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 some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2Rdependent 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 HSP90 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 receptors (also known as taste family 2 receptors, or T2Rs, encoded by TAS2R genes) are G protein-coupled receptors (GPCRs) used by the tongue to detect bitter compounds (1, 2). However, many of the 25 human T2R isoforms are also expressed in other organs (1-5), including the nose, sinuses, and lungs (6, 7). These receptors regulate diverse processes like airway smooth muscle contraction (8-11) and innate immune responses in the oral epithelium (12). T2R receptors are also expressed in immune cells like monocytes and macrophages (13-15), which are important players in airway innate immunity ( 16, 17 ). In the airway epithelium, T2R isoforms 4, 14, 16, 38, and possibly others are expressed in bronchial and nasal motile cilia ( 18-21 ). These T2Rs are activated in response to acyl-homoserine lactone (AHL) and quinolone quorum-sensing molecules secreted by the common airway pathogen Pseudomonas aeruginosa ( 18, 22, 23 ).

Activation of the T2Rs in sinonasal cilia or unprimed (M0) macrophages causes Ca2+dependent activation of nitric oxide (NO) synthase (NOS) ( 4, 18, 24-28 ), likely the endothelial NOS (eNOS) isoform expressed in both airway ciliated cells ( 29-32 ) and M0 macrophages (33). In ciliated cells, NO activates soluble guanylyl cyclase to produce cyclic GMP (cGMP). NO activates protein kinase G (PKG) to elevate ciliary frequency to increase mucociliary clearance, the major physical defense of the airway. The T2R-activated NO also directly diffuses into the airway surface liquid (ASL), where it can have antibacterial effects ( 18, 34 ). NO can damage bacterial cell walls and/or DNA ( 35, 36 ). NO can also inhibit the replication of many respiratory viruses, including influenza, parainfluenza, rhinovirus,( 37 ) and SARS-CoV-1 & -2 ( 38-41 ). In macrophages, T2R-stimulated NO production and cGMP production acutely increases phagocytosis ( 42 ). Thus, T2R to NO signaling may also be an important therapeutic target in infectious diseases beyond the airway. As NOS and NO have been implicated in chronic rhinosinusitis ( 43 ) as well as asthma and other lung diseases ( 44 ), better understanding of the mechanisms of NOS activation in airway epithelial cells may have implications beyond T2Rs.

The importance of T2Rs in upper airway defense is supported by observations that patients homozygous for the AVI TAS2R38 polymorphism, which renders the T2R38 receptor non-functional, have increased frequency of gram-negative bacterial infection (18), have higher levels of sinonasal bacteria in general ( 45, 46 ) and specifically have higher levels of biofilmforming bacteria ( 47 ), have higher frequency of chronic rhinosinusitis ( 48-51 ), and exhibit worse outcomes after functional endoscopic sinus surgery ( 52 ). One study has suggested that TAS2R38 genetics may also play a role in cystic fibrosis P. aeruginosa infection ( 53 ), though other studies have suggested that TAS2R38 may not be a modifier gene in CF (54, 55). Recently, an association between the TAS2R38 PAV (functional) genotype has been associated with a lower mortality of SARS-COV2 compared with the AVI (non-functional) genotype (56). Better understanding the role of T2R38 and other T2Rs in airway innate immunity is important for determining if and how to leverage these receptors as therapeutic targets or predictive biomarkers.

In this study, we explored the role of heat shock protein 90 (HSP90) in T2R function in two types of cells important for airway innate immunity: ciliated epithelial cells and macrophages. The members of the HSP90 class of molecular chaperones are highly conserved and ubiquitously expressed ( 57 ). In addition to promoting protein folding, HSP90 regulates signaling by facilitating trafficking or localization of signaling proteins and/or functioning as molecular scaffolds to bring signaling molecules together. Because HSP90 is critical for endothelial cell NO production via eNOS, we tested if HSP90 is involved in T2Rdependent NO generation. HSP90 proteins can facilitate eNOS activation via scaffolding of eNOS with activating kinases such as Akt or Ca2+-bound calmodulin (CaM) kinases ( 58-62 ). HSP90 has been localized to the base of airway cell cilia ( 63-65 ), suggesting HSP90 may be localized close to T2Rs in airway ciliated cells and may help facilitate their signal transduction to eNOS. Recently, it was proposed that HSP90 inhibition by geldanamycin can revert Th2- and Th17-induced airway epithelial goblet cell metaplasia (66). If HSP90 inhibition reduces T2R NO responses, this may have unwanted effects of reducing T2R/NO-mediated innate immunity.

