Bitter and sweet taste receptors regulate human upper respiratory innate immunity

Abstract

Bitter taste receptors (T2Rs) in the human airway detect harmful compounds, including secreted bacterial products. Here, using human primary sinonasal air-liquid interface cultures and tissue explants, we determined that activation of a subset of airway T2Rs expressed in nasal solitary chemosensory cells activates a calcium wave that propagates through gap junctions to the surrounding respiratory epithelial cells. The T2R-dependent calcium wave stimulated robust secretion of antimicrobial peptides into the mucus that was capable of killing a variety of respiratory pathogens. Furthermore, sweet taste receptor (T1R2/3) activation suppressed T2R-mediated antimicrobial peptide secretion, suggesting that T1R2/3-mediated inhibition of T2Rs prevents full antimicrobial peptide release during times of relative health. In contrast, during acute bacterial infection, T1R2/3 is likely deactivated in response to bacterial consumption of airway surface liquid glucose, alleviating T2R inhibition and resulting in antimicrobial peptide secretion. We found that patients with chronic rhinosinusitis have elevated glucose concentrations in their nasal secretions, and other reports have shown that patients with hyperglycemia likewise have elevated nasal glucose levels. These data suggest that increased glucose in respiratory secretions in pathologic states, such as chronic rhinosinusitis or hyperglycemia, promotes tonic activation of T1R2/3 and suppresses T2R-mediated innate defense. Furthermore, targeting T1R2/3-dependent suppression of T2Rs may have therapeutic potential for upper respiratory tract infections.

Figures

Figure 1. Calcium responses in sinonasal ALIs during stimulation with denatonium benzoate.

(A) Representative trace showing dose-dependent calcium elevations in response to denatonium. 0.1 D, 0.1 mM denatonium benzoate; 1 D, 1 mM denatonium benzoate; 10 D, 10 mM denatonium benzoate. (B) Dose response plot of data from experiments shown in A. Each red data point is the mean of results from 5 to 12 experiments. (C) Representative images showing propagation of calcium signal from single denatonium-responding cells compared to global calcium mobilization by ATP. denat., denatonium. (D and E) Signal propagation was blocked in the presence of (D) 100 μM carbenoxolone (cbx), a gap junction inhibitor, (E) but not apical apyrase. Arrows denote single denatonium-responsive cells. Images shown are representative of 5 to 12 experiments for each condition. Scale bar: 50 μm.

Figure 2. Human sinonasal chemosensory cells express both bitter and sweet taste receptors as well as IP3R3.

(A) Cells matching the morphology of SCCs exhibited immunofluorescence for both T2R47 and T1R3. These cells also appeared to express IP3R3, though IP3R3 expression appeared to be more widely distributed than that of T2R47 or T1R3 receptors. Merge shows T1R3 and T2R47 signals. (B) T2R47 staining was reduced when T2R47 antibody was preincubated with antigenic blocking peptide (2 hours) before staining. Scale bar: 10 μm.

Figure 3. Sweet receptor activation inhibits T2R-activated calcium signaling.

(A) Dose-dependent glucose (Glc) inhibition of the calcium response to 10 mM denatonium benzoate (mean ± SEM; n = 5–9 experiments using cultures from at least 3 patients each). (B) Inhibition is reversed by lactisole (mean ± SEM; n = 5–7 cultures from at least 3 patients each). (C) Peak calcium responses (mean ± SEM; 6–12 cultures from at least 3 patients each) and inhibition by glucose, sucrose, and sucralose (but not phloretin/phlorizin) as well as reversal of inhibition by lactisole. Red asterisks denote significance versus control (10 mM denatonium stimulation alone) determined by 1-way ANOVA with Dunnett’s post-test. Blue asterisks denote significance between bracketed bars determined by 1-way ANOVA with Bonferroni post-test. Experiments in A–C were performed without carbenoxolone. (D) In the presence of carbenoxolone, single denatonium-responsive cells exhibited glucose inhibition of both denatonium- and ATP-induced calcium signals (mean ± SEM; 18–20 cells from 3 patients for each condition). (E) Peak denatonium-induced calcium response (mean ± SEM) in the presence of carbenoxolone with or without glucose (significance determined by Student’s t test) from experiments in D. ATP response (mean ± SEM) in denatonium-responsive (solid bars) and nonresponsive (hatched bars) cells (significance determined by 1-way ANOVA with Bonferroni post-test). *P < 0.05; **P < 0.01.

