Alan D. Workman

Correlation of T2R38 taste phenotype and in vitro biofilm formation from nonpolypoid chronic rhinosinusitis patients

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.

The Role of Bitter and Sweet Taste Receptors in Upper Airway Immunity

Over the past several years, taste receptors have emerged as key players in the regulation of innate immune defenses in the mammalian respiratory tract. Several cell types in the airway, including ciliated epithelial cells, solitary chemosensory cells, and bronchial smooth muscle cells, all display chemoresponsive properties that utilize taste receptors. A variety of bitter products secreted by microbes are detected with resultant downstream inflammation, increased mucous clearance, antimicrobial peptide secretion, and direct bacterial killing. Genetic variation of bitter taste receptors also appears to play a role in the susceptibility to infection in respiratory disease states, including that of chronic rhinosinusitis. Ongoing taste receptor research may yield new therapeutics that harness innate immune defenses in the respiratory tract and may offer alternatives to antibiotic treatment. The present review discusses taste receptor-protective responses and analyzes the role these receptors play in mediating airway immune function.

The effect of drugs and other compounds on the ciliary beat frequency of human respiratory epithelium.

Background Cilia in the human respiratory tract play a critical role in clearing mucus and debris from the airways. Their function can be affected by a number of drugs or other substances, many of which alter ciliary beat frequency (CBF). This has implications for diseases of the respiratory tract and nasal drug delivery. This article is a systematic review of the literature that examines 229 substances and their effect on CBF. Methods MEDLINE was the primary database used for data collection. Eligibility criteria based on experimental design were established, and 152 studies were ultimately selected. Each individual trial for the substances tested was noted whenever possible, including concentration, time course, specific effect on CBF, and source of tissue. Results There was a high degree of heterogeneity between the various experiments examined in this article. Substances and their general effects (increase, no effect, decrease) were grouped into six categories: antimicrobials and antivirals, pharmacologics, human biological products, organisms and toxins, drug excipients, and natural compounds/other manipulations. Conclusion Organisms, toxins, and drug excipients tend to show a cilioinhibitory effect, whereas substances in all other categories had mixed effects. All studies examined were in vitro experiments, and application of the results in vivo is confounded by several factors. The data presented in this article should be useful in future respiratory research and examination of compounds for therapeutic and drug delivery purposes.

T2R38 genotype is correlated with sinonasal quality of life in homozygous ΔF508 cystic fibrosis patients

Chronic rhinosinusitis (CRS) is very prevalent in the cystic fibrosis (CF) patient population, and leads to high morbidity and markedly decreased quality of life (QOL). Identification of genetic markers that contribute to CRS symptoms in these patients can allow for risk stratification and tailoring of medical and surgical treatments. T2R38 is a bitter taste receptor expressed in the sinonasal tract, and nonfunctional alleles of this receptor have been implicated in treatment‐refractory CRS in non‐CF patients. The purpose of this study is to investigate the significance of T2R38 genotype in the variability of sinonasal QOL and CRS disease severity in a sample of CF patients.

Staphylococcus aureus triggers nitric oxide production in human upper airway epithelium.

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.

Fungal Aflatoxins Reduce Respiratory Mucosal Ciliary Function

Aflatoxins are mycotoxins secreted by Aspergillus flavus, which can colonize the respiratory tract and cause fungal rhinosinusitis or bronchopulmonary aspergillosis. A. flavus is the second leading cause of invasive aspergillosis worldwide. Because many respiratory pathogens secrete toxins to impair mucociliary immunity, we examined the effects of acute exposure to aflatoxins on airway cell physiology. Using air-liquid interface cultures of primary human sinonasal and bronchial cells, we imaged ciliary beat frequency (CBF), intracellular calcium, and nitric oxide (NO). Exposure to aflatoxins (0.1 to 10 μM; 5 to 10 minutes) reduced baseline (~6–12%) and agonist-stimulated CBF. Conditioned media (CM) from A. fumigatus, A. niger, and A. flavus cultures also reduced CBF by ~10% after 60 min exposure, but effects were blocked by an anti-aflatoxin antibody only with A. flavus CM. CBF reduction required protein kinase C but was not associated with changes in calcium or NO. However, AFB2 reduced NO production by ~50% during stimulation of the ciliary-localized T2R38 receptor. Using a fluorescent reporter construct expressed in A549 cells, we directly observed activation of PKC activity by AFB2. Aflatoxins secreted by respiratory A. flavus may impair motile and chemosensory functions of airway cilia, contributing to pathogenesis of fungal airway diseases.

