bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling

1 1 1,2 Indiwari Gopallawa , Jenna R. Freund , and Robert J. Lee

Departments of 1Otorhinolaryngology and 2Physiology, University of Pennsylvania Perelman School of Medicine

Correspondence to Robert J. Lee Department of Otorhinolaryngology—Head and Neck Surgery, Hospital of the University of Pennsylvania Ravdin, 5th Floor, Suite A 3400 Spruce Street, Philadelphia, PA 19104. Tel: 215-573-9766 Email: [email protected]

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 1 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Abstract Bitter taste receptors (T2Rs) are GPCRs involved in detection of bitter compounds by type 2 taste cells of the tongue, but are also expressed in other tissues throughout the body, including the airways, gastrointestinal tract, and brain. These T2Rs can be activated by several bacterial products and regulate innate immune responses in several cell types. Expression of T2Rs has been demonstrated in immune cells like neutrophils; however, the molecular details of their signaling are unknown. We examined mechanisms of T2R signaling in primary human monocyte-derived unprimed (M0) macrophages (MFs) using live cell imaging techniques. Known bitter compounds and bacterial T2R agonists activated low-level calcium signals through a pertussis toxin (PTX)-sensitive, phospholipase C-dependent, and inositol trisphosphate receptor-dependent calcium release pathway. These calcium signals activated low-level nitric oxide (NO) production via endothelial and neuronal NO synthase (NOS) isoforms. NO production increased cellular cGMP and enhanced acute phagocytosis ~3-fold over 30-60 min via protein kinase G. In parallel with calcium elevation, T2R activation lowered cAMP, also through a PTX-sensitive pathway. The cAMP decrease also contributed to enhanced phagocytosis. Moreover, a co-culture model with airway epithelial cells demonstrated that NO produced by epithelial cells can also acutely enhance MF phagocytosis. Together, these data define MF T2R signal transduction and support an immune recognition role for T2Rs in MF cell physiology.

Keywords: live cell imaging, innate immunity, quorum sensing, airway epithelium, G-protein-coupled receptors

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 2 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Introduction Taste family 2 receptors (T2Rs) are GPCRs involved in bitter taste perception on the tongue [1, 2]. However, T2Rs are expressed throughout the body, including the nose and sinuses [3, 4] and lung [5]. T2Rs canonically decrease cAMP via Ga-gustducin (Gagust) or Gai [6, 7] and Gbg activation of phospholipase C

(PLC), production of IP3, and elevation of calcium (Fig 1B) [3]. There are 25 T2Rs on the human tongue [1]. Nasal ciliated epithelial cells express T2Rs 4, 14, 16, 38, and possibly others. In addition to their canonical role in taste, mounting evidence suggests T2Rs are bona fide immune recognition receptors for chemosensation of bacteria. Cilia T2Rs detect bacterial products and activate local defense responses within seconds [7, 8], including Ca2+-dependent nitric oxide (NO) production that increases ciliary beating and has direct antibacterial effects [8, 9]. T2R38, T2R10, and T2R14 respond to bacterial acyl-homoserine lactone (AHL) quorum-sensing molecules [9-11]. T2Rs 4, 16, and 38 respond to Pseudomonas quinolone signal (PQS), and T2R14 responds to heptyl-hydroxyquinolone (HHQ) secreted by Pseudomonas [7]. Clinical importance of extraoral T2Rs in immunity is supported by correlation of polymorphisms rendering T2R38 non-functional with susceptibility to chronic rhinosinusitis CRS [9, 12-17]. A high number of T2R polymorphisms exist, contributing to individual taste preferences, but possibly also contributing to susceptibility to infection. It is critical to elucidate mechanisms of extraoral chemosensation by T2Rs to understand if and how to target T2Rs to activate innate immune responses. Dedicated immune cells also express T2Rs [18-21]. T2R38 detects AHLs in neutrophils [18, 19] and is expressed in both resting and activated lymphocytes [20]. Circulating human monocytes, natural killer (NK) cells, B cells, T cells, and polymorphonuclear (PMN) leukocytes also express T2Rs [21]. Stimulation of PMNs with saccharin, which activates both T2Rs and T1R2/3 sweet receptors, enhanced leukocyte migration [21]. However, our understanding of the details of T2R signaling and physiological consequences in immune cells are limited. Our goal was to determine if T2Rs in primary human macrophages (MFs) are coupled to calcium and if this activates NO as in airway epithelial cells. MFs are important players in early innate immune responses, and unprimed (M0) MFs express Ca2+-activated endothelial (e) and/or neuronal (n) nitric oxide synthase (NOS) isoforms, though studies of e/nNOS in MF function are limited. The majority of studies of NO in MFs focuses on higher level NO production via upregulation of inducible iNOS in LPS ± IFNg-activated (classically-activated or M1) MFs and the relationship of the NO to bacterial killing or metabolism [22]. However, iNOS is not expressed or is expressed at very low levels in monocytes and unstimulated M0 MFs [22], while eNOS and/or nNOS are expressed [23-28] and can be activated by calcium [23, 26, 28]. Although relatively few papers examine e/nNOS activation in MF function, evidence suggests a potential role for NO in phagocytosis. Stimulation of Fcg receptor (FcgR, not a GPCR) in M0 human MFs activates both eNOS and nNOS to enhances phagocytosis [23], and the NO produced upregulates nNOS expression [23]. IFNg also up-regulates phagocytosis in an NO-dependent manner in mouse MFs [29]. Thus,

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 3 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. we hypothesized that T2R activation of eNOS or nNOS may play a role in very early responses of MFs encountering secreted “bitter” bacterial metabolites.

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 4 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Results MFs express functional T2Rs tied to calcium signaling Human monocytes obtained from healthy apheresis donors were differentiated to MFs by adherence culture for 12 days, confirmed by functional expression of H1 receptors (Supplementary Fig. 1). We observed expression of several T2Rs in unprimed (M0) MFs. Immunofluorescence using previously validated antibodies [7, 8] directed against T2R4 (Fig. 1a) and T2R46 (Fig. 1b), but not T2R16 (Fig. 1c), exhibited plasma membrane staining similar to GLUT1, a major MF glucose transporter [30]. Reverse transcription (rt) PCR for T2Rs 4, 14, 38, and 46 demonstrated expression in MFs from 3 different individuals (Fig. 1d). We tested a variety of T2R agonists (listed in Supplementary Table 1) using live cell calcium imaging. We observed low-level but sustained calcium responses to denatonium benzoate, which activates eight T2Rs including T2R4 and T2R46 (Fig. 1e) [31, 32], and to quinine, which activates 11 T2Rs including T2R14 and 46 (Fig. 1f) [31, 32]. Bacterial T2R agonists PQS and HHQ also increased calcium (Fig. 1g-h). A panel of bitterants revealed responses to T2R14 agonists thujone and flufenamic acid (FFA), T2R38 agonist phenylthiocarbamide (PTC), T2R14/38 agonist 3-oxo-dodecanoylhomoserine lactone (3oxoC12HSL), and T2R14/46 agonist parthenolide [31, 32]. Peak responses are summarized in Fig. 1i and representative traces in Supplementary Fig. 2. No response was observed with T2R16 agonist salicin, fitting with observed lack of expression (Fig. 1c). Stimulation of Gaq-coupled purinergic receptors with ATP was a positive control. The responses to PTC were blocked by pertussis toxin (PTX; Fig. 1i), which ADP ribosylates and inactivates both Gagust and Gai. Calcium responses to FFA and HHQ were inhibited by PTX and PLC inhibitor U73122, which inhibits T2R responses in airway cells [7, 8], Gbg inhibitor gallein, which inhibits T2R responses in airway smooth muscle [33], and inositol trisphosphate receptor (IP3R) inhibitor xestospongin C (Fig. 1j). Moreover, responses to thujone, HHQ, or FFA were eliminated after intracellular calcium store depletion (thapsigargin in calcium-free solution) (Fig. 1j). Thus, T2R agonists activate calcium release from intracellular stores through GPCR signaling. Representative traces from experiments in Fig. 1i-j are in Supplementary Fig. 2 and 3. We found no differences in responses in MFs differentiated by adherence + cytokine M-CSF (Supplementary Fig. 4). To more concretely tie these results specifically to T2Rs, we used known T2R inhibitors; 4’-fluoro-6- methoxyflavanone is a T2R14 and T2R39 antagonist [34], while probenecid inhibits T2R16 and T2R38 [35]. Calcium responses to apigenin, which activates T2R14 and T2R39, were inhibited after pretreatment with 4’- fluoro-6-methoxyflavanone (Fig. 1k). Calcium responses to PTC were inhibited after pretreatment with probenecid (Fig. 1l). Representative experiments are in Supplementary Fig. 5. Knockdown of T2R14 with pooled siRNAs inhibited responses to T2R14 agonists niflumic acid (NFA; [31, 32]; Fig. 1m) and HHQ (Fig. 1m).

