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Background: The role of Akt in nasal immunity is unstudied. Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) expressed in epithelial ciliated cells. Nitric oxide (NO) production by ciliated cells can have antibacterial and antiviral effects. Increasing nasal NO may be a useful antipathogen strategy in chronic rhinosinusitis (CRS). We previously showed that small‐molecule Akt activator SC79 induces nasal cell NO production and suppresses IL‐8 via the transcription factor Nrf‐2. We hypothesized that SC79 NO production may additionally have antibacterial effects. Methods: NO production was measured using fluorescent dye DAF‐FM. We tested effects of SC79 during co‐culture of Pseudomonas aeruginosa with primary nasal epithelial cells, using CFU counting and live–dead staining to quantify bacterial killing. Pharmacology determined the mechanism of SC79‐induced NO production and tested dependence on Akt. Results: SC79 induced dose‐dependent, Akt‐dependent NO production in nasal epithelial cells. The NO production required eNOS and Akt. The NO released into the airway surface liquid killed P. aeruginosa. No toxicity (LDH release) or inflammatory effects (IL8 transcription) were observed over 24 h. Conclusions: Together, these data suggest multiple immune pathways are stimulated by SC79, with antipathogen effects. This in vitro pilot study suggests that a small‐molecule Akt activator may have clinical utility in CRS or respiratory other infection settings, warranting future in vivo studies. [ABSTRACT FROM AUTHOR]

Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two innate immune cell types are activated by bitter products, including those secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells. NO also increases phagocytosis by macrophages. Using biochemistry and live-cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). Immunofluorescence showed that H441 cells express eNOS and T2Rs and that the bitter agonist denatonium benzoate activates NO production in a Ca2+- and HSP90-dependent manner in cells grown either as submerged cultures or at the air–liquid interface. In primary sinonasal epithelial cells, we determined that HSP90 inhibition reduces T2R-stimulated NO production and ciliary beating, which likely limits pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves as an innate immune modulator by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream Ca2+ signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases such as chronic rhinosinusitis, asthma, or cystic fibrosis. [ABSTRACT FROM AUTHOR]

Background The bitter taste receptor T2R38 was recently described to play a role in upper airway innate mucosal defense. When activated by bacterial quorum-sensing molecules, T2R38 stimulates the ciliated epithelial cells to produce nitric oxide (NO), resulting in bactericidal activity and an increase in mucociliary clearance (MCC). Polymorphisms within the T2R38 gene ( TAS2R38) confer variability in activation of the receptor yielding dramatic differences in upper airway defensive responses (NO production and accelerated MCC) to microbial stimulation based on genotype. Our objective was to determine whether the nonprotective TAS2R38 polymorphisms, which render the receptor inactive, correlate with medically recalcitrant chronic rhinosinusitis (CRS) necessitating surgical intervention in the context of known risk factors, and thus identify whether the TAS2R38 genotype is an independent risk factor for patients undergoing functional endoscopic sinus surgery (FESS). Methods CRS patients undergoing primary FESS were prospectively genotyped for TAS2R38. Chi-square analysis was performed on the genotype distribution with respect to other risk factors, including allergies, asthma, nasal polyposis, aspirin sensitivity, diabetes, and smoking exposure. Results Seventy primary FESS patients were genotyped demonstrating a statistically significant skewing from the expected distribution of the general population ( p < 0.0383). CRS patients with a particular polymorphism seemed less likely to have allergies, asthma, nasal polyposis, aspirin sensitivity, and diabetes, but this did not demonstrate statistical significance. Conclusion Our investigation suggests that TAS2R38 genotype is an independent risk factor for patients failing medical therapy, necessitating surgical intervention. [ABSTRACT FROM AUTHOR]

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]

Background We recently demonstrated the bitter taste receptor T2R38 upregulates sinonasal mucosal innate defense in response to gram-negative quorum-sensing molecules through increased nitric oxide production and mucociliary clearance. T2R38 was initially identified in the quest to understand the variability in bitter taste perception to the compound phenylthiocarbamide (PTC) and demonstrated to have polymorphisms generating diplotypes dividing people into PTC supertasters, heterozygotes (with variable PTC detection), and nontasters. We have further demonstrated that sinonasal epithelial cultures derived from supertasters significantly increase innate defenses in response to gram-negative quorum-sensing molecules compared with sinonasal cultures derived from heterozygotes and nontaster individuals. Based on this data, we hypothesize that supertasters are less likely to require sinus surgery compared with heterozygous or nontasters and that supertasters have improved surgical outcomes. Methods Banked sinonasal tissue samples from patients who had undergone primary functional endoscopic sinus surgery at the University of Pennsylvania or the Philadelphia Veterans Affairs Medical Center were genotyped for T2R38 and compared to the expected population distribution. Necessity for additional antibiotic therapy following the postoperative healing time frame was evaluated. Results A total of 28 patients were included in the study. Only 1 supertaster was identified (expected 5.6, p < 0.043). Additionally, 14 heterozygous and 13 nontaster patients were identified. Conclusion This pilot study investigating the genetics of the bitter taste receptor T2R38 in the context of primary sinonasal surgery demonstrates supertaster patients are less likely to need surgical intervention for chronic rhinosinusitis. Additional study is necessary to ascertain postsurgical outcomes. [ABSTRACT FROM AUTHOR]