To test the requirement for HSP90 in T2R signaling, we combined biochemistry and live cell imaging with a human bronchiolar cell line, primary sinonasal epithelial cells, and primary monocyte-derived macrophages. Results below reveal important molecular insights into the T2R signaling pathway in both airway epithelial and immune cells and identify a specific role for HSP90 in airway epithelial NO-mediated innate immunity. (CellVis) as described ( 68 ). Our prior studies suggest no differences in T2R responses among macrophages differentiated by adherence alone or by adherence plus M-CSF ( 42 ), and thus adherence only was used for these studies.

Primary human nasal epithelial cells were obtained in accordance with The University of Pennsylvania guidelines regarding use of residual clinical material from patients undergoing sinonasal surgery at the University of Pennsylvania with institutional review board approval (#800614) and written informed consent from each patient in accordance with the U.S. Department of Health and Human Services code of federal regulation Title 45 CFR 46.116. Inclusion criteria were patients ≥18 years of age undergoing sinonasal surgery for sinonasal disease (CRS) or other procedures (e.g. trans-nasal approaches to the skull base). Exclusion criteria included history of systemic inheritable disease (eg, granulomatosis with polyangiitis, cystic fibrosis, systemic immunodeficiences) or use of antibiotics, oral corticosteroids, or antibiologics (e.g. Xolair) within one month of surgery. Individuals ≤18 years of age, pregnant women, and cognitively impaired persons were not included. Tissue was transported to the lab in saline on ice and mucosal tissue was immediately removed for cell isolation.

Sinonasal epithelial cells were enzymatically dissociated and grown to confluence in proliferation medium (50% DMEM/Ham’s F-12 plus 50% BEBM plus Lonza Singlequot supplements) for 7 days ( 18-20, 69 ). Confluent cells were dissociated and seeded on Corning transwells (0.33 cm2, 0.4 µm pore size; transparent; corning) coated with BSA, type I bovine collagen, and fibronectin (Corning). When culture medium was removed from the upper compartment, basolateral media was changed to differentiation medium as described above for H441 ALIs. Primary ALI cultures were genotyped for TAS2R38 PAV (functional) or AVI (nonfunctional) polymorphims ( 70, 71 ) as described (18-20). Cell identity was verified based airway epithelial morphology (formation of motile cilia, goblet cells, transepithelial electrical resistance, etc.) observed after differentiation.

Live cell imaging of Ca2+, NO, and cGMP

Unless indicated, all regents were from MilliporeSigma (St. Louis MO). Adherent, submerged HEK293Ts in 20 mM HEPES-buffered Hank’s Balanced Salt solution (HBSS) were simultaneously loaded and stimulated for 30 min in the presence of 10 µM DAF-FM-diacetate (ThermoFisher Scientific) ± SC79 (Cayman Chemical) as indicated. HEK293Ts were then immediately washed three times in HBSS and imaged as below. Submerged H441 cells were loaded for 30 minutes with 10 µM DAF-FM-diacetate (ThermoFisher Scientific) in 20 mM HEPES-buffered HBSS supplemented with 1x MEM amino acids followed by washing with

Measurement of ciliary beat frequency (CBF)

Whole-field CBF was measured using the Sisson-Ammons Video Analysis system ( 73 ) as previously described ( 18, 74, 75 ) at ~26-28 °C, with the exception of bacterial cHBSS experiments, which were carried out at room temperature. Cultures were imaged at 120 frames/second using a Leica DM-IL microscope (20x/0.8 NA objective) with Hoffman modulation contrast in a custom glass bottom chamber. Experiments utilized Dulbecco’s PBS (+ 1.8 mM Ca2+) on the apical side and 20 mM HEPES-buffered Hank’s Balanced Salt Solution supplemented with 1× MEM vitamins and amino acids on the basolateral side. As typically done with CBF measurements ( 74-77 ), changes in CBF were normalized to baseline CBF. This was validated by measurements of raw baseline CBF (in Hz) between control and experimental cultures showing no significant differences, as indicated in the text.

