Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages

IntroductionBitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses, possibly contributing to reduced innate immunity in CF.MethodsImmunofluorescence, qPCR, and live cell imaging were used to measure T2R localization, calcium and NO signaling, ciliary beating, and antimicrobial responses in air-liquid interface cultures of primary human nasal epithelial cells and immortalized bronchial cell lines. Immunofluorescence and live cell imaging was used to measure T2R signaling and phagocytosis in primary human monocyte-derived macrophages.ResultsPrimary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o- CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation.ConclusionsTogether, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate T2Rs

Neuropeptide Y Reduces Nasal Epithelial T2R Bitter Taste Receptor–Stimulated Nitric Oxide Production

Bitter taste receptors (T2Rs) are G-protein-coupled receptors (GPCRs) expressed on the tongue but also in various locations throughout the body, including on motile cilia within the upper and lower airways. Within the nasal airway, T2Rs detect secreted bacterial ligands and initiate bactericidal nitric oxide (NO) responses, which also increase ciliary beat frequency (CBF) and mucociliary clearance of pathogens. Various neuropeptides, including neuropeptide tyrosine (neuropeptide Y or NPY), control physiological processes in the airway including cytokine release, fluid secretion, and ciliary beating. NPY levels and/or density of NPYergic neurons may be increased in some sinonasal diseases. We hypothesized that NPY modulates cilia-localized T2R responses in nasal epithelia. Using primary sinonasal epithelial cells cultured at air–liquid interface (ALI), we demonstrate that NPY reduces CBF through NPY2R activation of protein kinase C (PKC) and attenuates responses to T2R14 agonist apigenin. We find that NPY does not alter T2R-induced calcium elevation but does reduce T2R-stimulated NO production via a PKC-dependent process. This study extends our understanding of how T2R responses are modulated within the inflammatory environment of sinonasal diseases, which may improve our ability to effectively treat these disorders

HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

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

Two- versus four-handed techniques for endonasal resection of orbital apex tumors

BACKGROUND: Open versus endonasal resection of orbital apex (OA) tumors is generally based on tumor size, location, and pathology. For endonasal resection, two- and four-handed techniques have been reported, but whether one technique is more optimal based on these tumor features has not been evaluated. OBJECTIVE: To determine whether two- versus four-handed techniques result in better outcomes after endoscopic resection of OA tumors, and whether either technique is better suited for intra- versus extraconal location and for benign versus malignant pathology. METHODS: A retrospective review of all expanded endonasal approaches for OA tumors was performed at a single institution from 2009 to 2013. A PubMed database search was also performed to review series published on endonasal OA tumor resection. Across all the cases reviewed, the following data were recorded: two- versus four-handed techniques, intra- versus extraconal tumor location, and benign versus malignant pathology. The relationship between these variables and resection extent was analyzed by the Fisher exact test. Postoperative visual status and complications were also reviewed. RESULTS: Ten cases from the institution and 94 cases from 17 publications were reviewed. Both two- and four-handed techniques were used to resect extra- and intraconal OA tumors, for both benign and malignant pathology. Four-handed techniques included a purely endonasal approach and a combined endonasal-orbital approach. On univariate analysis, the strongest predictor of complete resection was benign pathology (p = 0.005). No significant difference was found between the extent of resection and a two- versus a four-handed technique. Visual status was improved or unchanged in 94% of cases, and other complications were rare. CONCLUSION: Benign tumors that involve the medial extraconal and posterior inferomedial intraconal OA can be treated by either two- or four-handed endonasal techniques. Selecting two- versus four-handed techniques and endonasal versus endonasal-orbital four-handed techniques depends mainly on surgeons\u27 experience. Endonasal approaches for malignant OA tumors are less likely to result in complete resection

Sinus irrigations before and after surgery-Visualization through computational fluid dynamics simulations

OBJECTIVES/HYPOTHESIS: Topical sinus irrigations play a critical role in the management of sinonasal disease, and the improvement in irrigant penetration into the sinuses postoperatively greatly contributes to the success of endoscopic sinus surgery. Prior investigations on postoperative sinus irrigations have been mostly limited to cadaver studies, which are labor intensive and do not capture the full dynamics of the flows. A pilot study was conducted to investigate the impact of surgery on sinus irrigation through computational fluid dynamics (CFD) simulations. STUDY DESIGN: Retrospective computational study. METHODS: Pre- and postoperative computed tomography (CT) scans were obtained on a patient who underwent standard endoscopic surgeries for all sinuses, including a Draf III frontal sinusotomy. CT-based pre- and postoperative CFD models then simulated irrigations of 120 mL saline per nostril at 12 mL/s (typical of Sinugator) and 60 mL/s (SinusRinse Bottle), in two head positions: face parallel and at a 45° angle to the ground. RESULTS: Overall, surgery most significantly improved frontal sinus irrigation, but surprisingly resulted in less maxillary and ethmoid sinuses penetration. This may due to the partial removal of the septum during the Draf III, causing most fluid to exit prematurely across the resected septum. Higher flow rate slightly improved ethmoid sinus irrigation, but resulted in less preoperative contralateral maxillary sinus penetration. CONCLUSIONS: CFD modeling of sinonasal irrigations is a novel technique for evaluating irrigant penetration of individual sinus cavities. It may prove useful in determining the optimal degree of surgery or the ideal irrigation strategy to allow for maximal and targeted sinus irrigant penetration. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E90-E96, 2016

Modified Endoscopic Medial Maxillectomy for Zygomatic Implant Salvage

Objectives Odontogenic chronic rhinosinusitis (CRS) is an epidemiologically important disease process due, in part, to the increasingly commonplace use of dental restorative procedures such as zygomatic implantation. Traditional management of this clinical entity typically entails extraction of the infected hardware via an open or endoscopic approach. We describe a novel management strategy of odontogenic CRS following bilateral zygomatic implantation for oral rehabilitation that we surgically salvaged via a modified endoscopic medial maxillectomy. Methods We describe the presentation and management of a case of metachronous development of bilateral CRS subsequent to zygomatic implantation. Results The patient's postoperative course was characterized by marked endoscopic, radiologic, and symptomatic improvement as measured by the 22-item Sino-Nasal Outcome Test. Conclusion We describe a novel treatment strategy for the management of odontogenic sinusitis resulting from erroneous zygomatic implant placement. Modified endoscopic medial maxillectomy in this clinical context facilitates mucosal normalization of the affected sinus, while permitting preservation of oral function through salvage of the displaced implant

DataSheet_1_Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages.pdf

IntroductionBitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses, possibly contributing to reduced innate immunity in CF.MethodsImmunofluorescence, qPCR, and live cell imaging were used to measure T2R localization, calcium and NO signaling, ciliary beating, and antimicrobial responses in air-liquid interface cultures of primary human nasal epithelial cells and immortalized bronchial cell lines. Immunofluorescence and live cell imaging was used to measure T2R signaling and phagocytosis in primary human monocyte-derived macrophages.ResultsPrimary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o- CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation.ConclusionsTogether, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate T2Rs.</p