Solitary chemosensory cells are a primary epithelial source of IL-25 in patients with chronic rhinosinusitis with nasal polyps

Background: IL‐25 can function as an early signal for the respiratory type 2 response characteristic of allergic asthma and chronic rhinosinusitis with nasal polyps (CRSwNP). In the mouse gut, tuft cells are the epithelial source of IL‐25. However, the source of human airway epithelial IL‐25 has remained elusive. Objective: In this study we sought to determine whether the solitary chemosensory cell (SCC) is the predominant source of IL‐25 in the sinonasal epithelium. Method: Flow cytometry and immunofluorescence for SCCs and IL‐25 were used to interrogate polyp and turbinate tissue from patients with CRSwNP. Mucus was collected during acute inflammatory exacerbations from patients with CRSwNP or chronic rhinosinusitis without nasal polyps and IL‐25 levels determined by using ELISA. Lastly, sinonasal epithelial cultures derived from polyp and turbinate tissue were stimulated with IL‐13 and analyzed for SCC proliferation and IL‐25 production. Results: This study demonstrates that a discrete cell type, likely an SCC, characterized by expression of the taste‐associated G protein gustducin and the intestinal tuft cell marker doublecortin‐like kinase 1, is the predominant source of IL‐25 in the human upper airway. Additionally, we show that patients with CRSwNP have increased numbers of SCCs in nasal polyp tissue and that in vitro IL‐13 exposure both increased proliferation and induced apical secretion of IL‐25 into the mucosal layer. Conclusions: Inflammatory sinus polyps but not adjacent turbinate tissue show expansion of the SCC population, which is the source of epithelial IL‐25.

CALHM1-Mediated ATP Release and Ciliary Beat Frequency Modulation in Nasal Epithelial Cells

Mechanical stimulation of airway epithelial cells causes apical release of ATP, which increases ciliary beat frequency (CBF) and speeds up mucociliary clearance. The mechanisms responsible for this ATP release are poorly understood. CALHM1, a transmembrane protein with shared structural features to connexins and pannexins, has been implicated in ATP release from taste buds, but it has not been evaluated for a functional role in the airway. In the present study, Calhm1 knockout, Panx1 knockout, and wild-type mouse nasal septal epithelial cells were grown at an air-liquid interface (ALI) and subjected to light mechanical stimulation from an air puff. Apical ATP release was attenuated in Calhm1 knockout cultures following mechanical stimulation at a pressure of 55 mmHg for 50 milliseconds (p < 0.05). Addition of carbenoxolone, a PANX1 channel blocker, completely abolished ATP release in Calhm1 knockout cultures but not in wild type or Panx1 knockout cultures. An increase in CBF was observed in wild-type ALIs following mechanical stimulation, and this increase was significantly lower (p < 0.01) in Calhm1 knockout cultures. These results demonstrate that CALHM1 plays a newly defined role, complementary to PANX1, in ATP release and downstream CBF modulation following a mechanical stimulus in airway epithelial cells.

Relative susceptibility of airway organisms to antimicrobial effects of nitric oxide

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.

Denatonium-induced sinonasal bacterial killing may play a role in chronic rhinosinusitis outcomes: Denatonium-responsiveness and CRS outcomes

Sinonasal bitter taste receptors (T2Rs) contribute to upper airway innate immunity and correlate with chronic rhinosinusitis (CRS) clinical outcomes. A subset of T2Rs expressed on sinonasal solitary chemosensory cells (SCCs) are activated by denatonium, resulting in a calcium‐mediated secretion of bactericidal antimicrobial peptides (AMPs) in neighboring ciliated epithelial cells. We hypothesized that there is patient variability in the amount of bacterial killing induced by different concentrations of denatonium and that the differences correlate with CRS clinical outcomes.