Bei Chen

T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection

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.

Bitter and sweet taste receptors regulate human upper respiratory innate immunity

Bitter taste receptors (T2Rs) in the human airway detect harmful compounds, including secreted bacterial products. Here, using human primary sinonasal air-liquid interface cultures and tissue explants, we determined that activation of a subset of airway T2Rs expressed in nasal solitary chemosensory cells activates a calcium wave that propagates through gap junctions to the surrounding respiratory epithelial cells. The T2R-dependent calcium wave stimulated robust secretion of antimicrobial peptides into the mucus that was capable of killing a variety of respiratory pathogens. Furthermore, sweet taste receptor (T1R2/3) activation suppressed T2R-mediated antimicrobial peptide secretion, suggesting that T1R2/3-mediated inhibition of T2Rs prevents full antimicrobial peptide release during times of relative health. In contrast, during acute bacterial infection, T1R2/3 is likely deactivated in response to bacterial consumption of airway surface liquid glucose, alleviating T2R inhibition and resulting in antimicrobial peptide secretion. We found that patients with chronic rhinosinusitis have elevated glucose concentrations in their nasal secretions, and other reports have shown that patients with hyperglycemia likewise have elevated nasal glucose levels. These data suggest that increased glucose in respiratory secretions in pathologic states, such as chronic rhinosinusitis or hyperglycemia, promotes tonic activation of T1R2/3 and suppresses T2R-mediated innate defense. Furthermore, targeting T1R2/3-dependent suppression of T2Rs may have therapeutic potential for upper respiratory tract infections.

Cigarette smoke condensate inhibits transepithelial chloride transport and ciliary beat frequency

Although the pathophysiology leading to rhinosinusitis is complex, evidence indicates that decreased mucociliary clearance (MCC) is a major contributing feature. Normal respiratory epithelial MCC is an important host defense mechanism that is dependent on proper ciliary beating and the biological properties of the airway surface liquid (ASL). The role that tobacco smoke exposure plays as an inhibitor of MCC has yet to be elucidated. The present study investigates the consequences of cigarette smoke exposure on ciliary function and transepithelial chloride (Cl−) secretion, a major determinant of ASL.

Altered sinonasal ciliary dynamics in chronic rhinosinusitis.

Background Although multiple etiologies contribute to the development of rhinosinusitis, a common pathophysiological sequelae is ineffective sinonasal mucociliary clearance, resulting in stasis of sinonasal secretions, with subsequent infection, and persistent inflammation. The respiratory cilia beat continually at a basal rate, while during times of stress, such as exercise or infection, ciliary beat frequency (CBF) increases, accelerating mucus clearance. Previous investigations have led to conflicting results with some authors reporting decreased CBF while others have found normal values of CBF in patients with chronic rhinosinusitis (CRS). Additionally, these studies have only analyzed basal CBF. The goal of this study was to compare the basal as well as the stimulated sinonasal CBF in patients with CRS versus controls. Methods A dual temperature controlled perfusion chamber, differential interference contrast microscopy, and high-speed digital video were used to analyze both basal and adenosine triphosphate (100 μM)–stimulated CBF in human sinonasal mucosal explants. Results Although no difference in basal CBF was detected between control and CRS patients, a marked difference in stimulated CBF was noted. Exogenously applied adenosine triphosphate resulted in a 50–70% increase of CBF in control tissue with a minimally observed CBF increase in explants from CRS patients. Conclusion Dynamic regulation of respiratory ciliary activity is critical for the respiratory epithelium to adapt to varying environmental situations. Thus, diminished or absent adaptation could predispose the sinonasal cavity to accumulation of inhaled infectious and noxious particulate matter resulting in infection/inflammation. Our findings suggest that CRS patients have decreased sinonasal ciliary adaptation to environmental stimuli.