Bacteria culture

PAO-1 (ATCC 15692) and PAO-JP2 (ΔlasI, ΔrhlI; Tcr, HgCl2r; ( 78, 79 ) were cultured in LB media as described ( 18, 67 ). Conditioned HBSS (cHBSS) was prepared by taking the pellet of an overnight culture and resuspending to OD 0.1 in HBSS and incubating overnight with shaking. We used cHBSS over conditioned LB due to the slight stimulatory effects of LB alone on CBF at dillutions >10% (18). After centrifuging (5000 x g, 15 min, 4°C) to pellet bacteria, cHBSS was filtered through a 0.2 µm filter then diluted as indicated with unconditioned (unmodified) HBSS.

Immunofluorescence (IF) microscopy

IF was carried out as previously described (18-20). ALI cultures were fixed in 4% formaldehyde for 20 min at room temperature, followed by blocking and permeabilization in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), 5% normal donkey serum (NDS), 0.2% saponin, and 0.3% triton X-100 for 1 hour at 4°C. A549 or 16HBE cells were fixed in 4% formaldehyde for 20 min at room temp, followed by blocking and permeabilization in PBS containing 1% BSA, 5% NDS, 0.2% saponin, and 0.1% triton X-100 for 30 min at 4°C. Primary antibody incubation (1:100 for anti-T2R antibodies, 1:250 for tubulin antibodies) were carried out at 4°C overnight. Incubation with AlexaFluor (AF)-labeled donkey anti-mouse and rabbit secondary antibody incubation (1:1000) was carried out for 2 hours at 4°C. Transwell filters were removed from the plastic mounting ring and mounted with Fluoroshield with DAPI (Abcam; Cambridge, MA USA)). For co-staining of T2R14 and T2R38, Zenon antibody labeling kits (Thermo Fisher Scientific) were used to directly label primary antibodies with either AF546 or AF647 as described (18-20). Images of ALIs were taken on an Olympus Fluoview confocal system with IX-73 microscope and 60x (1.4 NA) objective and analyzed in FIJI (80). Images of submerged H441 cells were taken on an Olympus IX-83 microscope with 60x (1.4 NA) objective using Metamorph. Anti-T2R38 (ab130503; rabbit polyclonal; RRID:AB_11156286) and anti-beta-tubulin IV (ab11315; mouse monoclonal; RRID:AB_297919) antibodies were from Abcam. Anti-T2R14 (PA5-39710; rabbit polyclonal; RRID:AB_2556261) primary antibody and conjugated secondary antibodies (donkey anti-rabbit AlexaFluor 546 [RRID:AB_2534016] and donkey anti-mouse AlexaFluor 488 [RRID:AB_141607]) were from ThermoFisher Scientific. Alpha-tubulin antibody was from Developmental Studies Hybridoma Bank (12G10; mouse monoclonal; University of Iowa, Iowa City; RRID:AB_1157911). Anti-eNOS antibody (NB-300-605; rabbit polyclonal; RRID:AB_10002794) was from Novus (Littleton, CO). Immunofluorescence images were analyzed in FIJI (80) using only linear adjustments (min and max), set equally between images that are compared. Compared images were always taken with the same exposure, objective, and other camera and microscope settings. Both conventional (0 = black) and inverted (0 = white) lookup tables (LUTs) were used in this study to illustrate localizations as clearly as possible. Inverted LUTs were from ChrisLUTs FIJI package (https://github.com/cleterrier/ChrisLUTs; C. Leterrier, Neuropathophysiology Institute, Marseille University). fluoresce when particles are internalized into low pH endosomes (previously demonstrated in ( 42 )), this assay does not require washing or quenching of the extracellular pHrodo S. aureus. Macrophages were incubated with pHrodo-S. aureus for 30 min at 37°C as described ( 68 ) with excitation at 555 nm and emission at 595 nm measured on the Tecan Spark 10M plate reader. Background measurements were made using wells containing fluorescent S. aureus in the absence of macrophages. Representative images were taken as above except using a standard TRITC filter set (Semrock).

