithelial cells as a model to study the function of T1R3 on Ca2+ signaling dynamics. We found that both T1R1 and T1R3 detect amino acids and signal via cAMP, increasing the responsiveness of the cells to Ca2+ signaling stimuli. Either knocking down T1R1/3 or treating wild-type cells with MEM amino acids caused a reduction in ER Ca2+ content through a non-cAMP signaled pathway. Treatment with amino acids led to a reduction in downstream denatonium-induced Ca2+-signaled caspase activity. Thus, amino acids may be used to reduce unwanted apoptosis signaling in treatments containing bitter compounds.

Utilizing the Off-Target Effects of T1R3 Antagonist Lactisole to Enhance Nitric Oxide Production in Basal Airway Epithelial Cells.

Human airway sweet (T1R2 + T1R3), umami (T1R1 + T1R3), and bitter taste receptors (T2Rs) are critical components of the innate immune system, acting as sensors to monitor pathogenic growth. T2Rs detect bacterial products or bitter compounds to drive nitric oxide (NO) production in both healthy and diseased epithelial cell models. The NO enhances ciliary beating and also directly kills pathogens. Both sweet and umami receptors have been characterized to repress bitter taste receptor signaling in healthy and disease models. We hypothesized that the sweet/umami T1R3 antagonist lactisole may be used to alleviate bitter taste receptor repression in airway basal epithelial cells and enhance NO production. Here, we show that lactisole activates cAMP generation, though this occurs through a pathway independent of T1R3. This cAMP most likely signals through EPAC to increase ER Ca2+ efflux. Stimulation with denatonium benzoate, a bitter taste receptor agonist which activates largely nuclear and mitochondrial Ca2+ responses, resulted in a dramatically increased cytosolic Ca2+ response in cells treated with lactisole. This cytosolic Ca2+ signaling activated NO production in the presence of lactisole. Thus, lactisole may be useful coupled with bitter compounds as a therapeutic nasal rinse or spray to enhance beneficial antibacterial NO production in patients suffering from chronic inflammatory diseases such as chronic rhinosinusitis.

Cilia Stimulatory and Antibacterial Activities of T2R Bitter Taste Receptor Agonist Diphenhydramine: Insights into Repurposing Bitter Drugs for Nasal Infections.

T2R bitter taste receptors in airway motile cilia increase ciliary beat frequency (CBF) and nitric oxide (NO) production. Polymorphisms in some T2Rs are linked to disease outcomes in chronic rhinosinusitis (CRS) and cystic fibrosis (CF). We examined the expression of cilia T2Rs during the differentiation of human nasal epithelial cells grown at air-liquid interface (ALI). The T2R expression increased with differentiation but did not vary between CF and non-CF cultures. Treatment with Pseudomonas aeruginosa flagellin decreased the expression of diphenhydramine-responsive T2R14 and 40, among others. Diphenhydramine increased both NO production, measured by fluorescent dye DAF-FM, and CBF, measured via high-speed imaging. Increases in CBF were disrupted after flagellin treatment. Diphenhydramine impaired the growth of lab and clinical strains of P. aeruginosa, a major pathogen in CF and CF-related CRS. Diphenhydramine impaired biofilm formation of P. aeruginosa, measured via crystal violet staining, as well as the surface attachment of P. aeruginosa to CF airway epithelial cells, measured using colony-forming unit counting. Because the T2R agonist diphenhydramine increases NO production and CBF while also decreasing bacterial growth and biofilm production, diphenhydramine-derived compounds may have potential clinical usefulness in CF-related CRS as a topical therapy. However, utilizing T2R agonists as therapeutics within the context of P. aeruginosa infection may require co-treatment with anti-inflammatories to enhance T2R expression.

T2R bitter taste receptors regulate apoptosis and may be associated with survival in head and neck squamous cell carcinoma.

Better management of head and neck squamous cell carcinomas (HNSCCs) requires a clearer understanding of tumor biology and disease risk. Bitter taste receptors (T2Rs) have been studied in several cancers, including thyroid, salivary, and GI, but their role in HNSCC has not been explored. We found that HNSCC patient samples and cell lines expressed functional T2Rs on both the cell and nuclear membranes. Bitter compounds, including bacterial metabolites, activated T2R-mediated nuclear Ca2+ responses leading to mitochondrial depolarization, caspase activation, and ultimately apoptosis. Buffering nuclear Ca2+ elevation blocked caspase activation. Furthermore, increased expression of T2Rs in HNSCCs from The Cancer Genome Atlas is associated with improved overall survival. This work suggests that T2Rs are potential biomarkers to predict outcomes and guide treatment selection, may be leveraged as therapeutic targets to stimulate tumor apoptosis, and may mediate tumor-microbiome crosstalk in HNSCC.

The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells.

