key: cord-0290339-psezjpmt
authors: McMahon, Derek B.; Kuek, Li Eon; Johnson, Madeline E.; Johnson, Paige O.; Horn, Rachel L.J.; Carey, Ryan M.; Adappa, Nithin D.; Palmer, James N.; Lee, Robert J.
title: The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells
date: 2021-05-17
journal: bioRxiv
DOI: 10.1101/2021.05.16.444376
sha: aa1121b0ee9c5808c922cb39f3e95c260182fc41
doc_id: 290339
cord_uid: psezjpmt

Bitter taste receptors (T2Rs) localize to airway motile cilia and initiate innate immune responses in retaliation to bacterial quorum sensing molecules (acyl-homoserine lactones and quinolones). Activation of T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several airway diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by epithelial remodeling, including loss of motile cilia and/or squamous metaplasia. To understand the function of T2Rs within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells de-differentiated to a squamous phenotype. In differentiated cells, T2Rs localize to cilia, however in de-differentiated, non-ciliated cells they localize to the nucleus. Cilia and nuclear import utilize many shared proteins, thus in the absence of motile cilia some T2Rs may target to the nucleus. T2R agonists selectively elevated both nuclear and mitochondrial calcium through a G-protein-coupled receptor, phospholipase C, and InsP3 receptor-dependent mechanism. Additionally, T2R agonists decreased nuclear cAMP, increased nitric oxide, and increased cGMP, consistent with T2R signaling. Furthermore, exposure to T2R agonists led to nuclear calcium-induced mitochondrial depolarization and caspase activation. T2R agonists induced apoptosis in primary bronchial and nasal cells differentiated at air-liquid interface but then induced to a squamous phenotype by apical submersion. Air-exposed well-differentiated cells did not die. This T2R-induced apoptosis may be a last-resort defense against infection, possibly when bacteria have breached the epithelial barrier and reach non-ciliated cells below. However, it may also increase susceptibility of de-differentiated or remodeled epithelia to damage by bacterial metabolites. Moreover, the T2R-activated apoptosis pathway occurs in airway cancer cells. T2Rs may thus contribute to microbiome-tumor cell crosstalk in airway cancers. T2R agonists may also be useful topical therapeutics (e.g., delivered by nasal rinse or nebulizer) for activating airway cancer cell apoptosis without killing surrounding differentiated tissue.

Motile cilia defend the airways. Pathogens get trapped in airway mucus and motile cilia drive their clearance toward the oropharynx for swallowing [1] . Motile cilia are also immune sensors [2] . Cilia contain bitter taste receptors (T2Rs), G protein-coupled receptors (GPCRs) originally identified on the tongue which also detect bacterial N-acylhomoserine lactones and quinolones [3] [4] [5] . T2R isoforms 4, 14, 16, and 38 , are in human nasal [3, 6] and bronchial [7] cilia. Cilia T2Rs initiate Ca 2+ -triggered nitric oxide (NO) production to increase ciliary beating via cGMP and protein kinase G [3, 5] . NO also damages cell walls and DNA of bacteria [8] and inhibits replication of viruses [9] , including SARS-COV-1 and -2 [10, 11] .

Clinical data support importance of ciliated cell T2Rs in chronic rhinosinusitis (CRS) [1] .

Patients with polymorphisms that render the cilia-localized T2R38 isoform non-functional are at higher risk of CRS, are more likely to require sinus surgery, and may have poorer outcomes after surgery [5] . We hypothesize that pathologies resulting in cilia loss or airway remodeling may impair the T2R pathway and have detrimental effects on innate immunity.

Several airway diseases share phenotypes of acquired cilia defects or loss of cilia due to squamous metaplasia or other inflammatory remodeling, including CRS [1] , chronic obstructive pulmonary disease (COPD) [12, 13] , and cystic fibrosis (CF) [14] . Loss of cilia occurs with viral or bacterial infection [2] , type-2 inflammation-driven remodeling [15] , or smoking [16] .