Data analysis and statistics

Multiple comparisons were made with one-way ANOVA with Bonferroni (pre-selected pairwise comparisons), Tukey-Kramer (comparing all values), or Dunnett’s (comparing to control value) post-tests; p <0.05 was considered statistically significant. Asterisks (* and **) indicate p <0.05 and p <0.01, respectively. All data in bar graphs are shown as mean ± SEM with n derived from biological replicates (separate experiments conducted with different passage/patient cells on different days). Images shown for comparison were collected on the same day under identical conditions with identical min/max settings. No non-linear (e.g., gamma) adjustments were made to any images for either display or analysis. Raw unprocessed image data were analyzed in FIJI (80) and resulting numerical data were analyzed in Excel (Microsoft) and/or Prism (GraphPad software, La Jolla, CA). All data used to generate bar graphs and traces are available upon request.

HSP90 inhibition reduces heterologously-expressed eNOS function in HEK293Ts and A549s

To first determine if we could recapitulate results that HSP90 is important for eNOS function ( 62, 81-84 ) in a reductionist model, we expressed eNOS-RFP in HEK293Ts, an eNOS null cell line ( 85 ). We measured NO production using reactive nitrogen species (RNS)-sensitive dye DAF-FM over 30 min. One mechanism by which eNOS can be activated is phosphorylation at S1177 (S1179 in bovine eNOS). We found that expression of S1179D eNOS dramatically increased DAF-FM fluorescence compared with Wt eNOS-RFP or S1179A eNOS-RFP (Figure 1A-B). DAF-FM fluorescence increases likely reflected NO production as they were inhibited after 30 min pre-treatment and in the continued presence of 10 µM L-NAME but not inactive control D-NAME (Figure 1B). We also observed that HSP90 inhibitor geldanamycin (10 µM; 30 min pre-treatment then continued throughout the 30 min experiment) also reduced DAF-FM fluorescence in S1179D eNOS-RFP-expressing cells. Supporting that the effects of geldanamycin were mediated by HSP90 inhibition, we also found that co-transfection of a dominant negative (DN) HSP90 isoform (D88N) ( 84 ) reduced DAF-FM fluorescence while Wt HSP90 did not significantly change DAF-FM fluorescence (Figure 1B).

Small molecule Akt activator SC79 also induces eNOS phosphorylation and NO production in airway epithelial cells ( 86 ). We found that SC79 (10 µg/ml) activates DAF-FM fluorescence increases in HEK293Ts transfected with Wt eNOS but not untransfected cells (Figure 1C). In Wt eNOS-transfected cells, SC79-induced DAF-FM fluorescence increases were reduced by Akt inhibitor MK2206 or GSK690693 (10 µM; 30 min pre-treatment then continued throughout the 30 min experiment) as well as co-transfection with dominant negative (K179M) Akt ( 87 ) (Figure 1D). SC79-induced DAF-FM fluorescence increases were also blocked by HSP90 inhibitors geldanamycin or BIIB 021 (Figure 1D). Further supporting the role of HSP90 in eNOS function, we found that co-transfection of DN HSP90 in SC79-stimulated HEK293Ts reduced DAF-FM fluorescence (Figure 1E). All together, these data suggest that HSP90 is important for eNOS-mediated NO production.