MF T2R activation reduces baseline and stimulated cAMP signaling

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 5 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

As described above, T2R-stimulated Gai or Gagust can reduce cAMP through inhibition of adenylyl cyclase or activation of phosphodiesterase, respectively (Fig. 2a). Therefore, we also tested if bitter agonist stimulation reduced cAMP in MFs. We utilized an mNeonGreen-based cAMP biosensor (cADDis [36]) in a baculovirus modified for mammalian cells (BacMam) to monitor cAMP in real time (Fig. 2b). Stimulation with b-adrenergic agonist isoproterenol increased cAMP (Fig.2c), whereas stimulation with 3oxoC12HSL, FFA, or PQS reduced cAMP (Fig. 2d). The cAMP reductions were eliminated by PTX (Fig. 2d). Moreover, cAMP increases with isoproterenol were reduced in the presence of PQS or 3oxoC12HSL (Fig. 2e); this was likewise eliminated by PTX (Fig. 2f). We confirmed these results using another cAMP biosensor, the mTurquoise2- Venus FRET-based EPAC-SH187 [37]. MFs expressing EPAC-SH187 exhibited PTX-sensitive cAMP decreases during stimulation with quinine, FFA, or 3oxoC12HSL (Fig. 2g). Data demonstrate that T2Rs affect both basal and stimulated cAMP levels in MFs. Using a ratiometric Cerulean-Venus FRET-based PKA biosensor, AKAR4 [38], we observed that PKA activity was also reduced during stimulation with PQS or FFA, and this was eliminated by PTX (Fig. 2h). Together, these data indicate that both arms of canonical T2R signaling appear to be active in MFs when stimulated with bitter compounds.

MF T2R calcium responses drive NO production These T2R-activated low-level calcium responses were reminiscent of airway cells [3, 7, 8]. We tested if NO production was also downstream. M0 MFs express calcium-sensitive eNOS and/or nNOS [23-28]. We confirmed eNOS transcript by rtPCR (Fig. 3a) and detected eNOS and nNOS by Western (Fig. 3b). We used the fluorescent indicator DAF-FM to monitor production of reactive nitrogen species (RNS) in living MFs in real time. DAF-FM terminally reacts with NO and RNS derivatives, increasing fluorescence. Non-specific NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was used as a positive control. Several T2R agonists, but not T2R16 agonist salicin, caused increases in DAF-FM fluorescence (Fig. 3b) that were blocked by PTX (Fig. 3c) or U73122 (Fig. 3d), suggesting they are downstream of T2R calcium signaling. DAF-FM fluorescence increases in response to T2R stimulation, but not SNAP, were blocked by pretreatment with NOS inhibitor L-NG-niroarginine methyl ester (L-NAME), but not inactive D-NAME, and were reduced in the presence of NO scavenger carboxy-PTIO (cPTIO; Fig. 3e). DAF-FM results were confirmed by - - measuring NO decomposition products NO2 and NO3 in culture media (Supplementary Fig. 6). Thus, DAF- FM fluorescence increases reflect NOS activation.

MF T2R signaling increases cGMP To test the hypothesis that NO production would increase cGMP, we utilized a fluorescent cGMP biosensor (Green GENIe). We observed increases in cGMP production with denatonium benzoate or parthenolide (Fig. 4a), which activate calcium and NO responses, but not with salicin or sodium benzoate, which do not activate calcium or NO. The cGMP responses to FFA or PQS were blocked by L-NAME or

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 6 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. cPTIO (Fig. 4b), PTX (Fig. 4c), or elimination of calcium (Fig. 4d). The cGMP increase was enhanced when MFs were co-infected with BacMam to over express soluble guanylyl cyclase (sGC), further tying these responses to NO-induced cGMP. We used siRNA to confirm involvement of T2Rs. MFs treated with control siRNA exhibited cGMP responses to HHQ, PQS, thujone, and niflumic acid (NFA; Fig. 4f). Treatment with a cocktail of eNOS and nNOS siRNA blocked the response to each agonist. T2R14 siRNA, but not T2R10 siRNA significantly reduced the cGMP responses to T2R14 agonists HHQ and NFA, but the response to PQS, which does not activate T2R14 [7], remained intact (Fig. 4f). The response to thujone, which activates both T2R14 and T2R10 [31, 32], was slightly inhibited by either T2R14 or T2R10 siRNAs, and more fully inhibited with a cocktail of both siRNAs (Fig. 4f).

MF T2R activation may alter MF metabolism through combined elevations of calcium and NO. We briefly examined if T2R stimulation had acute effects on MF metabolic state, tied to MF activation [39], by live-cell imaging of NAD(P)H autofluorescence [40]. Ultraviolet autofluorescence increased with bitter stimulation in a PTX- and L-NAME-sensitive manner (Supplementary Fig. 7), suggesting an increase in NAD(P)H levels. Increases in autofluorescence were not observed with either thapsigargin or SNAP separately to non-specifically increase Ca2+ or NO, respectively. However, addition of thapsigargin plus SNAP robustly increased NAD(P)H autofluorescence, suggesting this is an effect of raising both Ca2+ and NO simultaneously (Supplementary Fig. 7). Together, data suggest that T2R stimulation may acutely alter MF metabolism, at least short term, supporting a role for T2Rs in activation of MF immune responses.

MF T2R-driven NO signaling enhances phagocytosis Activation of T2Rs in MFs increases intracellular calcium to activate e/nNOS and produce NO and cGMP. What consequences does this have for MF cell physiology? As mentioned above, NO was implicated in regulation of phagocytosis. We tested if T2R stimulation increased acute phagocytosis of FITC-labeled E. coli bioparticles. Incubation of MFs with FITC-E. coli in the presence of T2R bitter agonists that activated calcium and NO also increased phagocytosis by ~300% (Fig. 5a-c). Responses to FFA were inhibited by L- NAME, cPTIO, PKG inhibitor KT5823, or PTX (Figure 5C). Responses to denatonium benzoate were inhibited by PLC inhibitor U73122 or Gbg inhibitor gallein (Fig. 5d). A cocktail of pooled T2R14 siRNA inhibited responses to NFA or HHQ but not denatonium benzoate, which does not activate T2R14 (Fig. 5e). Responses to all three agonists were reduced with e/nNOS siRNAs (Fig. 5e). We confirmed that we were observing phagocytosis using Staphylococcus aureus bioparticles labeled with pHrodo, a dye that fluoresces at the low pHs existing in lysosomes and phagosomes (Fig. 6a). MFs phagocytosed pHrodo S. aureus, evidenced by increased fluorescence, when incubated at 37 °C but not 4 °C; this was enhanced by denatonium benzoate but not sodium benzoate (Fig. 6b and d). Similar results were

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 7 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. observed with FFA (Fig. 6c and e). Increased phagocytosis with FFA was inhibited by L-NAME, cPTIO (Fig. 6c and e), U73122, or PTX (Fig. 6f). Increased phagocytosis with denatonium benzoate or quinine was also inhibited by PKG inhibitor KT5823 but not PKA inhibitor H89 (Fig. 6g). Others have suggested that cAMP inhibits MF phagocytosis or activation [41-44]. We hypothesized that T2R-mediated cAMP decreases also facilitate phagocytosis. Baseline phagocytosis was inhibited by direct adenylyl cyclase activation by forskolin or cell permeant cAMP analogs, including 8-Br-cAMP and 8- CPT-cAMP, which activate both PKA and EPAC, and to a lesser extent by PKA-specific 6-Bnz-cAMP or EPAC- specific 8-CPT-2-O-Me-cAMP (Fig. 6h). In contrast, cell permeant cGMP analog 8-Br-cGMP increased phagocytosis via PKG; this was inhibited by KT5823. Thus, increased cAMP likely reduces phagocytosis partially through PKA and partially through EPAC, unlike cGMP which increases phagocytosis through PKG. Denatonium benzoate-induced phagocytosis was inhibited by forskolin or permeant cAMP analogs used above, but were not inhibited by 8-Br-cGMP (Fig. 6i). Together, these data suggest both the calcium/NO and cAMP arms of the T2R pathway facilitate MF phagocytosis.