Mouse nasal epithelial innate immune responses to Pseudomonas aeruginosa quorum-sensing molecules require taste signaling components

We previously observed that the human bitter taste receptor T2R38 is an important component of upper respiratory innate defense because it detects acyl homoserine lactone (AHL) quorum-sensing molecules secreted by Gram-negative bacteria. T2R38 activation in human sinonasal epithelial cells stimulates calcium and NO signals that increase mucociliary clearance, the major physical respiratory defense against inhaled pathogens. While mice do not have a clear T2R38 ortholog, they do have bitter taste receptors capable of responding to T2R38 agonists, suggesting that T2R-mediated innate immune mechanisms may be conserved in mice. We examined whether AHLs activate calcium and NO signaling in mouse nasal epithelial cells, and utilized pharmacology, as well as cells from knockout mice lacking important components of canonical taste signal transduction pathways, to determine if AHL-stimulated responses require taste signaling molecules. We found that AHLs stimulate calcium-dependent NO production that increases mucociliary clearance and thus likely serves an innate immune role against Gram-negative bacteria. These responses require PLCβ2 and TRPM5 taste signaling components, but not α-gustducin. These data suggest the mouse may be a useful model for further studies of T2R-mediated innate immunity.

Reversal of chronic rhinosinusitis-associated sinonasal ciliary dysfunction.

Background Although multiple etiologies contribute to the development of chronic rhinosinusitis (CRS), a common pathophysiological sequelae is ineffective sinonasal mucociliary clearance, leading to stasis of sinonasal secretions, with subsequent infection and/or persistent inflammation. Proper therapeutic intervention typically restores mucociliary activity, suggesting that the pathophysiological process(es) responsible for CRS-associated mucostasis may be reversible. We previously demonstrated a blunted response of CRS sinonasal cilia after purinergic stimulation. This study investigated whether the blunted ciliary response is unique to purinergic stimulation and addressed whether the blunted effect is primarily caused by local CRS-associated mediators or inherent genetic defects in ciliary function. Methods A dual temperature-controlled perfusion chamber, differential interference contrast microscopy, and high-speed digital video were used to analyze both basal as well as cholinergic, adrenergic, and purinergic stimulation of cilia in human sinonasal mucosal explants. Additionally, enzymically dissociated sinonasal ciliated cells were maintained ex vivo in submersion, on glass coverslips, and assessed daily for purinergic ciliary beat frequency stimulation. Results Cholinergic and adrenergic stimulation generally were blunted in mucosal explants obtained from CRS patients. Ex vivo maintenance of samples demonstrated that the majority of CRS samples developed a stimulatory phenotype within 36 hours of culturing. Conclusion CRS is a common debilitating disease principally affecting sinonasal epithelial function with a resultant diminution of mucociliary transport. Presently, little is known about how this disease process affects the sinonasal epithelial ciliated cells. Our data suggest that ciliary response to environmental insults is blunted in a reversible manner in CRS patients.

Vasoactive intestinal peptide regulates sinonasal mucociliary clearance and synergizes with histamine in stimulating sinonasal fluid secretion

Mucociliary clearance (MCC) is the primary physical airway defense against inhaled pathogens and particulates. MCC depends on both proper fluid/mucus homeostasis and epithelial ciliary beating. Vasoactive intestinal peptide (VIP) is a neurotransmitter expressed in the sinonasal epithelium that is up‐regulated in allergy. However, the effects of VIP on human sinonasal physiology are unknown, as are VIPs interactions with histamine, a major regulator of allergic disease. We imaged ciliary beat frequency, mucociliary transport, apical Cl‐ permeability, and airway surface liquid (ASL) height in primary human sinonasal air‐liquid‐interface cultures to investigate the effects of VIP and histamine. VIP stimulated an increase in ciliary beat frequency (EC50 0.5 μM; maximal increase ~40% compared with control) and cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent and Na+K+2Cl‐ cotransporter‐dependent fluid secretion, all requiring cAMP/PKA signaling. Histamine activated Ca2+ signaling that increased ASL height but not ciliary beating. Low concentrations of VIP and histamine had synergistic effects on CFTR‐dependent fluid secretion, revealed by increased ASL heights. An up‐regulation of VIP in histamine‐driven allergic rhinitis would likely enhance mucosal fluid secretion and contribute to allergic rhinorrhea. Conversely, a loss of VIP‐activated secretion in patients with CF may impair mucociliary transport, contributing to increased incidences of sinonasal infections and rhinosinusitis.—Lee, R. J., Chen, B., Doghramji, L., Adappa, N. D., Palmer, J. N., Kennedy, D. W., Cohen, N. A., Vasoactive intestinal peptide regulates sinonasal mucociliary clearance and synergizes with histamine in stimulating sinonasal fluid secretion. FASEB J. 27, 5094–5103 (2013). www.fasebj.org