When we transfected GFP-tagged eNOS ( 88 ) and mCherry-tagged HSP90 ( 89 ) into submerged A549 airway cells, chosen here because of their transfectability and their ability to stick well to glass. We saw punctate perinuclear localization of eNOS likely reflecting Golgi, as eNOS localizes partly to the Golgi in endothelial cells ( 85 ) (Figure 1F). We also saw some likely plasma membrane eNOS localization at cell-cell contact points (Figure 1F), also expected from studies of endothelial cells ( 88 ). HSP90 localization was more global (Figure 1F), though perinuclear puncta were observed. However, when we exited GFP and collected mCherry emission, we saw an mCherry emission signal that almost identically overlapped with GFPeNOS localization (Figure 1F). As GFP and mCherry are a donor-acceptor pair for Förster resonance energy transfer (FRET), we hypothesized that we were collecting emission from mCherry-HSP90 in close proximity to the excited GFP-eNOS. When this experiment was performed with GFP-eNOS and mCherry alone (no HSP90), no mCherry emission was detected with GFP excitation (Figure 1F). A549 cells endogenously express T2R bitter taste receptors activated by the bitter agonist denatonium benzoate ( 90 ). When we monitored GFP and mCherry emission, both with GFP excitation, we noted an increase in mCherry emission and concomitant decrease in GFP emission with denatonium benzoate stimulation (Figure 1G), suggesting that an increase in FRET occurs with bitter agonist stimulation. This increase in FRET was not observed during stimulation with Na benzoate and was reduced with HSP90 inhibitor geldanamycin (Figure 1H). Together, these A549 data suggest that heterologouslyexpressed HSP90 and eNOS are partly closely co-localized in an airway cell line, and this association or close co-localization may increase during T2R stimulation.

HSP90 inhibition reduces endogenous eNOS function in H441 cells

We next wanted to test if HSP90 activity affects endogenous eNOS function when activated by endogenous T2R receptors. We started by examining if T2R stimulation activates NO production in H441 small airway epithelial cells, a club cell-like cell line that expresses eNOS similarly to primary bronchial cells ( 31, 91 ). H441 cells produce NO in response to estrogen and other types of stimulation ( 31, 91-94 ). We observed positive immunofluorescence (IF) for eNOS in submerged H441s compared with rabbit serum and fluorescent secondary alone (Figure 2A-B). This eNOS signal was blocked by pre-treatment of H441 cells with eNOS siRNA (Figure 2C), confirming that H441s express eNOS as demonstrated previously by others using Western ( 31, 91 ).

We also noted positive T2R4 and T2R46 immunofluorescence in submerged H441s (Figure 2D-F). T2R4 staining was highly similar to the pattern observed for plasma membrane glucose transporter Glut1 (Figure 2E), while T2R46 appeared to be more diffusely localized but also possibly localized to the edges of H441 cell islands (Figure 2F). Like many GPCRs, a substantial amount of T2R46 immunofluorescence was located intracellularly, possibly representing ER and/or trafficking compartments. However, potential plasma membrane staining was observed at the cell periphery. The implications for these different staining patterns are unclear, but our goal here was to test for T2R expression and not perform detailed localization analysis. The rationale for examining T2R4 and 46 was that T2R4 localizes to nasal but not sodium benzoate, both increased apical DAF-2 fluorescence in a NOS-dependent manner, as responses were inhibited by L-NAME but not D-NAME (Figure 6B). These DAF-2 responses, likely reflecting NO diffusion into the ASL, were blocked by pretreatment (10 µM, 45 min) with GPCR G protein inhibitor YM-254890 ( 105-107 ) or HSP90 inhibitors geldanamycin, 17-AAG, or BIIB 021 (Figure 6C) but were not blocked by HSP70 inhibitor VER-15508. Like submerged H441s, we saw inhibition with eNOS siRNA but not with scramble, nNOS, or PAR-2 siRNA (Figure 6D).