NO crosstalk from airway epithelial cells may also enhance MF phagocytosis Airway epithelial cells also make NO in response to various stimuli including T2R [3, 7] and estrogen receptor activation [45]. We hypothesized that MFs close to the airway surface may be influenced by local intercellular NO production. We designed an assay to test epithelial-MF crosstalk using PTX-treated MFs and H441 small airway epithelial cells, which produce NO in response to 17b-estradiol (E2) (Supplementary Fig. 8). Although MFs exhibited low-level calcium transients to 10 nM E2, these were eliminated by PTX pretreatment (Supplementary Fig. 9). DAF-FM-loaded, PTX-treated MFs were incubated with unloaded H441s separated by a transwell filter in close proximity (≤1 mm; Fig. 7a). Stimulation of H441 cells with E2 resulted in increased MF DAF-FM fluorescence (Fig. 7b), suggesting NO produced by the H441s translated to an increase in RNS within the MFs. Thus, epithelial NO/RNS could act as an intercellular signal. Inclusion of cPTIO or pretreatment of H441 cells with PTX or L-NAME blocked the MF DAF-FM response (Fig. 7c), but when MFs were pretreated with L- NAME, there was no inhibition (Fig. 7c). Thus, the NO/RNS originated in the H441 cells. E2 had no effect when MFs were incubated in the absence of H441s (Fig. 7c). Together, these data support the potential of airway epithelial cells to produce enough NO to be sensed by MFs in close proximity. To test if airway epithelial NO production could act as a signal to stimulate MF phagocytosis, we measured changes in pHrodo S. aureus phagocytosis by PTX-treated MFs when H441s were stimulated with E2 (Fig. 7d). E2 stimulation of H441s increased MF phagocytosis (Fig. 7e and f), and this was blocked by H441 pretreatment with PTX or L-NAME or inclusion of cPTIO in the media (Fig. 7f). E2 had no effect when PTX-treated MFs were incubated in the absence of H441 cells (Fig. 7f).

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 8 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Discussion We only understand the “tip of the iceberg” of extraoral T2Rs and their signaling. We demonstrated that T2Rs in MFs may enhance phagocytosis through calcium-driven NO-activation of guanylyl cyclase to increase cGMP (Fig. 8). Interestingly, this is a similar pathway to airway epithelial cells [3, 7-9, 46], but the physiological output is different. In ciliated cells, the pathway controls cilia beating; in MFs, it regulates phagocytosis. Both processes are critical for innate defense, supporting a role for T2Rs as immune receptors. In MFs encountering bitter bacterial agonists at sites of infection, activated T2Rs may “rev up” acute phagocytic activity while likely simultaneous toll-like receptor (TLR) stimulation up-regulates expression of iNOS and antimicrobial proteins such as lysozyme to further combat infection. Of note, T2Rs also respond to a variety of natural plant compounds associated with complementary medicines, including flavonoids [8, 47-49], as well as common clinical drugs [50, 51]. Activation of extraoral T2Rs in MFs may underlie some effects of homeopathic plant-based treatments or off-target drug effects. This study also suggests that NO produced by airway epithelial cells could increase MF phagocytosis in a paracrine fashion (Fig. 8). NO produced during activation of T2Rs in airway epithelial cells [3, 7] may influence immune cells or amplify immune responses. NO may regulate phagocytosis via several mechanisms, including ADP-ribosylation of actin to regulate cytoskeletal polymerization, and pseudopodia formation, and phagocytosis in mouse peritoneal MFs [52]. In Raw 264.7 mouse MF-like cells, NO acutely regulates actin organization through cGMP in combination with Ca2+/calmodulin [53], and cGMP/PKG may be important in phagocytic responses during TLR2 stimulation in THP-1 monocytic leukemia cells [54]. It is important to better understand e/nNOS and cGMP signaling in Mfs beyond phagocytosis. Production of cGMP was linked to protection against toxicity of peroxynitrite (ONOO-) or oxidized LDL [55], reduction of cytokine release during LPS stimulation [56] or high fat exposure [57, 58], and promotion of wound healing [59]. NO itself may be anti-inflammatory by activation of PPARg, antagonizing NOX2 production of reactive oxygen species [60], or attenuation of NF-kB and activation of Nrf2 transcription factors [61]. However, MFs from eNOS knockout mice exhibit reduced NO production, NF-kB activation, and iNOS upregulation during LPS stimulation [28]. Interestingly, T2R-induced lowering of cAMP levels may be another mechanism to enhance phagocytosis; cAMP elevation through prostaglandins decreases phagocytosis in human MFs [41] and other inhibitory effects of cAMP have been reported [42-44]. Canonical T2R signaling is driven by Ca2+ downstream of Gbg activation of PLC. The decrease in cAMP by Ggust was proposed to accentuate calcium signaling via type III IP3Rs in taste cells [62, 63], but data supporting this mechanism are limited. Increased cAMP and PKA phosphorylation of IP3R is more often reported to enhance calcium signaling [64], though PKA phosphorylation of type III IP3R has been suggested to slow kinetics of calcium release in pancreatic acinar cells [65]. If taste cell signaling is primarily driven by calcium [66], why have T2Rs not evolved to couple to Gq instead of Ggust or

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 9 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Gi? This may be because, as observed here, decreases in cAMP have important biological effects in extraoral tissues.

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 10 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Materials and Methods

Reagents and solutions Unless indicated, all reagents and solutions were as described [7, 8, 67, 68]. Full details of materials, reagents, and solution compositions are in the Supplementary Material. Bitter agonists used and known cognate T2Rs are in Supplementary Table 1. Immunofluorescence (IF) microscopy was carried out as described [7, 8, 67, 68], with more details in the Supplementary Material.

MF culture Monocytes were isolated by the University of Pennsylvania Human Immunology Core from healthy apheresis donors with institutional review board approval by RosetteSepTM human monocyte enrichment cocktail (Stem Cell Technologies, Vancouver, Canada), and MFs were differentiated by 12-days adherence culture in RPMI 1640 media containing 10% human serum and 1x cell culture pen/strep antibiotic mix (Gibco). All cells were deidentified before receipt. No investigator had contact with personal identifying information or demographics. Between 5-15 million cells were obtained from each individual. Cells from 29 individuals were used; some individuals donated multiple times during the course of the study. Details of siRNA protocols are in the Supplementary Material.

Live cell imaging of intracellular calcium, reactive nitrogen species (RNS) production, and cAMP/cGMP signaling Intracellular calcium and RNS were imaged using Fura-2 or Fluo-4 and DAF-FM, respectively, as described [7, 8, 67]. MFs were infected with BacMams containing green downward cADDis or green downward GENie (both from Montana Molecular, Bozeman MT). For AKAR4 or Epac-SH187, MFs on 8-well chambered coverglasses were transfected with 0.5 µg/well plasmid using Effectene (MilliporeSigma, Burlington, MA) per the manufacturer’s protocol. Full details are in the Supplementary Material.

Phagocytosis assays For microscopy, MFs on chambered coverglasses were incubated with heat-killed FITC-labeled E. coli bioparticles at 250 µg/ml (strain K-12; Vybrant phagocytosis assay kit; ThermoFisher Scientific) in phenol red- free, low glucose DMEM ± bitter agonists or inhibitors for 15 min at 37°C. For microscopy, extracellular FITC was quenched with trypan blue and cells were washed ≥5x in PBS to remove residual extracellular FITC-E. coli. Remaining adherent MFs were fixed in 4% formaldehyde (Electron Microscopy Sciences) for 10 min followed by DAPI staining. For pHrodo Red-labeled S. aureus (strain Wood 46; ThermoFisher A10010), living MFs on chambered coverglass were visualized by fluorescence microscopy at room temperature (which drastically reduces any further phagocytosis) immediately after the assay incubation at 37°C.