Elucidating how T2R signaling changes as the airway epithelium changes is required to understand airway pathophysiology.

We studied how T2R signaling changes when ciliated cells are de-differentiated or replaced with squamous cells in airway disease. We used airway cell lines as well as primary nasal and bronchial cells. While squamous epithelial cells still express T2Rs, altered intracellular localization of T2R-induced Ca 2+ responses, and possibly the T2Rs themselves, T2R regulation of nuclear calcium and apoptosis 4 contributes to activation of alternative apoptotic signaling involving Ca 2+ signaling from the nucleus to mitochondria.

All reagents are shown in Supplementary Table S1 . More detailed methods are in Supplementary Material.

Primary sinonasal culture was carried out as described [6, 17] . Institutional review board approval (#800614) and written informed consent was obtained. Tissue was collected from patients ≥18 years of age undergoing surgery for sinonasal disease or trans-nasal approaches to the skull base. Primary cells were obtained through dissociation (1.4 mg/ml pronase; 0.1 mg/ml DNase; 1 hour; 37°C) [3] . Primary normal human bronchial (HBE) cells were from Lonza (Walkerville, MD) cultured in PneumaCult (Stemcell Technologies) plus penicillin/streptomycin. Immortalization with BMI-1 (indicated for some experiments) was carried out as in [18] . For deciliation, (HBE) air liquid interface (ALI) cultures were differentiated for 3 weeks and then either exposed to air or apical submersion for 4 days [17] . Age-matched cultures were compared. TAS2R38 genotyping was previously described [3] .

Cell culture was as described [6, 17] in Minimal Essential Media with Earl's salts (Gibco; Gaithersburg, MD USA) plus 10% FBS and 1% penicillin/streptomycin (Gibco). RPMI 2650 (nasal squamous carcinoma), BEAS-2B (adenovirus 12-SV40 hybrid immortalized bronchial), A549 (alveolar type II-like carcinoma), HEK293T, NCI-H292 (lung mucoepidermoid carcinoma expressing T2R14 [19] ), and Caco-2 (colorectal adenocarcinoma) cells were from ATCC (Manassas, VA USA). 16HBE (SV-40 immortalized bronchial) cells [20] were from D. Gruenert (University of California San Francisco, San Francisco, CA USA). Transfections used Lipofectamine 3000 (ThermoFisher Scientific, Waltham MA). After transfection with shRNA plasmids for either T2R8, T2R10, T2R14, or scramble shRNA for stable expression, Beas2B T2R regulation of nuclear calcium and apoptosis cells were supplemented with 1 μg/mL puromycin for 1 week and then maintained in 0.2 μg/mL puromycin.

Unless noted, imaging was as described [6, 17] . For Ca 2+ , cells were loaded with 5 μM Fura-2-AM or Fluo-8-AM for 1 hour in HEPES-buffered Hank's Balanced Salt Solution (HBSS) at room temperature in the dark. Cells were loaded with 10 μM DAF-FM diacetate for 1.5 hours. Fura-2 was imaged using an Olympus IX-83 microscope (20x 0.75 NA objective), fluorescence xenon lamp with excitation and emission filter wheels (Sutter Instruments, Novato, CA USA), Orca Imaging of Fluo-8 or DAF-FM used a FITC filter set (49002-ET, Chroma). For nuclear Ca 2+ or cAMP, plasmids for G-GECO, R-GECO-nls, Flamindo2 or nls-Flamindo2 were transfected 48 hours prior to imaging. Images were taken with FITC or TRITC filters. Annexin V-FITC was imaged at 10x (0.4 NA).

XTT was added to sub-confluent cells immediately before absorbance measurements at 475nm (specific absorbance) and 660nm (reference) with Spark 10M plate reader (Tecan; Männedorf, Switzerland). JC-1 dye was added 10 min prior to recording (ex.488/em.535 and em.590) while CellEvent Caspase 3/7 (ThermoFisher) was added immediately prior to recording (ex.495/em.540).