Figure 4. Denatonium/absinthin-sensitive T2Rs activate sinonasal epithelial cells to secrete compounds with potent and broad-spectrum antimicrobial activity.

(A) Bacterial CFUs when Pseudomonas were mixed with ASL from ALI cultures stimulated with PBS or 10 mM denatonium. (B) Confirmation of bacterial kill by live-dead staining of planktonic Pseudomonas. Syto 9 and propidium iodide (PI) indicate live and permeabilized (dead) bacteria, respectively (10). The images are representative of 5 experiments using ASL from 5 patients. Scale bar: 15 μm. (C and D) Percentage Pseudomonas CFUs remaining (mean ± SEM) when bacteria were mixed with ASL from ALIs stimulated under indicated conditions. Secretion of antimicrobial products was inhibited by inhibition of calcium signaling (BAPTA/EGTA), gap junction communication (carbenoxolone, CBX), or PLCβ2 (U73122) as well as by glucose. Glucose inhibition was reversed by lactisole (lact.). Inhibition was not observed with the TRPM5 inhibitor triphenylphosphine oxide (TPPO) (mean ± SEM; data from 12–66 cultures from at least 3 different patients for each condition). Concentrations shown are in mM. **P < 0.01, vs. control (PBS) determined by 1-way ANOVA with Dunnett’s post-test. (E) Activity of ASL secretions against other bacteria species (mean ± SEM; n = 6–16 cultures from at least 3 different patients for each condition). **P < 0.01, each species vs. control (PBS) determined by 1-way ANOVA with Bonferroni post-test.

Figure 5. Kinetics of the denatonium-induced antimicrobial response.

(A) ALI cultures were exposed to denatonium for various times, and ASLs were then incubated to Pseudomonas. Resulting CFUs remaining (normalized to PBS) are shown (mean ± SEM; n = 6–8 experiments from 6 to 8 cultures from at least 3 patients for each condition). Decreased CFU count equals a reduced number of viable bacteria. Human sinonasal ALI cultures secreted antimicrobials over the course of 5 to 10 minutes. (B) ALI cultures were stimulated with denatonium for 30 minutes, and ASL was mixed with Pseudomonas for varying periods of time before dilution and spotting on LB plates. Resulting CFUs remaining (normalized to PBS) are shown (mean ± SEM; n = ALI cultures from 4 patients for each condition). Maximal killing was achieved only after >90 minutes of exposure of bacteria to ASL. (C) Cultures were stimulated with 10 mM denatonium benzoate and then either stimulated the next day (1-day recover) or given 3 or 6 days to recover. A second stimulation of maximal killing of Pseudomonas was achieved only after cultures were allowed to recover for >3 days. CFUs remaining (mean ± SEM) are shown (n = 4–12 ALI cultures from at least 4 patients for each condition). *P < 0.05; **P < 0.01, determined via 1-way ANOVA with Bonferroni post-test.

Figure 6. T2R agonists stimulate secretion of β-defensins from HSECs.