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 11 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

For plate reader assays, MFs on microplates were incubated similarly as above with 500 µg/ml FITC-E. coli. As phagocytosis was eliminated at 4 °C and almost completely eliminated at room temperature, we recorded fluorescence from living cells at room temperature immediately after the 15 min incubation with FITC- E. coli. Extracellular FITC was quenched with trypan blue, and fluorescence recorded on a Spark 10M plate reader (Tecan; 485 ex, 535 em). Phagocytosis assays were carried out similarly using 125 µg/ml pHrodo-S. aureus for 30 min at 37°C.

Airway epithelial cell culture and coculture H441 cells (ATCC HTB-174) were cultured in MEM with Earle’s salts (Gibco) containing 10% FetalPlex (Gemini Biosciences) in T75 flasks, lifted with trypsin when 75% confluent and transferred to 24 well plates and grown to confluence for MFs co-culture experiments or seeded onto glass chamber slides and imaged for acute calcium and NO measurements. For phagocytosis and NO co-culture, MFs were seeded separately on transparent Falcon filters (#353095; 0.3 cm2; 0.4 µm pores), which come very close (0.8 mm) to the bottom of standard 24 well plates. Filters containing 100 µL HBSS on the apical side were transferred into the 24 well plates containing H441 cells and a small amount of HEPES-buffered HBSS (200 µL) at the time of the assay. MFs were pretreated with PTX (100 ng/ml) for ~18 hrs in media (37°C, 5% CO2), followed by copious washing with HBSS to remove residual PTX before co-incubation with H441s. Similarly, DAF-FM loading of MFs was performed prior to co- incubation with H441s. Addition of fluorescently-labeled bacteria to MFs on transwells was done at the time of assay start. Immediately after, 100 µL of a 3x E2 solution in HBSS was added to the basolateral side to stimulate H441 NO production and the 24 well plate was then incubated at 37°C.

Data analysis and statistics Statistical significance was determined in GraphPad Prism as indicated in figure legends; p <0.05 was considered statistically significant. Other analyses were performed in Excel. Data are presented as mean ± SEM. Data points in bar graphs are ≥3 independent experiments using cells from ≥2-3 donors.

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 12 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Acknowledgements This study was supported by National Institutes of Health grants (R01DC016309, R21AI137484). Content is solely the responsibility of the authors and does not represent official views of the National Institutes of Health. The authors thank J. Riley (University of Pennsylvania Human Immunology Core, supported by P30-CA016520 and P30-AI045008) for access to monocytes and M. Victoria (University of Pennsylvania) for excellent assistance with MF culture and molecular biology and helpful comments on the manuscript. We thank N. Cohen (University of Pennsylvania) for sharing reagents. The authors have no conflicts of interest to declare.

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 13 WhilebioRxiv c preprintirculating doi: BMDMshttps://doi.org/10.1101/776344 differentiate into ; Mthisφs version that are posted not September 26, 2019. The copyright holder for this preprint (which was exactly the same as thenot alveolar certified by M peerφs that review) populate is the author/funder. the airways All at rights reserved. No reuse allowed without permission. baseline,(31-33) BMDM-derived Mφs are often used as surrogates for alveolar Mφs and are nonetheless themselves important for infec- tions all over the body. BMDMs migrate to sites of infection and inflammationFigures, including the airway. BMDM-derived Mφs are par- ticularly importanta inT2R4 conditions of chronic inflammation, like chron- b T2R46 ic rhinosinusitis. DysfunctionDAPI of both alveolar and BMDM-derived DAPI Mφs is implicated in pathogenesis of chronic obstructive pulmonary disease, asthma, and cystic fibrosis.(34-36) Our data suggest that T1Rs and T2Rs may stimulate immune responses from Mφs, and suggest that T1Rs/T2R-targeted therapeutics (which could be de- livered via inhaler or nasal spray) may be beneficial against infec- tions associated with these diseases. However, we need to do the d experiments outlined in this proposal to better understand how ND512 ND486 ND522 c secondary only T2R16 Glut1 Φ Φ Φ T1Rs and T2Rs contributeDAPI to Mφs function.DAPI Because of theDAPI differ- BEAS-2B16HBE14o-M M M ences in numbers of T2Rs in human (~25 T2Rs) and mouse (~35 T2Rs) and lack of clear othologues,(19) experiments need to be T2R4 done in human cells as proposed here. T2R14 We have access to large amounts of primary human T2R38 BMDMs at Penn and began studying M0 (unpolarized/unprimed) Mφs (Fig. 2A). Studies proposed here will also examine T1R/T2R T2R46 ATP 1.2 physiology in M1e and1.2 M2 Mφs and dendriticf cells differentiated i (37-41) 1.5 water-soluble DMSO-soluble liquid from BMDMs using defined cytokines. 1.0 BMDMs are differenti- 1.0 bitterants bitterants bitterant 1.0 1 mM 100 µM denat. ATP 0.5 ated to M0 Mφs by 100.8-day culture in media0.8 + human serum (Fig. benz 670 µM 0.3 ** ## 2A). We have found no differences between0.6 quinine M s differentiated by ## ** (Fura-2 340/380) 0.6 φ (Fura-2 340/380) ## i Fura-2 340/380) i

] **

] #

(42-44) Δ # 2+

2+ 0.2

( # adherence ± the cytokine0.4 M-CSF (as reported0.4 by others ), but i 3 min 3 min ] [Ca [Ca we are acquiring data from both conditions. For simplicity, data 2+ 0.1 [Ca shown here are from M s differentiated by adherence only. Differ- Δ g φ 100 µM h 1.6 entiation to Mφs was 1.4confirmedATP by functional expression of mark- 0.0 1.2 (45, 46)1.2 100 µM Peak ers, including histamine H1 receptors (monocytesATP express H2 1.0 M FFAM FFAM PQS M PTC M thuj. M ATP 2+ M parth.µ µ µ M HHQM HHQµ µ µ 100 µM 50 µM M quinine µ µ µ receptors), determined0.8 by Ca imaging and specific antagonists µ 20 1 mM thuj. HHQ 0.8 3 mM salicin0.1% DMSO 200 50 20 500 300 100 PQS 300 100 M PTC + PTX (Fura-2 340/380) (Fura-2 340/380) i i 1 mM Na benz.670 µ

] M 3oxoC12HSL ] 0.6 (cetirizine and ranitidine, respectively; not shown). 1 mM denat benz. µ 2+ 2+ 500 0.4 0.4 100 3 min 3 min M 3oxoC12HSL + PTX [Ca M0 Mφs express[Ca several T2Rs (Fig. 2B-C) and T1R3 (Fig. µ 2D) localized to the plasma membrane. When Mφs were loaded 100 2+ ) with fura-2 to track changes in intracellular Ca , we noted low- ) o o

2+ ) )

o 1.0 level Ca responsesj 2.0 to a variety of bitter agonists (Fig. 3A-B; nextk l o 1.0 ** m 1.5 NFA ** HHQ page). Responses were blocked by pertussis toxin (PTX; Fig 3C), 0.8 0.8 1.5 ** ** Fluo4 F/F ** Fluo4 F/F 1.0

Δ 0.6 which ADP-ribosylates and inactivates Gαs that can couple to 0.6 Δ Fluo4 F/F (

( ** i Fluo4 F/F i

] 1.0 ** (47) (48-50) ] Δ ** Δ (

2+ 0.4 i

T2Rs (G and/or G ). Responses to bacterial T2R14 2+ i gustducin ( 0.4 i ] ** ] 0.5 2+

0.5 2+ [Ca agonist HHQ were blocked by T2R14 antagonist** 4-fluoro-6- 0.2 0.2 [Ca Δ Δ

(15, 51) 2+ [Ca [Ca methoxy-flavanone (Fig. 3D). Similar Ca responses were Δ 0.0 0.0 Δ (52) 0.0 0.0