Molecular modulation of airway epithelial ciliary response to sneezing

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.—Zhao, K.‐Q., Cowan, A. T., Lee, R. J., Goldstein, N., Droguett, K., Chen, B., Zheng, C., Villalon, M., Palmer, J. N., Kreindler, J. L., Cohen, N. A. Molecular modulation of airway epithelial ciliary response to sneezing. FASEB J. 26, 3178–3187 (2012). www.fasebj.org

In vivo effects of citric acid/zwitterionic surfactant cleansing solution on rabbit sinus mucosa.

Background Chronic rhinosinusitis that is refractory to medical or surgical intervention may involve a particularly resistant form of infection known as a bacterial biofilm that is recalcitrant to antibiotics secondary to physical barrier characteristics. Recently, a novel sinus cleansing solution, citric acid/zwitterionic surfactant (CAZS) was shown to be extremely effective in disrupting biofilms in vitro. The purpose of this study was to determine the effects of CAZS on sinonasal epithelium in vivo compared with normal saline. Methods Indwelling catheters were placed into the right maxillary sinus of New Zealand white rabbits. CAZS solution or normal saline (10 mL) was instilled at a rate of 20 mL/minute into the sinus followed by aspiration. Rabbits were killed 1, 3, and 6 days after treatment. Mucosa from both maxillary sinuses was harvested and evaluated for physiological activity (ciliary beating) as well as morphological integrity of the epithelium by scanning electron microscopy. Results One day after treatment, beating cilia was evident with morphological analysis shown intact epithelium with 80–85% denudation of cilia compared with saline. Three days after treatment, ciliary activity was again noted with morphological evidence of persistent denuded cilia. By day 6 after treatment, the epithelium had regenerated cilia over the apical surface. Throughout the recovery period beating cilia was evident in CAZS-treated sinuses. Conclusion This study shows that although CAZS acutely denudes respiratory cilia, the remaining cilia are active. Additionally, the epithelial barrier appears intact with active ciliogenesis, and reciliation of the mucosal surface occurring 6 days after treatment.

The effects of retinoic acid on ciliary function of regenerated sinus mucosa.

Background Retinoic acid (RA) has been shown to enhance ciliary ultrastructure in regenerated sinus mucosa compared with controls. However, the functional status of the regenerated cilia has not been studied. Our objective was to evaluate the ciliary beat frequency (CBF) of regenerated sinus mucosa treated with topical RA in a rabbit model. Methods Twelve rabbits underwent bilateral surgical stripping of the maxillary sinuses, followed by treatment with RA gel in the right side and an inert gel control in the left side. The rabbits were then killed at either 2 or 4 weeks, and CBF analysis of the regenerated mucosa was performed. Three unoperated rabbits were used to establish normative CBF data. Results Functional cilia were recovered from 11/12 RA-treated sinuses and 12/12 gel control sinuses. At 2 weeks postoperatively, the RA-treated sinuses showed an average CBF of 19.78 Hz, which was statistically comparable with the normal unoperated controls (p < 0.26). The inert gel-treated mucosa showed a CBF of 29.24 Hz, which was significantly elevated compared with normals (p < 0.05). At 4 weeks, ciliary activity persisted, but both RA-treated sinuses and gel controls showed elevated CBF compared with normals (p < 0.03). Conclusion Topical RA placed in a demucosalized maxillary sinus yields functional cilia. RA appears to have a normalizing effect on CBF early in the mucosal wound healing process compared with control. This effect appears to be mitigated in later stages of wound healing. RA may be beneficial in enhancing morphological and functional aspects of regenerating cilia.