Cultures were fixed (4% paraformaldehyde, 20 min) followed by incubation in phosphate saline buffer containing 5% normal donkey serum, 1% bovine serum albumen, 0.2% saponin, and 0.1% Triton X-100 for 45 min. Cultures were incubated 1:100 dilutions of T2R or α-gustducin antibodies at 4°C overnight, then AlexaFluor-labeled donkey anti-mouse or anti-rabbit (1:1000) at 4°C for 1 hour. Images were taken on using 60x objective (1.4 NA oil). MitoTracker Deep T2R regulation of nuclear calcium and apoptosis Red FM was used at 10 nM for 15 min. Co-staining of T2R14 and T2R38 used AlexaFluor 488 and 546 Zenon antibody labeling kits.

For endogenous T2Rs, cells were lysed and run on a NuPage 4-12% Bis-Tris gel, transferred to nitrocellulose, then blocked in 5% milk in 50 mM Tris, 150 mM NaCl, and 0.025% Tween-20 (Tris-Tween) for 1 hour. Primary antibody (1:1000) in Tris-Tween with 5% BSA for 1.5 hours.

Goat anti-rabbit or anti-mouse IgG-horseradish peroxidase secondary antibodies (1:5000) for 1 hour. Blots were visualized with Clarity ECL on an imager with Image Lab Software (BioRad).

Nuclei were isolated using the REAP method [21] then either used for biochemistry or fixed on slides.

Subconfluent cultures were resuspended in TRIzol (ThermoFisher Scientific). Purified RNA (Direct-zol RNA kit; Zymo Research) was transcribed to cDNA via High-Capacity cDNA RT Kit (ThermoFisher Scientific). Taqman Q-PCR probes were used in a QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific).

T-tests (two comparisons only) and one-way ANOVA (>2 comparisons) were calculated using GraphPad PRISM with appropriate post-tests. In all figures, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). Fig. S1 ) and T2R expression was observed (Supplementary Fig. S2 ). Cognate T2Rs for agonists used are in Table S2 . T2R agonist-induced Ca 2+ i appeared most intense in the nucleus of airway (Fig. 1A,   Supplementary Fig. S3-S5 ) and non-airway Caco-2 cells ( Figure S6 ). Other GPCRs elicited more global Ca 2+ i responses (Supplementary Fig. S2, S4) . We saw similar bitterant-induced nuclear Ca 2+ i in primary nasal cells from turbinate brushing cultured in submersion ( Supplementary Fig. S7 ).

To directly investigate if bitterants elevate nuclear Ca 2+ (Ca 2+ nuc), Beas-2Bs were cotransfected with genetically-encoded Ca 2+ biosensors: green (G)-GECO and red (R)-GECO-nls

[24] to differentiate between global Ca 2+ i and Ca 2+ nuc. Denatonium benzoate increased R-GECO-nls more than G-GECO fluorescence ( Fig. 1B) suggesting T2Rs preferentially increase Ca 2+ nuc. In contrast, histamine elevated both similarly, (Fig. 1B) . Ca 2+ nuc also increased in A549s, RPMI2650s, and submerged primary nasal cells from 3 patients in response to bitterants ( Supplementary Fig. S7-S8 ). Pseudomonas aeruginosa 3-oxo-C12-HSL activates multiple T2Rs [4, 5] ; 50 μM 3-oxo-C12-HSL activated both Ca 2+ i (Fluo-8) and Ca 2+ nuc (R-GECO-nls) in

Beas-2Bs ( Fig. 1C-E) . Together, these results demonstrate the novel finding that diverse T2R

agonists specifically elevate Ca 2+ nuc. Although the cells lack cilia, the T2Rs nonetheless are functional.

