HSP90 modulates T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages

Abstract

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 airway innate immune cell types are activated by bitter products, including those secreted by common airway pathogens like Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO production leads to enhanced mucociliary clearance and direct antibacterial effects by ciliated epithelial cells as well as increased 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). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for 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 an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis.

Bitter taste receptor-stimulated nitric oxide innate immune responses are reduced by loss of CFTR function in nasal epithelial cells and macrophages

“…Next, we examined the ability of CF and non-CF nasal ALIs to kill both lab (strain PAO1) and clinical (strain P11006) P. aeruginosa. In this assay, incubation of P. aeruginosa with nasal ALIs results in bacterial killing over 2 hours that requires T2R38 and NO production, as previously demonstrated (39,72). PAV/PAV (functional T2R38) non-CF cultures killed both strains of P. aeruginosa to levels comparable with antibiotic We next tested if this was a general effect of CFTR function on T2R signaling or if this was specific to nasal epithelial cells.…”

“…To answer this, we first tested ciliary beat frequency (CBF), which drives mucociliary transport and clearance of inhaled/inspired pathogens. T2R activation and stimulation of NO production activates guanylyl cyclase to increase CBF via protein kinase G (22, 72, 86). CF ALIs exhibited reduced CBF increases with 3oxoC12HSL ( Fig 6A ), apigenin ( Fig 6B ), and DPD ( Fig 6C ) compared with non-CF ALIs.…”

“…Compounds were added to the apical side in glucose-free HBSS. Macrophages were loaded with 5 μM fura-2-AM or DAF-FM DA for 45 min as previously described (71, 72) and imaged with 20x 0.75 NA PlanApo objective.…”

“…Phagocytosis assay (as descried (71, 72)) were carried out by incubating macrophages with heat-killed FITC-labeled Escherichia coli strain K-12 bioparticles (ThermoFisher Scientific Vybrant phagocytosis assay kit) at 250 μg/ml in phenol red-free, low glucose DMEM (15 min, 37°C) followed by immediate recording of fluorescence from living cells after quenching extracellular FITC with trypan blue per the manufacturer’s instructions. Fluorescence was recorded on a Spark 10M plate reader (Tecan) with 485 nm excitation and 535 nm emission.…”

“…CF cells used here were identified as homozygous for phenyalanine 508 deletion (F508del/F508del) or to have one copy of F508del plus one copy of a minimally functional CFTR variant (e.g., G542X) based on their patient medical record. Because Primary human monocyte-derived macrophages were cultured as previously reported (71,72). Deidentified human monocytes isolated from healthy apheresis donors were obtained by the University of Pennsylvania Human Immunology core.…”