Peak 4'-fluoro-6- observed with T1R2/3 (sweet receptor) agonist sucralose and Fig. 2: (A) Primaryprobenecid: human- BMDMs Peak are obtained (53) methoxy- - + + 20 FFA 20 HHQ were blocked by T1R3 antagonist100 FFA lactisole (Fig100 HHQ3E). flavanone:and differentiated in vitro to unprimed (M0) Mφs. 0.1% DMSO 500 µM PTC Further100 “priming µM ” by LPS ± IFNγ or IL-control4 yields siRNAT2R14 siRNA iT2R10n- siRNA As would be expected100 FFA +from PTX a G100 iFFA- or+ XeC G100gust HHQ-coupled + PTX 100 response HHQ + XeC 100 FFA + gallein 100 FFA100 + FFA U73122 + U73343 100 HHQ + gallein apigenin (Fig 1b), a cAMP decrease was also100 FFA after observed thaps. 100 in HHQ100 MHHQ+ U73343φ +100 sU73122 HHQtransfected after thaps flammatory M1 Mφs or anti-inflammatory ( “tissue repair”) M2 Mφs, respectively. M1 Mφs secrete high with Figa cAMP. 1 Unprimed-sensitive (M0) fluorescent monocyte protein-derived biosensor MFs express (EPAC -functional bitter taste receptors (T2Rs). a-c Plasma membrane SH187(18, 54)). This cAMP decrease was blocked by PTX pretreat- levels of IL-12, while M2 Mφs make IL-10 (which staining observed for T2R4 (a) and T2R46 (b) reminiscent of plasmawill be membrane used to verify-localized polarization) glucose. BMDMs transporter can GLUT1 (c). ment (Fig. 3F). Similar results were observed with a mammalian- also be differentiated into dendritic cells. (B) M0 modifiedNo immunofluorescence baculovirus (BacMam was; Autographa observed withcalifornica secondary [AcMNPV] only control or T2R16 antibody (c). d T2Rs detected in MFs by rtPCR. Airway lines were used as control. e-h Representative lowMφs- levelexpress Ca T2Rs2+ responses at the mRNA (fura level-2) thr observedough in response to pseudotyped to infect mammalian cells)-expressed cAMP biosen- rtPCR (T2Rs 8, 10, and 14 also detected; not (55) sor (cADDisdenatonium; Montana benzoate Molecula (e), quininer, Bozeman (f), HHQ M (Tg;), not and shown). PQS (h ). Subsequentshown). T2R4 stimulation detects bacterial with purinergicPQS while T2R38 receptor agonist 2+ BacMamATP resultedused as controlin high fortransduction viability. iefficiency Peak change for M φins Ca(~30 -(fura50%- 2 340/30detects bacterial ratio) during AHLs. 516HBE min stimulation& Beas2B airway with water soluble, vs 1-DMSO2% with-soluble, Effectene and or liquid FuGene bitter and compounds. pcDNA). Future ATP response experi- showncells for shown comparison. as positive control. Also shown (C-D) IF is of inhibition T2R4 of responses (56) (C; Glut1 as plasma membrane control) and T1R3 mentsto willPTC take and advantage 3oxoC12HSL of BacMam by pertussis vectors. toxin (PTX; 100 ng/ml, 18 hrs pretreatment). Significance by one-way ANOVA with (D; CD44 as plasma membrane control). Bonferroni As Mφ T2Rposttest responses with preselected were reminiscent paired comparisons of airway cells; **,(6,p <0.01 between bracketed bars and #p <0.05 and ##p <0.01 15, 18) 2+ denote we tested sign ifificance NO was compared downstream with. DMSOMost M onlyφ studies for DMSO focus- solubleon NO productionbitterants. viaj P mRNAeak Ca/protein responses upregul (fluoa- -4 F/Fo) with tion ofT2R14 inducible agonists iNOS FFA during and M HHQφ activation (concentrations by LPS ± are IFN µM)γ.(57 -in59) the Howe presencever, iNOS of inhibitors is not expressed of GPCR insignaling. mono- Significance by cytesone and-way unstimulated ANOVA, Dunnett’s M0 Mφs ( posttest(58-60) and comparing our own data, each not value shown). to DMSO Constitutive only. k ePeakxpression Ca2+ responsesof Ca2+-sensitive (fluo-4) to T2R14/39 agonist apigenin ± T2R14/39 antagonist 4’-fluoro-6-methoxy-flavanone (50 µM). Significance by Student’s t test; **p <0.01. l Bar graph showing peak Ca2+ (fluo-4) in response to T2R38 agonist PTC ± antagonist probenecid (1 mM). Significance by Student’s t test; **p <0.01. m Peak Ca2+ (fluo-4) during stimulation with T2R14 agonists NFA or HHQ in MFs pre-treated with ON-TARGET plus SMARTpool siRNAs. Significance by one-way ANOVA with Bonferroni posttest; **p <0.01 vs control siRNA condition for each respective agonist. Representative traces are averages of 10-30 MFs from single experiments. Data points in bar graphs are independent experiments using cells from ≥6 experiments (≥3 separate donors; ≥2 experiments per each donor)

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 14 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a b 0 10 20 50 80 sec sec sec sec sec

Gi G AC gust PDE PLC

IP3

òcAMP ñCa2+ cADDis c d ** ** ** o o 0.2 1 µM isoprot. 10 µM 1.4

fsk increase)

0.4 o 0.4 0.6 100 µM 1.2 0.6 Bottom panel 3oxoC12HSL 0.8 Middle panel 1.0 baseline decrease Peak cAMP 0.8 Top panel 1.0 (cADDis F/F control FFA PQS 1.0 1 min 1.2 + PTX

Downward cADDis F/F 2 min Downward cADDis F/F FFA + PTX PQS + PTX 3oxoC12HSL

3oxoC12HSL + PTX 0.8 100 nM isoprot. o o e 0.2 0.2 o 0.2 100 nM isoprot. f isoprot. 100 nM isoprot. ± PQS ± 3oxoC12HSL 0.6 * 0.4 100 nM 0.4 0.4 control decrease) o control 0.4 ** 0.6 0.6 0.6 10 nM PQS C12HSL 0.2 0.8 0.8 0.8 1 nM Peak cAMP increase Peak cAMP

(cADDis F/F 0.0 1.0 2 min 1.0 2 min 1.0 2 min PTX: - + - - Downward cADDis F/F + + Downward cADDis F/F Downward cADDis F/F

control + PQS + AHL

2.5 2.5 + PTX 187 g 187 0.15 2.0 10 µM forskolin 2.0 10 µM forskolin ** ** ** 1.5 1.5 500 µM 500 µM quinine 0.10 1.1 quinine 1.1

baseline baseline CFP/FRET decrease) 0.05

1.0 1.0 187 5 min 5 min Peak cAMP decrease Peak cAMP CFP/FRET em ratio CFP/FRET em ratio 0.9 0.9 0.00 Normalized EPAC-SH Normalized EPAC-SH

FFA (EPAC-SH quinine FFA + PTX 1 µM 3oxoC12HSL h 1.8 0.08 ** quinine + PTX isoprot. 1.6 0.06 3oxoC12HSL + PTX 1.4 ** 1.2 PQS 0.04 1.10 + PTX control 1.05 0.02 baseline

1.00 decrease Peak PKA FRET/CFP em ratio FRET/CFP Normalized AKAR4 Normalized 0.00 0.95 2 min (AKAR4 FRET/CFP decrease) (AKAR4 FRET/CFP PQS FFA PQS + PTX FFA + PTX Fig. 2 T2R signaling in MFs decreases cAMP. a Diagram (created using Biorender.com) showing canonical T2R transduction pathway by which Gai (Gi) or Ga gustducin (Ggust) decreases cAMP. b-c Representative image series from 3 separate experiments showing MFs expressing green downward cADDis (b) and graphs (c) showing reproducibility of fluorescence changes in response to cAMP-elevating isoproterenol. Decrease in fluorescence equals an increase in cAMP, thus shown as an upward deflection (note inverse y axis). d Representative traces (left) and bar graph (right) showing cAMP decreases with 3oxoC12HSL, FFA, and PQS in control MFs (blue) but not PTX-treated MFs (pink); adenylyl cyclase-activating forskolin used as control. Traces are mean ± SEM of ≥6 independent experiments. e Traces of cADDis fluorescence (mean ± SEM; ≥6 independent experiments) during stimulation with isoproterenol ± PQS or 3oxoC12HSL. f Bar graph of peak cAMP increases with 100 nM isoproterenol ± PQS or 3oxoC12HSL (AHL) ± PTX. Graph shows mean ± SEM; each data point equals one independent experiment (n = ≥6 total from ≥3 donors). Significance by one-way ANOVA with Dunnett’s posttest (control is isoproterenol only, no PTX). g Traces (left and middle) of changes EPAC-SH187 CFP/FRET fluorescence emission ratio with quinine ± PTX. Each representative trace is a single experiment. Downward deflection equals decrease in cAMP. Right is bar graph of peak cAMP decreases with quinine, FFA, and 3oxoC12HSL ± PTX (mean ± SEM; ≥6 experiments each condition from ≥3 donors). h Trace of AKAR4 FRET/CFP fluorescence emission ratio (left; mean ± SEM of independent experiments)with PQS (100 µM) ± PTX. Downward deflection equals a decrease in PKA activity. Bar graph (right) shows peak PKA decrease with PQS or FFA ± PTX. Data points in bar graphs use cells from ≥3 donors (≥ 6 experiments total, ≥2 per donor). Significance in d, g, and h by one-way ANOVA with Bonferroni posttest. Significance in f by one-way ANOVA with Dunnett’s posttest comparing values to control (no PTX); *p<0.05 and **p<0.01