We utilized a protocol to permeabilize plasma membrane but not intracellular organelles, previously used to study ER Ca 2+ release [25] . In permeabilized A549s, quinine still increased Ca 2+ nuc (Fig. S9) , suggesting Ca 2+ nuc originates from T2R signaling on intracellular membranes.

In Beas-2Bs, Ca 2+ nuc was slightly reduced but still intact with 0-Ca 2+ o (no added Ca 2+ plus 2 mM EGTA) buffer but fully eliminated when cells were preloaded with Ca 2+ chelator BAPTA (Fig.   1F ). Thus, T2R-induced Ca 2+ nuc originates largely from internal Ca 2+ stores, supported by an ER-localized Ca 2+ biosensor (Fig. S10) . Fig. S12A-B) . HEK's express a several T2Rs by qPCR (Supplementary Fig. S12C) , and a TAS2R14 promoter GFP reporter revealed bright GFP fluorescence (Supplementary Fig. S12D ). Staining of HEK293Ts with two different T2R14 antibodies was plasma membrane localized at cell-cell contact points but also partly nuclear (Supplementary Fig. S12E) . Thus, However, T2R elevation of Ca 2+ nuc may have implications in many cell types and caution should be exercised with HEK's as a model for T2R

expression.

T2Rs can signal through Gα-gustducin [5] or Gαi [29] to lower cAMP. T2R signaling in ciliated cells is gustducin-independent [3] , but nonetheless decreases cAMP [5] . To assess changes nuclear cAMP (cAMPnuc), we used cAMP biosensor Flamindo2 and nls-Flamindo2 [30]. T2R agonist diphenidol decreased global intracellular cAMP in Beas-2Bs ( Fig. 2A) . Diphenidol and quinine both decreased cAMPnuc concomitant with increasing Ca 2+ nuc (Fig. 2B-D) . It remains to be determined if this requires gustducin or a Gαi. However, over expression of Wt gustducin reduced Ca 2+ nuc by >50%, while non-functional gustducin had no effect (Fig. 2E ). There may be partial coupling of Gαq to T2Rs; competing away Gαq with gustducin may lower Ca 2+ responses.

Many GPCRs are G protein promiscuous [31] . Nonetheless, the effects of overexpressing functional gustducin supports T2R involvement.

T2Rs in differentiated ciliated cells produce NO downstream of Ca 2+ via endothelial nitric oxide synthase (eNOS) [3, 6] . Both FFA and denatonium benzoate, but not sodium benzoate, increased DAF-FM (NO indicator dye) fluorescence in 16HBEs that was reduced by NO scavenger cPTIO ( Fig. 3A-B) or NOS inhibitor L-NAME ( Fig. 3C-D) . GENIe cGMP biosensor showed cGMP increases with FFA and denatonium benzoate ( Fig. 3E-H) . Thus, the T2R Ca 2+ response, even though nuclear, can still increase NO/cGMP. Pretreatment with L-NAME did not alter Fluo-8 responses (Fig. 3I) , suggesting the NO is downstream of Ca 2+ .

Intrigued by strong Ca 2+ nuc responses and the partial nuclear staining of T2Rs above, we examined T2R localization in airway cells. GPCRs and associated proteins can localize to and function on the outer nuclear membrane or within internal nucleoplasmic reticulum membranes [32, 33] . A previous study reported an altered staining of T2R38 from cilia in normal tissue to nuclear localization in inflamed and de-ciliated CRS tissue [34], though antibody specificity was not verified nor were functional consequences reported. We hypothesized that certain T2Rs may localize to the nucleus or surrounding ER in the absence of proper trafficking to cilia.

Poor trafficking of T2Rs to the cell surface was reported in the context of potential requirement for b2 adrenergic receptors (b2ARs) to "chaperone" T2Rs [19] . However, most airway epithelial cells have robust b2AR expression; cAMP imaging with b2AR agonists in Beas-2Bs, 16HBEs, NCI-H292s, and A549s (Supplementary Fig. S13 ) suggests results here are not due to lack of b2ARs.

