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Bitter taste receptors (taste family 2 bitter receptor proteins; T2Rs), discovered in many tissues outside the tongue, have recently become potential therapeutic targets. We have shown previously that airway epithelial cells express several T2Rs that activate innate immune responses that may be important for treatment of airway diseases such as chronic rhinosinusitis. It is imperative to more clearly understand what compounds activate airway T2Rs as well as their full range of functions. T2R isoforms in airway motile cilia (T2R4, -14, -16, and -38) produce bactericidal levels of nitric oxide (NO) that also increase ciliary beating, promoting clearance of mucus and trapped pathogens. Bacterial quorum-sensing acyl-homoserine lactones activate T2Rs and stimulate these responses in primary airway cells. Quinolones are another type of quorum-sensing molecule used by Pseudomonas aeruginosa To elucidate whether bacterial quinolones activate airway T2Rs, we analyzed calcium, cAMP, and NO dynamics using a combination of fluorescent indicator dyes and FRET-based protein biosensors. T2R-transfected HEK293T cells, several lung epithelial cell lines, and primary sinonasal cells grown and differentiated at the air-liquid interface were tested with 2-heptyl-3-hydroxy-4-quinolone (known as Pseudomonas quinolone signal; PQS), 2,4-dihydroxyquinolone, and 4-hydroxy-2-heptylquinolone (HHQ). In HEK293T cells, PQS activated T2R4, -16, and -38, whereas HHQ activated T2R14. 2,4-Dihydroxyquinolone had no effect. PQS and HHQ increased calcium and decreased both baseline and stimulated cAMP levels in cultured and primary airway cells. In primary cells, PQS and HHQ activated levels of NO synthesis previously shown to be bactericidal. This study suggests that airway T2R-mediated immune responses are activated by bacterial quinolones as well as acyl-homoserine lactones., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

Innate and adaptive defense mechanisms protect the respiratory system from attack by microbes. Here, we present evidence that the bitter taste receptor T2R38 regulates the mucosal innate defense of the human upper airway. Utilizing immunofluorescent and live cell imaging techniques in polarized primary human sinonasal cells, we demonstrate that T2R38 is expressed in human upper respiratory epithelium and is activated in response to acyl-homoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Receptor activation regulates calcium-dependent NO production, resulting in stimulation of mucociliary clearance and direct antibacterial effects. Moreover, common polymorphisms of the TAS2R38 gene were linked to significant differences in the ability of upper respiratory cells to clear and kill bacteria. Lastly, TAS2R38 genotype correlated with human sinonasal gram-negative bacterial infection. These data suggest that T2R38 is an upper airway sentinel in innate defense and that genetic variation contributes to individual differences in susceptibility to respiratory infection.

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. [ABSTRACT FROM AUTHOR]

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 90s due to KCl efflux reflecting Ca2+-activated Cl- channel (CaCC, likely TMEM16A)-dependent secretion. At lower levels of PAR-2 stimulation (50 for Ca2+), shrinkage was not evident due to failure to activate CaCC. Low levels of cAMP-elevating VIP receptor (VIPR) stimulation, also insufficient to activate secretion alone, synergized with low-level PAR-2 stimulation to elicit fluid secretion dependent on both cAMP and Ca2+ to activate CFTR and K+ channels, respectively. Polarized cultures of primary serous cells also exhibited synergistic fluid secretion. Pre-exposure to Pseudomonas aeruginosa conditioned media inhibited PAR-2 activation by proteases but not peptide agonists in primary nasal serous cells, Calu-3 bronchial cells, and primary nasal ciliated cells. Disruption of synergistic CFTR-dependent PAR-2/VIPR secretion may contribute to reduced airway surface liquid in CF. Further disruption of the CFTR-independent component of PAR-2-activated secretion by P. aeruginosa may also be important to CF pathophysiology. [ABSTRACT FROM AUTHOR]