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 15 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

water soluble DMSO soluble a airway 1 2 3 b c φ φ φ T2R agonists T2R agonists cells M M M SNAP 148 1500 rt: + - + - + - + - nNOS ** 98 FFA SNAP ** 1000 eNOS Quinine SNAP 3 min (1000 a.u.) 500 1.NTC 148 DAF-FM fluor. PQS 2. Beas2B-RT 98 eNOS

after 5 min (arbitrary units) 0 3. Beas2B SNAP 64 DAF-FM fluorescence increase HHQ 4. Mφ ND522-RT HHQ PQS FFA Salicin Quinine 5. Mφ ND522 3 4 5 Φ Φ Φ Denatonium 6. Mφ ND512-RT M M M 0.1% DMSO only 7. Mφ ND512 8. Mφ ND486-RT SNAP d + vehicle e 9. Mφ ND486 1500 SNAP 1500 +U73343 10. 100bp Ladder FFA ** ** 1000 FFA 1000 ** ** ** eNos (top) & nNOS (bottom)

500 500

1000 units 3 min +U73122 + PTX SNAP 0 FFA 0 after 5 min (arbitrary units) FFA SNAP after 5 min (arbitrary units) U73343: + – + – + – PTX: - + - + DAF-FM fluorescence increase DAF-FM fluorescence increase U73122: – + – + – + FFA Denat. FFA Denat. PQS

** SNAP 2000 n.s. f + D-NAME SNAP g 1500 + D-NAME 1500 Denatonium # # FFA ** benzoate ** 1000 1000 * * 500 * 500 * ** + L-NAME ** ** + L-NAME after 5 min (arbitrary units) 0 after 5 min (arbitrary units) 0 SNAP Denatonium DAF-FM fluorescence increase DAF-FM fluorescence increase FFA benzoate SNAP eNOS nNOS control control e+nNOS FFA + cPTIO

FFA + L-NAME SNAP + cPTIO eNOSsiRNA nNOSsiRNA FFA + D-NAME Denat. + cPTIO T2R14siRNA T2R14siRNA SNAPSNAP + D-NAME + L-NAME Denat.Denat. + D-NAME + L-NAME e+nNOSsiRNA NFA denatonium

Fig. 3 T2R stimulation activates NO production in MFs. a Reverse transcription (rt) PCR of MFs from 3 donors showing expression of eNOS (NOS3). b Westerns of eNOS and nNOS using MF lysates from 3 donors. c Traces and bar graph showing DAF-FM fluorescence increases during stimulation with FFA (100 µM), quinine (500 µM), PQS (100 µM), HHQ (100 mM), or salicin (3 mM). Non-specific NO donor SNAP (10 µM) shown as a control. d Traces and bar graph showing DAF-FM fluorescence increase in response to FFA (100 µM) or denatonium benzoate (1 mM) ± PTX. e Traces and bar graph showing DAF-FM fluorescence increases in response to FFA (100 µM), denatonium benzoate (1 mM), or PQS (100 µM) in the presence of PLC inhibitor U73122 or inactive analog U73343 (30 min pretreatment, 10 µM). f Traces and bar graph showing DAF-FM fluorescence increases in response to FFA (100 µM) or denatonium benzoate (1 mM) in the presence of L-NAME or inactive D-NAME (100 µM; 30 min pretreatment) as well as with NO scavenger cPTIO (10 µM). g DAF-FM increases in response to 100 µM NFA or 1 mM denatonium benzoate in MFs treated with Accell SMARTpool siRNAs as indicated. Traces are mean ± SEM from 20-30 MFs from single representative experiments. Bar graphs are mean ± SEM with data points shown from independent experiments using cells from ≥3 donors (≥6 experiments total, ≥2 per donor). Significance determined by one-way ANOVA with Bonferroni posttest; **p>0.01 vs control, #p<0.05 vs bracketed group, n.s. = no statistical significance

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 16 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

25 40 ) ) o a o ** ** b 0.7 20 0.6 ) ) o o 300 µM denat. benz. 30 or Na benz. 0.7 0.8 15 100 µM FFA 20 10 0.8 0.9 FFA + D-NAME 10 ** 5 0.9 FFA + L-NAME ** ** cGMP level cGMP cGMP level cGMP ** (% change in F/F 1.0 denatonium benzoate (% change in F/F 0 1.0 0 (Green GENIe F/F (Green GENIe F/F sodium benzoate cGMP incresae over 10 min cGMP 1.1 2 min incresae over 10 min cGMP 1.1 2 min FFA PQS salicin M parth. µ M Na benz. µ 0.1% DMSO FFA + cPTIO M denat benz. 500 PQS + cPTIO µ FFA +FFA L-NAME + D-NAME PQS + L-NAME 500 500

25 30 c d ) )

0.6 o o ** ** ** ** ** ) ) o 100 µM 20 o 0.8 0.7 3oxoC12HSL 500 µM 20 15 0.8 quinine 0.9 10 0.9 10 cGMP level cGMP 5 level cGMP 1.0

1.0 (% change in F/F (% change in F/F

(Green GENIe F/F 2+ C12HSL (Green GENIe F/F control (+ Ca ) 0 0 2 min C12HSL + PTX 1.1 2 min 0-Ca2+ (BAPTA/EGTA) cGMP incresae over 10 min cGMP

cGMP incresae over 10 min cGMP 2+ 2+ 2+ FFA FFA Quinine FFA + PTX FFA 0-Ca 3oxoC12HSL 3oxoC12HSL Quinine 0-Ca

3oxoC12HSL + PTX 3oxoC12HSL 0-Ca e 0.6 f 40 ) ) 30 HHQ PQS thujone NFA

) ** ** o 100 µM o o 0.7 HHQ 30 0.8 20 # 20 0.9 * * ** cGMP level cGMP 10 * * ** ** 1.0 HHQ 10

(Green GENIe F/F HHQ + sGC ** (% change in F/F 1.1 (% change in F/F 2 min 0 0 cGMP incresae over 10 min cGMP cGMP incresae over 10 min cGMP HHQ PQS

HHQ + sGC PQS + sGC controlT2R14 siRNAT2R10 siRNA siRNA controlT2R14 siRNAT2R10 siRNA siRNA controlT2R14 siRNAT2R10 siRNA siRNA controlT2R14 siRNAT2R10 siRNA siRNA e+nNOS siRNA e+nNOS siRNA e+nNOS siRNA e+nNOS siRNA

T2R14 + T2R10 siRNA

Fig. 4 T2R-induced NO production increases cGMP. a Traces and bar graph showing changes in Green GENIe cGMP indicator with T2R agonists. Decrease in fluorescence equals increase in cGMP, plotted as upward deflection (note inverse y axis). b Traces and bar graph of cGMP increases with FFA or PQS (100 µM each) ± L-NAME or D-NAME (100 µM 30 min pretreatment) or cPTIO (10 µM). c Traces and bar graph of cGMP increases with 3oxoC12HSL or FFA (100 µm each) ± PTX (100 ng/ml pretreatment for 18 hrs). d Traces and bar graph of cGMP increases with quinine, FFA (100 µM), or 3oxoC12HSL (100 µM) ± Ca2+ signaling; 0-Ca2+ conditions are 1 µM BAPTA-AM loading for 60 min with stimulation in HBSS containing no added Ca2+ and 1 mM EGTA. e Traces and bar graph of cGMP increases with HHQ or PQS (100 µM each) ± co-infection with BacMam expressing soluble guanylyl cyclase (sGC). f Bar graph of cGMP increases in response to HHQ (100 µM), PQS (100 µM), thujone (600 µM), or NFA (100 µM) in MFs pre-incubated with ON-TARGET plus SMARTpool siRNAs directed against T2R14 or T2R10, a cocktail of eNOS and nNOS, or control siRNA. All traces are mean ± SEM of ≥6 independent experiments. Bar graphs are mean ± SEM with data points shown from independent experiments using cells from at least 2 separate donors (≥ 5 experiments total, ≥2 experiments per donor). Significance determined by one-way ANOVA with Bonferroni posttest; * or #p<0.05 and **p<0.01

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 17 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was T2Rs are also expressed innot immunecertified by peer review) cells is the author/funder. All rights reserved. No reuse allowed without permission.