Background: The non-cancerous functions of Akt in the airway are understudied. In some tissues, Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) that has anti-inflammatory effects. NO production has antibacterial and antiviral effects in the airway, and increasing NO may be a useful anti-pathogen strategy. Akt also stimulates the nuclear factor erythroid 2-related factor 2 (Nrf-2) transcription factor, which transcribes antioxidant genes. Therefore, we hypothesized that activation of the Akt/eNOS pathway, which also activates Nrf-2, may have protective effects in human airway cells against injury.Methods: To directly test the effects of Akt signaling in the airway, we treated A549 and 16HBE cells as well as primary bronchial, nasal, and type II alveolar epithelial cells with small molecule Akt activator SC79. We examined the effects of SC79 on eNOS activation, NO production, Nrf-2 target levels, and interleukin-8 (IL-8) transcription during exposure to TNF-α or Pseudomonas flagellin (TLR5 agonist). Additionally, air-liquid interface bronchial cultures were treated with cadmium, an oxidative stressor that causes airway barrier breakdown.Results: SC79 induced a ~ twofold induction of p-eNOS and Nrf-2 protein levels blocked by PI3K inhibitor LY294002. Live cell imaging revealed SC79 increased acute NO production. Quantitative RT-PCR showed a ~ twofold increase in Nrf-2 target gene transcription. TNF-α or flagellin-induced IL-8 levels were also significantly reduced with SC79 treatment. Moreover, the transepithelial electrical resistance decrease observed with cadmium was ameliorated by SC79, likely by an acute increase in tight junction protein ZO-1 levels.Conclusions: Together, the data presented here demonstrate SC79 activation of Akt induces potentially anti-pathogenic NO production, antioxidant gene transcription, reduces IL-8 transcription, and may protect against oxidative barrier dysfunction in a wide range of airway epithelial cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background Nitric oxide (NO) is released in the airway as a critical component of innate immune defense against invading pathogenic organisms. It is well documented that bacteriostatic and bactericidal effects of NO are concentration-dependent. However, few data exist comparing relative susceptibility of common pathogens to NO at physiologic concentrations. In this study we evaluated the effects of NO on 4 common airway bacteria and 1 fungus, and examined the potential implications of discrepancies in sensitivity. Methods Staphylococcus epidermis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans cultures were adjusted to a uniform optical density (OD) and grown in log phase at 37°C with varying concentrations of NO formed by DETA NONOate. Both OD readings and colony forming units (CFUs) were measured at varying time-points to evaluate for inhibitory effects of NO. Results P aeruginosa and C albicans were significantly more sensitive to NO at physiologic concentrations typical of the human airway. S aureus was attenuated by NO to a lesser degree, and K pneumoniae and S epidermis were more resistant to NO at all concentrations tested. Air surface liquid from cultured human sinonasal epithelial cells had an additive effect in bacterial killing of P aeruginosa, but not in S aureus. Conclusion Common airway pathogens have varying levels of susceptibility to NO at physiologic concentrations of innate immune defense. Relative sensitivity of P aeruginosa and relative resistance of S epidermis may help explain the composition of the healthy microbiome, as well as opportunistic infection in the absence of induced NO release. [ABSTRACT FROM AUTHOR]

Background Nitric oxide (NO) is produced by sinonasal epithelial cells as part of the innate immune response against bacteria. We previously described bitter-taste-receptor-dependent and -independent NO responses to product(s) secreted by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. We hypothesized that sinonasal epithelium would be able to detect the gram-positive, coagulase-negative bacteria Staphylococcus epidermidis and mount a similar NO response. Methods Sinonasal air-liquid interface cultures were treated with conditioned medium (CM) from lab strains and clinical isolates of coagulase-negative staphylococci and S aureus. NO production was quantified by fluorescence imaging. Bitter taste receptor signaling inhibitors were utilized to characterize the pathway responsible for NO production in response to S epidermidis CM. Results S epidermidis CM contains a low-molecular-weight, heat, and protease-stabile product that induces an NO synthase (NOS)-mediated NO production that is less robust than the response triggered by S aureus CM. The S epidermidis CM-stimulated NO response is not inhibited by antagonists of phospholipase C isoform β-2 nor the transient receptor potential melastatin isoform 5 ion channel, both critical to bitter taste signaling. Conclusion This study identifies an NO-mediated innate defense response in sinonasal epithelium elicited by S epidermidis product(s). The active bacterial product is likely a small, nonpeptide molecule that stimulates a pathway independent of bitter taste receptors. Although the NO response to S epidermidis is less vigorous compared with S aureus, the product(s) share similar characteristics. Together, the responses to staphylococci species may help explain the pathophysiology of upper respiratory infections. [ABSTRACT FROM AUTHOR]