Immunofluorescence in multiple cells showed nuclear staining for T2R14 (responds to FFA, DPD, and quinine) and T2R39 (responds to denatonium benzoate and quinine) ( Fig. 4A-F,   Supplementary Fig. S14 ). The antibody against T2R14 was previously validated [6] . To test if the T2R39 antibody was specific, CRISPR-Cas9 was used to create a frameshift in TAS2R39 in A549s. No T2R39 staining was observed in these knockout cells, supporting nuclear localization ( Fig. 4B) . We also saw nuclear staining with an antibody against α-gustducin ( Figure In contrast, other GPCRs exhibited plasma membrane or cytoplasmic localization while T2R14 was nuclear ( Fig. 4E-F) . Denatonium-responsive T2R4, 8, and 39 are located at least partly to the nucleus via Western (Supplementary Fig. S15 ). The same T2R14 antibody showed T2R14 localization to cilia in differentiated primary nasal epithelial cells (Fig. 4G) , as described [6] . T2R39 is likewise expressed in differentiated bronchial cilia [7] . Thus, in airway cells without cilia, such as de-differentiated squamous or cancer cells, T2R14 and T2R39 may instead localize at least partly to the nucleus.

Many studies utilize T2R expression constructs containing N-terminal rat somatostatin type 3 receptor (SSTR3) or bovine rhodopsin sequences to enhance plasma membrane localization (e.g., [19, 26] ). While these constructs are useful for discovering T2R agonists, we tested localization of expressed T2Rs with minimal tagging (single N-terminal myc). We observed nuclear localization of myc-T2R14 and myc-T2R39, but not myc-T2R10 (Fig. 4H) .

Coupled with the endogenous differential T2R38 vs T2R14 localization (Fig. 4E) , there may be different localizations of T2Rs within the same cells and between different cells ( Supplementary Fig. S15 ).

We also expressed either N-terminal or C-terminal GFP fusions. N-terminally tagged GFP-T2R39 co-localized partly with nuclear membrane Lamin-B1, while C-terminally tagged T2R39-GFP appeared less nuclear. C-terminal sequences in mGluR5 are important for nuclear localization [36] . Similar C-terminal sequences may be important for T2R39 and C-terminal GFP may block interactions conferring nuclear localization. GFP-T2R38 did not appear nuclear, suggesting that the nuclear localization of GFP-T2R39 is an effect of the T2R39 sequence and not the N-terminal GFP (Fig. 4I) . To further test this, we co-expressed either GFP-T2R39 or T2R39-GFP with mCherry-Lamin A in HEK293Ts. Consistent with above, GFP-T2R39 was localized to the membrane of isolated HEK293T nuclei (labeled with mCherry-Lamin A) while T2R39-GFP was not (Fig. 4J) , supporting a role for C-terminal sequences in T2R39 nuclear localization.

What are the consequences of T2R-induced Ca 2+ nuc in non-ciliated airway cells? In airway smooth muscle cells, T2R agonists induce phosphorylation of p38 MAPK [37], important for both cell survival. Western revealed that DPD, FFA, and denatonium benzoate promoted a 10-25-fold increase in p38 phosphorylation (Supplementary Fig. S16A-C) . However, buffering

Ca 2+ with BAPTA did not block p38 MAPK phosphorylation (Supplementary Fig. S16D ),

suggesting that Ca 2+ i and p38 MAPK activation are independent.