Bitter taste receptors detectbacterial bacterial products quinolones

a 15 5min min unstimulated control 515 min min +T2R 100 µ agonistM FFA b c 600 ** 600 ** FITC E. E. coli coli ** DAPI ** ** DAPI 400 400

200 200 % baseline phagocytosis 0 % baseline phagocytosis 0 (mean fluorescence per cell) (mean fluorescence per cell)

FFA

M quinineM thujone M Na benz.µ µ FFA + PTX unstimulatedµ 3 mM salicin unstimulated FFA + cPTIO FFA + KT5823 M denat. benz. 500 600 FFA + FFAD-NAME + L-NAME ? e/nNOS µ 250 250 NO cGMP? d 600 ** ** e unstimulated NFA HHQ denat. benz. "phagocytosis 400

400 300 * * 200 * 200 ** ** 100 Downloaded from (total well fluorescence) % baseline phagocytosis 0 % baseline phagocytosis 0 (mean fluorescence per cell)

unstim

denat. benz. iNOS siRNA iNOS siRNA iNOS siRNA iNOS siRNA controlT2R14 siRNA siRNA controlT2R14 siRNA siRNA controlT2R14 siRNA siRNA controlT2R14 siRNA siRNA denat. + gallein denat.denat. + U73122 + U73343 e+nNOS siRNA e+nNOS siRNA e+nNOS siRNA e+nNOS siRNA

http://www.jbc.org/ Fig. 5 T2R-induced NO/cGMP acutely increases MF phagocytosis of FITC-labeled E. coli. a Representative image of fixed MFs after 15 min with FITC E. coli ± FFA. b Bar graphs of normalized phagocytosis (quantified by microscopy) ± 2؉ Figure 3. PQS activates Ca signals in cultured airway cell lines.variousa, diagramT2R agonists. of canonical c Normalized T2R receptor phagocytosis signaling, (quantified which involves by microscopy) G␣-gustducin ± FFA (G␣gust (100) µM) ± D/L-NAME (100 µM, 30 2ϩ activation of PDE or G␣i inhibition of adenylyl cyclase and loweringmin of pretreatment) cAMP in addition, cPTIO to G (10␤␥ activationµM), KT5823 of PLC (1 ␤µM),2, which or PTX generates (100 ng/ml; IP3 and 18 hrs activates pretreatment). Ca d Bar graphs showing 2ϩ release from the IP3 receptor ER Ca release channel. b, mean tracesnormalized and bar graphphagocytosis of quinine-induced ± denatonium (cyan benzoate), PQS-induced (1 mM) (100± U73122␮M; red (10), or µM, vehicle-induced 30 min pretreatment), U73343 (10 µM, 30 2ϩ (0.1% DMSO; gray) [Ca ]i (n ϭ 3–4 independent experiments/condition).min pretreatment) Significance or gallein was determined(100 µM). e by Bar one-way graph ANOVA,of phagocytosis Dunnett’s ± NFA post-test. (100 cµM),, mean HHQ, (100 µM), or denatonium

2ϩ 2ϩ at University of Pennsylvania Library on June 25, 2018 [Ca ]i in BEAS-2B cells in response to thujone (1 mM; green) or 100 ␮M PQS (red); n ϭ 4–6 independent experiments/condition. d, mean [Ca ]i responses to benzoate (1 mM) in MFs treated with Accell SMARTpool siRNAs2ϩ as indicated. Results in b, c, and d were quantified by thujone or PQS in NCI-H292 (H292) cells; n ϭ 4–6 independent experimentsmicroscopy; for each each independent condition. e, meanexperiment traces andis average bar graph of 10 of [Cafields ]fromi responses a single to well. PQS after Results in e were quantified by plate pretreatment with PLC␤2 inhibitor U73122 (yellow) or inactive U73343 (pink;1␮M each); n ϭ 4 independent experiments/condition. f, mean traces showing 2ϩ reader; each independent experiment is average of 2 wells. Bar graphs are mean ± SEM with data points shown from lack of [Ca ]i response to DHQ in BEAS-2B cells (a) and H292 cells (independentb); n ϭ 3–5 independent experiments experiments using cells each.from ≥ Significance3 separate donors was determined (≥2 independent by Student’s experimentst test per donor) (b and e): **, p Ͻ 0.01. Error bars, S.E.

2ϩ PQS and HHQ decrease baseline and stimulated cAMP levels in the [Ca ]i elevation with PQS (Fig. S3d), supporting a role for G␤␥ signaling in this response. lung epithelial cells ϩ To confirm the Ca2 responses to quinolones observed with ϩ The other arm of the canonical T2R signaling pathway (Fig. 2 ␣ ␣ small-molecule Ca indicators, we transfected BEAS-2B cells 3a) is G gust,aG protein of the transducin family (80–83) 2ϩ with a genetically encoded green Ca -sensitive protein, which activates phosphodiesterase (PDE). Several airway cell GCaMP6s (79). BEAS-2B cells exhibited an increase in ␣ ϩ lines have been reported to express G gust (72, 84), although 2 ␣ GCaMP6s fluorescence, signaling an increase in Ca , in G i isoforms, which inhibit adenylyl cyclases, couple to T2Rs in response to PQS, HHQ, and the T2R10/14 agonist thujone (45) airway smooth muscle (85). To test whether quinolones acti- 2ϩ ␣ ␣ (Fig. S4, a–c). The PQS Ca response observed with vated aG gust/G i pathway, we monitored cAMP dynamics in GCaMP6s was likewise inhibited by U73122 (Fig. S4d), sup- real time in living cells using a high-affinity fourth-generation porting a requirement for receptor-mediated activation of Turquoise (CFP)/Venus (YFP) FRET-based EPAC cAMP indi- PLC. cator, EPAC-SH187 (86). This construct was previously used to Because HHQ appeared to activate only one knownGopallawa, airway- et al. image“Bitter tastea receptors decrease stimulate in baselinephagocytosis cAMP in human macrophages levels in HEK293Tthrough calcium, cells nitric oxide, and cyclic-GMP signaling” 18 ␣ ␮ expressed T2R isoform, T2R14, we examined the HHQ expressing G i/o-coupled -opioid receptors (87). BEAS-2B response in T2R14-expressing (73) H292 cells co-transfected cells transfected with EPAC-SH187 and stimulated with PQS or 2ϩ with GCaMP6s (to identify transfected cells and measure Ca quinine exhibited decreases in baseline CFP/FRET emission responses) and two different sets of two shRNA constructs ratio, signaling decreases in cAMP concentration (Fig. 5a). The 2ϩ directed against T2R14. HHQ stimulated robust Ca adenylyl cyclase activator forskolin was used as a control. PQS responses in H292 cells transfected with GCaMP6s only (Fig. also blunted cAMP increases during the simultaneous addition ϩ ␣ ␤ 4a) or GCaMP6s scramble shRNA (Fig. 4b). Both sets of the G s-coupled -adrenergic receptor agonist isoprotere- of distinct T2R14-directed shRNAs markedly reduced the nol (100 ␮M; Fig. 5b). Similar PQS-induced decreases in base- response (Fig. 4, c and d), whereas the purinergic or protease- line cAMP were observed in A549 cells (Fig. 5c). When we activated receptor 2 (PAR-2) GPCR responses were not signif- tested HHQ and DHQ in H292 cells, we observed a decrease in icantly affected by these shRNA vectors (Fig. 4e). These results baseline cAMP with HHQ but not DHQ (Fig. 5e), as would be support a role for endogenous T2R14 in detection of HHQ by expected if HHQ activates T2Rs and DHQ does not. PQS-me- lung epithelial cells. diated cAMP decreases were not observed when BEAS-2B cells