Background Sinonasal biofilms have been demonstrated in specimens collected from chronic rhinosinusitis (CRS) patients. Mounting evidence suggests that biofilms contribute to therapeutically recalcitrant CRS. Recently, the bitter taste receptor T2R38 has been implicated in the regulation of the sinonasal mucosal innate immune response. TAS2R38 gene polymorphisms affect receptor functionality and contribute to variations seen in sinonasal innate defense as well as taste perception reflected in gustatory sensitivity to the bitter compound phenylthiocarbamide (PTC). In a population of CRS patients with active infection or inflammation, we sought to determine if a correlation between T2R38 phenotype and in vitro biofilm formation existed. Methods Endoscopically guided sinonasal swabs were obtained prospectively from CRS (±polyp) patients with evidence of persistent inflammation or mucopurulence. In vitro biofilm formation was assessed with a modified Calgary Biofilm Detection Assay. Patients' phenotypic (functional) expression of the bitter taste receptor T2R38 was evaluated with a taste test including the compound PTC. Linear regression was used to determine the level of significance between mean in vitro biofilm formation levels and mean PTC taste test intensity ratings across CRS patients. Results Sinonasal swabs were obtained from 59 patients, with 42 of the 59 samples demonstrating in vitro biofilm formation. Analysis revealed an inverse linear association between in vitro biofilm formation and PTC taste intensity ratings ( p = 0.019) for all patients. This association was exclusively driven by nonpolypoid CRS patients ( p = 0.0026). Conclusion In vitro biofilm formation from sinonasal clinical isolates is inversely correlated with PTC taste sensitivity in nonpolypoid CRS patients. [ABSTRACT FROM AUTHOR]

Background Nitric oxide (NO) is an important antibacterial defense molecule produced by upper airway (sinonasal) epithelial cells. We previously showed that a bitter taste receptor expressed in airway epithelium detects quorum-sensing molecules secreted by Gram-negative bacteria and subsequently triggers bactericidal NO production. We hypothesized that the upper airway epithelium may also be able to detect the Gram-positive aerobe Staphylococcus aureus and mount an NO response. Methods Human sinonasal air-liquid interface (ALI) cultures were treated with methicillin-resistant S. aureus (MRSA)-conditioned medium (CM), and NO production was measured using fluorescence imaging. Inhibitors of bitter taste receptor signaling were used to pharmacologically determine if this pathway was involved in the production of NO. Results A low-molecular-weight, heat, and protease-stabile product found in MRSA CM induced differential, NO synthase (NOS)-mediated NO production. This response varied markedly between individual patients. The MRSA-stimulated NO production was not dependent on 2 important components of bitter taste signaling: phospholipase C isoform β-2 or the transient receptor potential melastatin isoform 5 (TRPM5) ion channel. Conclusion This study shows that a S. aureus product elicits an NO-mediated innate defense response in human upper airway epithelium. The active bacterial product is likely a small, nonpeptide molecule that triggers a pathway independent of bitter taste receptors. Patient variation in the NO response to MRSA product(s), potentially due to genetic differences, might play a role in pathophysiology of Gram-positive upper respiratory infections and/or pathogenesis of chronic rhinosinusitis. [ABSTRACT FROM AUTHOR]

Our purpose was to evaluate the effect of the mechanical force of a sneeze on sinonasal cilia function and determine the molecular mechanism responsible for eliciting the ciliary response to a sneeze. A novel model was developed to deliver a stimulation simulating a sneeze (55 mmHg for 50 ms) at 26°C to the apical surface of mouse and human nasal epithelial cells. Ciliary beating was visualized, and changes in ciliary beat frequency (CBF) were determined. To interrogate the molecular cascades driving sneeze-induced changes of CBF, pharmacologic manipulation of intra- and extracellular calcium, purinergic, PKA, and nitric oxide (NO) signaling were performed. CBF rapidly increases by ≥150% in response to a sneeze, which is dependent on the release of adenosine triphosphate (ATP), calcium influx, and PKA activation. Furthermore, apical release of ATP is independent of calcium influx, but calcium influx and subsequent increase in CBF are dependent on the ATP release. Lastly, we observed a blunted ciliary response in surgical specimens derived from patients with chronic rhinosinusitis compared to control patients. Apical ATP release with subsequent calcium mobilization and PKA activation are involved in sinonasal ciliary response to sneezing, which is blunted in patients with upper-airway disease. [ABSTRACT FROM AUTHOR]