Bitter taste receptors (T2Rs) localize to airway motile cilia and initiate innate immune responses in retaliation to bacterial quorum sensing molecules. Activation of cilia T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by loss of motile cilia and/or squamous metaplasia. To understand T2R function within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells. Several T2Rs localize to the nucleus in de-differentiated cells that typically localize to cilia in differentiated cells. As cilia and nuclear import utilize shared proteins, some T2Rs may target to the nucleus in the absence of motile cilia. T2R agonists selectively elevated nuclear and mitochondrial calcium through a G-protein-coupled receptor phospholipase C mechanism. Additionally, T2R agonists decreased nuclear cAMP, increased nitric oxide, and increased cGMP, consistent with T2R signaling. Furthermore, exposure to T2R agonists led to nuclear calcium-induced mitochondrial depolarization and caspase activation. T2R agonists induced apoptosis in primary bronchial and nasal cells differentiated at air-liquid interface but then induced to a squamous phenotype by apical submersion. Air-exposed well-differentiated cells did not die. This may be a last-resort defense against bacterial infection. However, it may also increase susceptibility of de-differentiated or remodeled epithelia to damage by bacterial metabolites. Moreover, the T2R-activated apoptosis pathway occurs in airway cancer cells. T2Rs may thus contribute to microbiome-tumor cell crosstalk in airway cancers. Targeting T2Rs may be useful for activating cancer cell apoptosis while sparing surrounding tissue.

PAR-2-activated secretion by airway gland serous cells: role for CFTR and inhibition by Pseudomonas aeruginosa.

Airway submucosal gland serous cells are important sites of fluid secretion in conducting airways. Serous cells also express the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor that activates secretion from intact airway glands. We tested if and how human nasal serous cells secrete fluid in response to PAR-2 stimulation using Ca2+ imaging and simultaneous differential interference contrast imaging to track isosmotic cell shrinking and swelling reflecting activation of solute efflux and influx pathways, respectively. During stimulation of PAR-2, serous cells exhibited dose-dependent increases in intracellular Ca2+. At stimulation levels >EC50 for Ca2+, serous cells simultaneously shrank ∼20% over ∼90 s due to KCl efflux reflecting Ca2+-activated Cl- channel (CaCC, likely TMEM16A)-dependent secretion. At lower levels of PAR-2 stimulation (

Neuropeptide regulation of secretion and inflammation in human airway gland serous cells.

Airway submucosal gland serous cells are sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and are important for fluid secretion in conducting airways. To elucidate how neuropeptides regulate serous cells, we tested if human nasal turbinate serous cells secrete bicarbonate (HCO3 -), important for mucus polymerisation and antimicrobial peptide function, during stimulation with cAMP-elevating vasoactive intestinal peptide (VIP) and if this requires CFTR. Serous cells stimulated with VIP exhibited a ∼15-20% cAMP-dependent decrease in cell volume and a ∼0.15 unit decrease in intracellular pH (pHi), reflecting activation of Cl- and HCO3 - secretion, respectively. HCO3 - secretion was directly dependent on CFTR and was absent in cells from CF patients. In contrast, neuropeptide Y (NPY) reduced VIP-evoked cAMP increases, CFTR activation, and Cl-/HCO3 - secretion. Culture of primary serous cells in a model that maintained a serous phenotype confirmed the activating and inhibiting effects of VIP and NPY, respectively, on fluid and HCO3 - secretion. Moreover, VIP enhanced antimicrobial peptide secretion and antimicrobial efficacy of secretions while NPY reduced antimicrobial efficacy. In contrast, NPY enhanced cytokine release while VIP reduced cytokine release through a mechanism requiring CFTR. As levels of VIP and NPY are up-regulated in diseases like allergy, asthma, and chronic rhinosinusitis, the balance of these two peptides in the airway may control mucus rheology and inflammatory responses in serous cells. Furthermore, the loss of CFTR conductance in serous cells may contribute to CF pathophysiology by increasing serous cells inflammatory responses in addition to directly impairing Cl- and HCO3 - secretion.

Protease-activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating.

Mucociliary clearance, driven by the engine of ciliary beating, is the primary physical airway defense against inhaled pathogens and irritants. A better understanding of the regulation of ciliary beating and mucociliary transport is necessary for identifying new receptor targets to stimulate improved clearance in airway diseases, such as cystic fibrosis and chronic rhinosinusitis. In this study, we examined the protease-activated receptor (PAR)-2, a GPCR previously shown to regulate airway cell cytokine and mucus secretion, and transepithelial Cl- current. PAR-2 is activated by proteases secreted by airway neutrophils and pathogens. We cultured various airway cell lines, primary human and mouse sinonasal cells, and human bronchial cells at air-liquid interface and examined them using molecular biology, biochemistry, and live-cell imaging. We found that PAR-2 is expressed basolaterally, where it stimulates both intracellular Ca2+ release and Ca2+ influx, which activates low-level nitric oxide production, increases apical membrane Cl- permeability ∼3-5-fold, and increases ciliary beating ∼20-50%. No molecular or functional evidence of PAR-4 was observed. These data suggest a novel and previously overlooked role of PAR-2 in airway physiology, adding to our understanding of the role of this receptor in airway Ca2+ signaling and innate immunity.-McMahon, D. B., Workman, A. D., Kohanski, M. A., Carey, R. M., Freund, J. R., Hariri, B. M., Chen, B., Doghramji, L. J., Adappa, N. D., Palmer, J. N., Kennedy, D. W., Lee, R. J. Protease-activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating.