9828 J. Biol. Chem. (2018) 293(25) 9824–9840 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a b MΦs only pHrodo MΦs + S. aureus MΦs + S. aureus MΦs + S. aureus pHrodo S. aureus 37°C S. aureus only 4°C 37°C + 250 µM denat. 30000 ex. 530 nm 37°C 37°C

20000

10000 pH 5.0 5.5 c DMSO 100 µM FFA 100 µM FFA 100 µM FFA 100 µM FFA 6.0 (vehicle control) + L-NAME + D-NAME + cPTIO 0 6.5 Fluorescence intensity (a.u.) 580 600 620 640 7.0 Emission wavelength (nm) 7.2 7.4 30000 7.6 7.8 PBS 20000 only 100 µM FFA 100 µM FFA 100 µM FFA 100 µM FFA + U73122 + U73343 + PTX 10000

Max fluorescence intensity 0 5 6 7 8 pH

n.s. g d e f n.s.** 2000 ** 2500 2500 3000 ** ** ** 2000 ** 2000 ** 1500 2000 1500 1500 1000 ** 1000 1000 1000 500 500 500

0 0 0 0 Whole field fluorescence intensity Whole field fluorescence intensity Whole field fluorescence intensity Whole field fluorescence intensity FFA FFA (4°C) denat. quinine (37°C) FFA + PTX FFA + cPTIO denat. + H89 s only (37°C) only (37°C) quinine + H89 FFA + L-NAMEFFA + D-NAME FFA + U73122FFA + U73343 Φ S. aureus DMSO (vehicle) denat. + KT5823 M S. aureus quinine + KT5823

S. aureus + NaBenzoate (37°C) MΦs + pHrodo S. aureus; 37°C MΦs + pHrodo S. aureus; 37°C MΦs + pHrodo S. aureus; 37°C

s + pHrodo Φ s + pHrodo pHrodo Φ M S. aureus M + Denatonium Benzoateh (37°C) ** i ** 10000 ** S. aureus 6000 8000 s + pHrodo ** Φ ** M 6000 2000 s + pHrodo ** Φ 4000 M 1000 2000

0 0 Whole field fluorescence intensity Whole field fluorescence intensity

forskolin 8-Br-cAMP 8-Br-cGMP denat. benz. unstimulated 6-Bnz-cAMP8-CPT-cAMP unstimulated denat. + forskolin denat. + 8-Br-cAMP denat. + 8-Br-cGMP 8-Br-cGMP + KT5823 denat. + 6-Bnz-cAMPdenat. + 8-CPT-cAMP

denat. + 8-CPT-2-O-Me-cAMP denat. + 8-CPT-2-O-Me-cAMP

MΦs + pHrodo S. aureus; 37°C MΦs + pHrodo S. aureus; 37°C

Fig. 6 T2R-induced NO and cGMP acutely increases MF phagocytosis of pHrodo-labeled S. aureus. a pHrodo-labeled S aureus were resuspended in PBS buffered to various pHs as indicated to confirm increase in fluorescence with decreasing pH. Experiment in top graph representative of 3 independent replicates, plotted on bottom graph. b Representative images (20x; scale bar 20 µm) of pHrodo labeled S. aureus and MFs showing phagocytosis occurring only when both were combined and only at 37°C. c Representative images of increased phagocytosis with FFA and inhibition with L-NAME (100 µM, 30 min pretreatment), cPTIO (10 µM), U73122 (10 µM, 30 min pretreatment), and PTX (100 ng/ml; 14 hrs pretreatment). d Quantification of experiments performed as in b. Significance by Bonferroni posttest; ** p<0.01. e-f Quantification of experiments performed as in c. g Similar experiments were performed with denatonium benzoate (2 mM) and quinine (500 µM); phagocytosis increase was inhibited by KT5823 but not H89 (5 µM, 10 min preincubation each). Significance by one-way ANOVA with Dunnett’s posttest; ** p<0.01. h Quantification of pHrodo S. aureus phagocytosis assays ± 10 µM forskolin or cell permeant cAMP or cGMP analogs as indicated. Forskolin or cAMP analogs inhibited phagocytosis; however 8-Br-cGMP increased phagocytosis via PKG, as it was blocked by KT5823 (5 µM, 10 min preincubation). i Quantification of pHrodo S. aureus assays with 1 mM denatonium benzoate ± forskolin or cAMP analogs or cGMP analog. Significance by 1-way ANOVA with Bonferroni posttest. Data points in d-i are independent experiments (≥6 from ≥2 individual donors, all taken with identical microscope settings), each experiment is average of 10 fields from a single well. Incubations for b-g were 20 min. Incubations for h and i were 60 min

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 19 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

PTX + a b c PTX-treated MΦs L-NAME- PTX-treated 2500 treated MΦs MΦs PTX-treated 2000 ** ** DAF-FM–loaded ** MΦs 1500 SNAP 17β-estradiol (E2) or 1000 HBSS only 500

after 5 min (arbitrary units) 0

H441 club cell-like DAF-FM fluorescence increase 0.4 µm pore filter 2 min E2 E2 E2 small airway cells DAF-FM 1000 units E2 + PTX E2+ HBSSonly HBSSonly HBSSonly

d cPTIO E2+ E2 + L-NAMEE2+ E2 + D-NAMEE2+ pHrodo-labeled + H441 cells in lower chamber no H441 cells S. aureus PTX-treated MΦs PTX-treated MΦs f 3000 0.4 µm pore filter ** ** H441 cells 2000 e HBSS 17β-estradiol (E2) E2 + L-NAME E2 + D-NAME 1000 Whole field

fluorescence intensity 0 E2 E2 E2 + PTX E2+ HBSSonly HBSSonly E2 + cPTIO E2+ E2 + L-NAMEE2+

50 µm D-NAMEE2+

+ H441 cells in lower chamber no H441 cells

Fig. 7 Airway epithelial cells can acutely increase MF phagocytosis via intercellular NO. a Diagram of experimental design for b-c. PTX-pretreated DAF-FM-loaded MFs were placed in close proximity (<1 mm) to unloaded H441s separated by a permeable plastic filter. b Stimulation of H441s with 10 nM 17b-estradiol (E2) resulted in an increase in MF DAF-FM fluorescence. SNAP (10 µM) added at the end as a positive control. c Bar graph of DAF-FM increases from experiments performed as in a-b. DAF-FM increases were inhibited by treatment of H441s with PTX (100 ng/ml; 18 hrs) or L-NAME (10 µM, 30 min) or in the presence of cPTIO (10 µM). Treatment of MFs with L-NAME did not alter responses, and there was no response to E2 in the absence of H441s. d Diagram of experimental design for e-f. PTX- pretreated MFs incubated with pHrodo-labeled S. aureus were placed in close proximity (<1 mm) to H441s. e Representative images of fluorescence increases with PTX-pretreated MFs with H441s stimulated as indicated for 30 min. f Bar graph of MFs fluorescence increases quantified by microscopy with stimulations as indicated. E2 increased MF phagocytosis that was blocked by PTX, L-NAME, cPTIO, or absence of H441s. Bar graphs are mean ± SEM with data points shown from independent experiments using cells from at least 2 individual donors (≥2 independent experiments per donor). Significance by one-way ANOVA with Bonferonni posttest with each bar compared with its respective HBSS only control; ** p<0.01. Figures in a and d created with Biorender.com

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 20 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

enhanced phagocytosis

T2Rs

ñCa2+ ñNO òcAMP ñcGMP

M0 MΦ NO

Bacterial products Hormonal stimulation

Airway epithelium

Fig. 8 Model of T2R signaling in M0 MFs. Activation of T2Rs increases Ca2+, lowers cAMP, activates eNOS and nNOS, increases cGMP, and enhances phagocytosis. Acute increase in phagocytosis may also be activated by airway epithelial cell NO production. Figure created with Biorender.com

Gopallawa, et al. “Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling” 21 bioRxiv preprint doi: https://doi.org/10.1101/776344; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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