HSP90 inhibition reduces T2R-driven NO production in primary nasal air-liquid interface cultures

We examined NO production using DAF-FM in primary sinonasal cells grown from residual surgical material and differentiated at ALI as described ( 18, 19, 69 ). These cells express T2R receptors in apical motile cilia (Figure 7A and ( 18-21 )). Note that, unlike H441 assays, denatonium benzoate was not used in primary nasal cell assays. While primary bronchial ciliated cells respond to denatonium benzoate (5), we find that nasal ciliated cells do not ( 18, 26 ), likely due to differential T2R expression between bronchial and nasal cells. For primary nasal cells, we instead used the T2R38-specific agonist phenylthiocarbamide (PTC) ( 70, 71 ) in primary ALIs genotyped for functional (PAV) or non-functional (AVI) polymorphisms in TAS2R38 encoding the T2R38 receptor ( 18, 71, 108 ). Homozygous PAV/PAV TAS2R38 cells produced NO in response to 1 mM PTC while AVI/AVI cells did not (Figure 7B). The NO produced during PTC stimulation was inhibited by geldanamycin (10 µM, 45 min pre-treatment; Figure 7B). We also tested the plant flavonoids apigenin and quercetin. Apigenin, a T2R14 and 39 agonist ( 20, 109 ), stimulates NO production and ciliary beat frequency increases in primary nasal ALIs via T2R14 (20). Apigenin (100 µM) stimulation increased DAF-FM fluorescence in primary nasal ALIs that was reduced by pretreatment with T2R14/39 antagonist 4’-fluoro-6-methoxyflavanone (50 µM; 45 min; ( 20, 110 )) as well as HSP90 inhibitor geldanamycin (10 µM 45 min; Figure 7C). We also tested quercetin, another plant flavonoid shown to be a T2R14 agonist in heterologous expression assays ( 111 ). While quercetin was previously shown to increase (CBF) ( 112 ) and reduce cAMP signaling ( 113 ), a mechanism for these effects was not elucidated. As T2Rs also decrease cAMP through inhibitory G protein signaling in airway cells (19, 114), we hypothesized that quercetin may act as a T2R agonist in airway epithelial cells. Quercetin (50 µM) stimulation likewise increased DAF-FM fluorescence that was blocked by 4’-fluoro-6-methoxyflavanone or geldanamycin (Figure 7D). Apigenin and quercetin-stimulated DAF-FM fluorescence responses are summarized in Figure 7E.

We also performed similar assays as in H441s in Figure 5 to measure NO diffusion into the ASL using 30 µL of DAF-2 solution overlaid onto the primary nasal ALIs. Both PTC (1 mM) and P. aeruginosa quorum-sensing molecule 3oxoC12HSL (100 µM) increased apical surface DAF-2 fluorescence in a manner that was T2R38 dependent as it occurred in PAV/PAV (functional T2R38 homozygous) cultures but not AVI/AVI (non-functional T2R38 homozygous) cultures (Figure 7F). PTC- and 3oxoC12HSL-induced increases in DAF-2 fluorescence were inhibited by geldanamycin, 17-AAG, or BIIB 021 but not VER-15008 (all 10 µM for 45 min pretreatment; Figure 7F). Apigenin (100 µM) increased apical DAF-2 fluorescence in a manner that was inhibited by T2R14 antagonist 4’-fluoro-6-methoxyflavanone ( 110 ) (50 µM for 45 min pre-treatment; Figure 7G) or PLC inhibitor U73122 (10 µM for 45 min pre-treatment; Figure 7G). Apigenin-stimulated DAF-2 increases were also reduced by HSP90 inhibitors geldanamycin or 17-AAG but not by HSP70 inhibitor VER 155008 (all 10 µM for 45 min pretreatment) (Figure 7G). Phospholipase C (PLC) inhibitor U73122 (10 µM for 45 min pretreatment) also inhibited the apigenin response while inactive analogue U74434 had no effect. As a control, when apigenin or vehicle was incubated in DAF-2 solution in the absence of cells (just transwells), no differences in DAF-2 fluorescence were observed (Figure 7G). HSP90 inhibition reduces T2R/NO-driven nasal ciliary beating