Plant flavones enhance antimicrobial activity of respiratory epithelial cell secretions against Pseudomonas aeruginosa.

Flavones are a class of natural plant secondary metabolites that have anti-inflammatory and anti-bacterial effects. Some flavones also activate the T2R14 bitter taste receptor, which is expressed in motile cilia of the sinonasal epithelium and activates innate immune nitric oxide (NO) production. Flavones may thus be potential therapeutics for respiratory infections. Our objective was to examine the anti-microbial effects of flavones on the common sinonasal pathogens Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, evaluating both planktonic and biofilm growth. Flavones had only very low-level antibacterial activity alone. They did not reduce biofilm formation, but did reduce production of the important P. aeruginosa inflammatory mediator and ciliotoxin pyocyanin. However, flavones exhibited synergy against P. aeruginosa in the presence of antibiotics or recombinant human lysozyme. They also enhanced the efficacy of antimicrobials secreted by cultured and primary human airway cells grown at air-liquid interface. This suggests that flavones may have anti-gram-negative potential as topical therapeutics when combined with antibiotics or in the context of innate antimicrobials secreted by the respiratory or other epithelia. This may have an additive effect when combined with T2R14-activated NO production. Additional studies are necessary to understand which flavone compounds or mixtures are the most efficacious.

Bacterial d-amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells.

In the upper respiratory epithelium, bitter and sweet taste receptors present in solitary chemosensory cells influence antimicrobial innate immune defense responses. Whereas activation of bitter taste receptors (T2Rs) stimulates surrounding epithelial cells to release antimicrobial peptides, activation of the sweet taste receptor (T1R) in the same cells inhibits this response. This mechanism is thought to control the magnitude of antimicrobial peptide release based on the sugar content of airway surface liquid. We hypothesized that d-amino acids, which are produced by various bacteria and activate T1R in taste receptor cells in the mouth, may also activate T1R in the airway. We showed that both the T1R2 and T1R3 subunits of the sweet taste receptor (T1R2/3) were present in the same chemosensory cells of primary human sinonasal epithelial cultures. Respiratory isolates of Staphylococcus species, but not Pseudomonas aeruginosa, produced at least two d-amino acids that activate the sweet taste receptor. In addition to inhibiting P. aeruginosa biofilm formation, d-amino acids derived from Staphylococcus inhibited T2R-mediated signaling and defensin secretion in sinonasal cells by activating T1R2/3. d-Amino acid-mediated activation of T1R2/3 also enhanced epithelial cell death during challenge with Staphylococcus aureus in the presence of the bitter receptor-activating compound denatonium benzoate. These data establish a potential mechanism for interkingdom signaling in the airway mediated by bacterial d-amino acids and the mammalian sweet taste receptor in airway chemosensory cells.

Flavones modulate respiratory epithelial innate immunity: Anti-inflammatory effects and activation of the T2R14 receptor.

Chronic rhinosinusitis has a significant impact on patient quality of life, creates billions of dollars of annual healthcare costs, and accounts for ∼20% of adult antibiotic prescriptions in the United States. Because of the rise of resistant microorganisms, there is a critical need to better understand how to stimulate and/or enhance innate immune responses as a therapeutic modality to treat respiratory infections. We recently identified bitter taste receptors (taste family type 2 receptors, or T2Rs) as important regulators of sinonasal immune responses and potentially important therapeutic targets. Here, we examined the immunomodulatory potential of flavones, a class of flavonoids previously demonstrated to have antibacterial and anti-inflammatory effects. Some flavones are also T2R agonists. We found that several flavones inhibit Muc5AC and inducible NOS up-regulation as well as cytokine release in primary and cultured airway cells in response to several inflammatory stimuli. This occurs at least partly through inhibition of protein kinase C and receptor tyrosine kinase activity. We also demonstrate that sinonasal ciliated epithelial cells express T2R14, which closely co-localizes (<7 nm) with the T2R38 isoform. Heterologously expressed T2R14 responds to multiple flavones. These flavones also activate T2R14-driven calcium signals in primary cells that activate nitric oxide production to increase ciliary beating and mucociliary clearance. TAS2R38 polymorphisms encode functional (PAV: proline, alanine, and valine at positions 49, 262, and 296, respectively) or non-functional (AVI: alanine, valine, isoleucine at positions 49, 262, and 296, respectively) T2R38. Our data demonstrate that T2R14 in sinonasal cilia is a potential therapeutic target for upper respiratory infections and that flavones may have clinical potential as topical therapeutics, particularly in T2R38 AVI/AVI individuals.