(A) Coomassie blue–stained gel showing a low-molecular-weight band in denatonium-stimulated ASL (representative of 3 gels; 6 patients). (B) Denatonium-stimulated secretions were run through a Centricon filter (30-kDa MWCO); antimicrobial activity was retained in the lower molecular weight fraction. Antibacterial activity was retained when ASL from denatonium-treated cultures was dialyzed against 3-kDa MWCO, was reduced with a 12-kDa MWCO, and was fully lost with a 50-kDa MWCO (n = 3–5 ALIs from at least 3 patients each). (C) Antimicrobial function was inhibited by increasing [NaCl] (n = 7 patients) (significance vs. PBS ASL with same [NaCl]). (D and E) T2R agonists denatonium, absinthin, parthenolide, and amarogentin stimulate secretion of β-defensins 1 (green) and 2 (white) (n = 5–25 cultures from >3 patients each). *P < 0.05; **P < 0.01, compared with PBS ASL, by 1-way ANOVA with Dunnett’s post-test. (F) Intracellular β-defensin decreased after 30-minute stimulation with 10 mM denatonium and returned to baseline levels by 6 days (n = 5 cultures from 5 patients each). (G) Bactericidal activity of denatonium-stimulated ASL was blocked by antibodies to β-defensins (30-minute preincubation) or immunodepletion of β-defensins (n = 5 cultures from 5 patients each). (B, C, and G) *P < 0.05, **P < 0.01, determined by 1-way ANOVA with Bonferroni post-test. (H) Denatonium (10 mM) stimulated β-defensin 1 and 2 secretion by ex vivo human turbinate that was significantly inhibited by 1.5 mM glucose. Defensin concentration normalized per mg of wet-weight tissue. *P < 0.05, **P < 0.01. All data are mean ± SEM.

Figure 7. Glucose concentrations are elevated in the nasal secretions of patients with CRS.

(A) Glucose was measured in nasal secretions from patients with CRS and control individuals. Glucose was 0.37 ± 0.06 mM vs. 1.63 ± 0.12 mM in control patients (n = 17) and patients with CRS (n = 32), respectively. A significant difference was observed between patients with CRS with (n = 20) and without (n = 12) polyps (1.4 ± 0.1 mM vs. 2.0 ± 0.2 mM, respectively), but nonetheless both groups had significantly higher glucose than control patients (mean ± SEM). *P < 0.05, **P < 0.01, determined via 1-way ANOVA with Tukey-Kramer post-test. (B) In patients with CRS, there was no correlation between blood and nasal secretion glucose concentrations (n = 32 patients for which nasal and blood glucose values could be obtained). Additionally, none of the patients with CRS used in this study had a prior diagnosis of hyperglycemia, prediabetes, or diabetes. Previous studies have shown that hyperglycemia is correlated with increased nasal secretion glucose (51, 74, 75). However, these data suggest that the patients with CRS in this study exhibited higher nasal glucose levels due to another mechanism. (C) Glucose was measured in ASL from ALI cultures, with no difference observed among the populations (mean ± SEM; n = 1 culture each from 5 control patients and 15 patients with CRS, including 6 with polyps and 9 without polyps). No significant differences were detected by 1-way ANOVA.

Figure 8. Proposed model of T2R bitter receptor– and T1R sweet receptor–based regulation of AMP secretion in the human nose.

(A) From left to right, bitter chemicals released by microbes during infection activate T2Rs in the sinonasal epithelium (10), likely including those expressed in nonciliated chemosensory epithelial cells, likely the SCCs previously described (–14). This results in a calcium response that propagates to the surrounding epithelial cells, causing secretion of multiple AMPs, including β-defensins 1 and 2, that are capable of direct bacterial killing. Glucose in the ASL normally governs the T2R-mediated response through T1R2/3 activation. However, during acute infections, bacteria may consume glucose (Supplemental Figure 18) and decrease the ASL glucose concentration, which relieves the T1R2/3-mediated inhibition of T2Rs and allows the activation of the antimicrobial response. (B) Proposed mechanism for T2R and T1R signaling in sinonasal chemosensory cells. T2R signaling is dependent upon Gα-gustducin and IP3R calcium release channels that likely include the IP3R3 isoform. T1R signaling likely uses an alternative G protein that acts through cAMP/PKA and may inhibit IP3R3-mediated calcium signaling.