Data in Figure 7 suggest that HSP90 function is required for NO production during T2R38 or T2R14 activation in primary nasal epithelial cells. We tested if this affected ciliary beat frequency (CBF) using the T2R14 agonist apigenin. As previously described (20), apigenin increased ciliary beat frequency ~10-15% over 10 min. This was blocked by T2R14 antagonist 4’-fluoro-6-methoxyfavanone or HSP90 inhibitors geldanamycin or BIIB 021 (10 minute pretreatment, 10 µM) but not by HSP70 inhibitor VER 1555008 (Figure 8A-B). Thus, HSP90 inhibitors reduced apigenin-stimulated T2R14 CBF responses. We also observed a ~30% increase in CBF with apical application of 25 µM quercetin (Figure 8C) that was reduced by the T2R14 inhibitor 4’-fluoro-6-methoxyflavanone ( 110 ) or geldanamycin. There was no inhibition of CBF increases in response to purinergic agonist ATP (50 µM; Figure 8A and C). Quercetinstimulated increases in CBF were also inhibited by blocking NO signaling with L-NAME (10 µM; Figure 8D). These data suggest quercetin activation of CBF may occur through T2R activation and NO production. Importantly, we observed that geldanamycin has no significant effect on baseline CBF after ≥20 min (Figure 8E), in contrast to prior studies in mouse tracheal cells, where geldanamycin rapidly reduced CBF to ~75% of basal values, postulated to be due to reduced stability of tubulin polymerization upon HSP90 inhibition ( 65 ). We did not see these effects.

We also tested CBF response to HBSS that had been conditioned by overnight exposure to P. aeruginosa. We previously performed similar experiments with conditioned LB media and showed that CBF increases in response to dilute (6.25%-12%) P. aeruginosa media were dependent on bitter receptor T2R38, which is expressed in cilia and detects acylhomoserine lactone (AHL) quorum sensing molecules (18). Here, P. aeruginosa Wt strain PAO-1 was incubated in HBSS for 24 hrs, and the resulting conditioned HBSS (cHBSS) was diluted and used to stimulated cells. We found that 5-15% cHBSS stimulated robust ciliary responses in nasal ALIs homozygous for the functional polymorphism (PAV) of the TAS2R38 gene encoding the T2R38 receptor (Figure 8F). Cells homozygous for the non-functional (AVI) polymorphism of TAS2R38 responded with much lower CBF increases (Figure 8F), showing the responses were dependent on T2R38. With cHBSS from strain PAO-JP2, which is unable to produce AHLs ( 78, 79, 115 ), we observed minimal CBF responses in PAV/PAV cells compared with PAO-1 Wt cHBSS (Figure 8G), showing the response were dependent on AHL signaling. Notably, AHL signaling also control production of quinolone quorum sensing molecules ( 116 ), which can also function as T2R agonists (19). Fitting with a role for HSP90 in T2R function, we observed that geldanamycin reduced the CBF response to PAO-1 cHBSS in PAV/PAV cells (Figure 8H). These data are summarized in Figure 8I and together suggest that geldanamycin can reduce the ability of nasal ALIs to detect P. aeruginosa through T2Rs and increase ciliary beating.

HSP90 inhibition reduces T2R NO production and phagocytosis in primary human macrophages

We wanted to examine T2R signaling to eNOS in another human primary cell model to test if it requires HSP90 function. Like epithelial cells, macrophages are important players in early innate immunity. Unprimed (M0) monocyte-derived macrophages also express eNOS involved in enhancement of phagocytosis during immune receptor activation (33). While isolated monocytes differentiate into Mφs that are not exactly the same as alveolar Mφs that populate the airways at baseline ( 117-119 ), monocyte-derived Mφs are often used as surrogates for alveolar Mφs and are nonetheless themselves important for infections, including during chronic airway inflammation like CRS, chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis ( 17, 120, 121 ). We previously observed that T2R stimulation in human M0 monocyte-derived macrophages also activates low-level Ca2+ responses that drive NO production to enhance phagocytosis ( 68 ). Macrophage DAF-FM responses to denatonium

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Carey, et al. T2R-stimulated nitric oxide production requires HSP90 103. Aarbiou J, Copreni E, Buijs-Offerman RM, van der Wegen P, Castellani S, Carbone A, Tilesi F, Fradiani P, Hiemstra PS, Yueksekdag G, Diana A, Rosenecker J, Ascenzioni F, Conese M, and Scholte BJ. Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells. J Gene Med 14: 733-745, 2012.

