Molecular modulation of airway epithelial ciliary response to sneezing

Abstract

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.

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

“…Because taste receptors are linked to downstream calcium signaling, we loaded mouse septal ALI cultures with the calcium-sensitive fluorophore Fluo-4 to measure relative changes in cytoplasmic free calcium concentration as previously reported. 36 …”

“…Calcium and nitric oxide imaging were performed as previously described 14, 36 using the calcium sensitive fluorophore Fluo-4 and reactive nitrogen species indicator DAF-FM, respectively. The validation of DAF-FM use as a method for measuring NO produced by sinonasal ALI cultures was previously described.…”

“…Images were analyzed in Olympus Fluoview software and/or ImageJ as previously described. 14, 36 No offset or gamma alterations were used. Fluo-4 fluorescence changes were normalized after subtraction of the background fluorescence, which was estimated for each experiment by measuring unloaded ALIs at identical settings.…”

New Insights into Upper Airway Innate Immunity

“…22 Other receptors also regulate mucociliary clearance, including, e.g., the bitter taste receptor (T2R) T2R38 (discussed below in Regulation of Antimicrobial Compounds) that binds bacterial metabolites 23 and those receptors that respond to mechanical stimulation. 24 Studies found that dehydrated mucus, as occurs in patients with cystic fibrosis, 22,25,26 and increased mucus production 27 can lead to mucociliary clearance dysfunction. Abnormalities that affect epithelial cilia function may also result in impaired mucociliary clearance.…”

In vitroEffects of Anthocyanidins on Sinonasal Epithelial Nitric Oxide Production and Bacterial Physiology

“…Stock solutions of chemicals were dissolved in dimethyl sulfoxide before dissolving in Dulbecco's phosphate-buffered saline (PBS), with the highest working concentrations reflecting no more than 0.1% dimethyl sulfoxide, a level that does not affect sinonasal ALIs. 19,20,42,43 In the experiments, PBS was primarily used on the apical side and Hank's Balanced Salt Solution (which contained 10 mM HEPES and Minimal Essential Medium amino acids) basolaterally, as described elsewhere. 19,20,[42][43][44] 4-Amino-5-methylamino-2Ј,7Ј-difluroescein (DAF-FM) was from Life Technologies, Inc. (Carlsbad, CA).…”