Mitochondria are in close proximity to nuclei (Fig. 5A) (Fig. 5B) . Denatonium benzoate and FFA also increased Ca 2+ mito in Beas-2Bs ( Fig. 5C-D) and 16HBEs ( Fig. 5F ) but not HEK293Ts (Fig. 5E) , which do not exhibit Ca 2+ i increases to denatonium. This was blocked by U73122 or IP3 receptor antagonist Xestospongin C ( Fig. 5G-H) Fig. 6A ) at concentrations ≥1 mM in A549's ( Fig. 6B,C) . T2R agonist chrysin also impaired XTT reduction ( Fig. 6C) . In 16HBEs and Beas-2Bs, denatonium benzoate and quinine, but not sodium benzoate, reduced mitochondrial potential (ΔΨm), measured by JC-1 ( Fig. 6D-G) . To test physiological relevance, we examined bitterant-induced apoptosis in de-ciliated squamous primary human bronchial epithelial (HBE) cells exposed to apical submersion and fully differentiated ciliated cultures exposed to apical air ( Fig. 7A and Supplementary Fig.   S17 ). Bitter agonists increased Annexin V staining over 3-6 hrs in squamous but not ciliated cultures ( Fig. 7B-C) . To tie these responses to T2Rs, similar differentiated vs squamous primary nasal cultures were stimulated with T2R38 agonist PTC. PTC increased annexin V staining only in squamous cultures homozygous for the functional T2R38 (PAV polymorphism).

Cultures homozygous for non-functional T2R38 (AVI polymorphism) did not exhibit increased annexin V staining ( Fig. 7D-E) , suggesting increased Annexin V-staining is dependent on functional T2R38. Submerged squamous HBEs also showed staining with propodium iodide (PI; reflecting permeabilization) 6 hours after bitterant stimulation ( Fig. 7F-G) , likely reflecting secondary necrosis in the absence of phagocytes [41] .. The earlier onset of Annexin V versus PI staining supports apoptosis. These data suggest that T2R agonists activate apoptosis in squamous but not well-differentiated epithelial cells.

Some T2Rs are localized to cilia of differentiated cells [3, 7] [55] . We hypothesize that, in de-ciliated airway cells, the nuclear membrane may become a reservoir for GPCRs that would normally traffic to the cilia under normal conditions. Non-ciliated squamous airway cell T2Rs retain the ability to activate NO production, as observed during activation of T2Rs in ciliated cells [3, 5] . While eNOS is localized to the cilia base in airway epithelial cells [56, 57] , eNOS localizes to the nucleus in some cells [58, 59] . 

The authors declare no competing interests.

T2R regulation of nuclear calcium and apoptosis benzoate activated NO production that was blocked. By NO scavenger cPTIO. C NOS inhibitor, L-NAME, blocks denatonium-induced NO production. Negative control D-NAME had no effect. Representative intensity pseudocolored images shown. Scale bar 100 µm. Sodium benzoate (control for denatonium benzoate) had no effect. C Bar graphs of relative fluorescence intensity (mean ± SEM) from 4 cultures (2 each from 2 donors) per condition stained at 3 (left) or 6 hours (right). Green bars show air-exposed; magenta bars show submerged. Significance by 1-way ANOVA, Bonferroni posttest; **p<0.01 between bracketed bars (submerged vs air-exposed) and ## p<0.01 vs HBSS only. D Experiments carried out similarly to A-B in TAS2R38-genotyped primary nasal cultures ± 500 µM PTC. TAS2R38 (encoding T2R38) has two polymorphisms Mendelianly-distributed in the Philadelphia population [72] . The PAV allele encodes a functional receptor while the AVI allele encodes a non-functional receptor [73] . ALIs from PAV/PAV patient cells exhibit Ca 2+ responses to T2R38-specific agonist PTC while ALIs from AVI/AVI homozygous patient cells do not [3] . Representative images show Annexin V-FITC staining at 6 hrs, which increased in PAV/PAV cells exposed to submersion but not air-exposed cells.

Staining did not increase in AVI/AVI cells under either condition. E Bar graph of mean ± SEM of Annexin V FITC staining from 4 cultures per condition, each from a separate PAV/PAV or AVI/AVI patient. Significance by 1-way ANOVA with Bonferroni posttest; **p<0.01 between bracketed bars (submerged vs air-exposed) and ## p<0.01 vs HBSS only. 

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T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection

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Taste Receptors in Upper Airway Innate Immunity

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

BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity

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