Polarization of protease-activated receptor 2 (PAR-2) signaling is altered during airway epithelial remodeling and deciliation

Abstract

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

Targeting the phosphoinositide-3-kinase/protein kinase B pathway in airway innate immunity

“…The primary physical innate defense mechanism of the airways is mucociliary clearance (MCC) (Figure 2 ). The main functional components of MCC are mucus production by airway secretory cells[ 23 ] and mucus transport by airway ciliated cells[ 11 , 24 - 26 ]. Cilia are specialized organelles lining airway epithelial cells.…”

“…Both CF and PCD patients are more susceptible to airway infections[ 30 - 32 ], supporting the importance of effective MCC to airway defense. A reduction of ciliated cells is also observed in patients with inflammatory diseases like chronic rhinosinusitis[ 32 , 33 ] as well as after exposure to compounds in cigarette smoke[ 24 ].…”

The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells

“… A Primary HBE cells grown on transwells and differentiated at air liquid interface (ALI) were exposed to 4 days of air or apical submersion (as described in the Methods and [17]). Apical submersion increases squamous differentiation ([17, 70, 71] and Supplementary Fig. S17 ).…”

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

“…Air-liquid interface (ALI) cultures were generated as previously described [24,86,87]. Briefly, enzymatically dissociated human sinonasal epithelial cells were then grown to confluence in proliferation medium (DMEM/Ham's F-12 plus BEBM; Lonza, Walkersville, MD, USA) for one week.…”

“…Cultures were washed with PBS to remove media and maintained at~28-30 • C. Dulbecco's phosphate-buffered saline (PBS) (1.8 mM calcium) on the apical side and HEPES-buffered Hank's balanced salt solution supplemented with 1× MEM vitamins and amino acids (ThermoFisher Scientific, Waltham, MA, USA) on the basolateral side. The Sisson-Ammons Video Analysis system (141) was used to measure whole-field ciliary beat frequency (CBF) as previously described [24,86,87]. A Leica (Wetzlar, Germany) DM-IL microscope (20× objective (0.8 NA)) with Leica IMC Hoffman modulation contrast was used for obtaining images at 100 frames/second.…”