Carey, et al. T2R-stimulated nitric oxide production requires HSP90 114. Kim D, Woo JA, Geffken E, An SS, and Liggett SB. Coupling of Airway Smooth Muscle Bitter Taste Receptors to Intracellular Signaling and Relaxation Is via Galphai1,2,3. Am J Respir Cell Mol Biol 56: 762-771, 2017. 120. Assani K, Shrestha CL, Robledo-Avila F, Rajaram MV, Partida-Sanchez S, Schlesinger LS, and Kopp BT. Human Cystic Fibrosis Macrophages Have Defective CalciumDependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia. J Immunol 198: 1985-1994, 2017. 123. Vega VL, Charles W, and Crotty Alexander LE. Rescuing of deficient killing and phagocytic activities of macrophages derived from non-obese diabetic mice by treatment with geldanamycin or heat shock: potential clinical implications. Cell Stress Chaperones 16: 573581, 2011.

Carey, et al. T2R-stimulated nitric oxide production requires HSP90 130. Purnell PR, Addicks BL, Zalzal HG, Shapiro S, Wen S, Ramadan HH, Setola V, and Siderovski DP. Single Nucleotide Polymorphisms in Chemosensory Pathway Genes GNB3, TAS2R19, and TAS2R38 Are Associated with Chronic Rhinosinusitis. Int Arch Allergy Immunol 1-6, 2019. 131. Lin C, Civantos AM, Arnold M, Stevens EM, Cowart BJ, Colquitt LR, Mansfield C, Kennedy DW, Brooks SG, Workman AD, Blasetti MT, Kohanski MA, Doghramji L, Douglas JE, Maina IW, Palmer JN, Adappa ND, Reed DR, and Cohen NA. Divergent bitter and sweet taste perception intensity in chronic rhinosinusitis patients. Int Forum Allergy Rhinol 2020. 132. Adappa ND, Workman AD, Hadjiliadis D, Dorgan DJ, Frame D, Brooks S, Doghramji L, Palmer JN, Mansfield C, Reed DR, and Cohen NA. T2R38 genotype is correlated with sinonasal quality of life in homozygous DeltaF508 cystic fibrosis patients. Int Forum Allergy Rhinol 6: 356-361, 2016. 133. Prodromou C. Mechanisms of Hsp90 regulation. Biochem J 473: 2439-2452, 2016. 135. Kupatt C, Dessy C, Hinkel R, Raake P, Daneau G, Bouzin C, Boekstegers P, and Feron O. Heat shock protein 90 transfection reduces ischemia-reperfusion-induced myocardial dysfunction via reciprocal endothelial NO synthase serine 1177 phosphorylation and threonine 495 dephosphorylation. Arterioscler Thromb Vasc Biol 24: 1435-1441, 2004.

Carey, et al. T2R-stimulated nitric oxide production requires HSP90 153. Davis BJ, Xie Z, Viollet B, and Zou MH. Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase. Diabetes 55: 496505, 2006. 154. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, and Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285-289, 1999.

Carey, et al. T2R-stimulated nitric oxide production requires HSP90 155. Thors B, Halldorsson H, and Thorgeirsson G. eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1. Biochim Biophys Acta 1813: 322-331, 2011. 161. Michell BJ, Chen Z, Tiganis T, Stapleton D, Katsis F, Power DA, Sim AT, and Kemp BE. Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem 276: 17625-17628, 2001. m 033000 r e fa2000 t M -F1000 F A D 0 Wt eNOS: - - + +

SC79: - + - +