T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection

Abstract

Innate and adaptive defense mechanisms protect the respiratory system from attack by microbes. Here, we present evidence that the bitter taste receptor T2R38 regulates the mucosal innate defense of the human upper airway. Utilizing immunofluorescent and live cell imaging techniques in polarized primary human sinonasal cells, we demonstrate that T2R38 is expressed in human upper respiratory epithelium and is activated in response to acyl-homoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Receptor activation regulates calcium-dependent NO production, resulting in stimulation of mucociliary clearance and direct antibacterial effects. Moreover, common polymorphisms of the TAS2R38 gene were linked to significant differences in the ability of upper respiratory cells to clear and kill bacteria. Lastly, TAS2R38 genotype correlated with human sinonasal gram-negative bacterial infection. These data suggest that T2R38 is an upper airway sentinel in innate defense and that genetic variation contributes to individual differences in susceptibility to respiratory infection.

Figures

Figure 1. T2R38 is expressed at the apical membrane and cilia of sinonasal airway epithelial cells in both human tissue explants and primary human sinonasal ALI cultures.

(A–H) Representative images of β-tubulin (green; a ciliary marker), T2R38 (red), and Hoechst (blue; a nuclear stain) in primary human sinonasal tissue (A–D) and in a human sinonasal ALI culture (E–H). Scale bars: 20 μm. The height of the cross-section is stretched to illustrate the co-localization pattern. D and H show cross-sections of z-projections from 4 other tissue samples (D) and from cultures (H) illustrating the co-localization pattern. (I) 32 regions of ciliated cells were analyzed for red and green fluorescence (left to right: basolateral to apical) over approximately 40 pixels.

Figure 2. PTC and Pseudomonas AHLs induce T2R38-dependent Ca2+ responses in sinonasal ALIs and in a heterologous expression system.

(A) Ca2+ responses to PTC and ATP stimulation (mean ± SEM; 12 PAV/PAV, 16 PAV/AVI, and 8 AVI/AVI cultures, n = 4 patients each). Inset: PTC responses on a larger scale. (B) Peak Ca2+ responses from patients in A. Results from individual patients were pooled and averaged; each independent observation represents 1 patient. Fluo-4 fluorescence after 5 minutes of PTC: 1.30 ± 0.027 (PAV/PAV), 1.14 ± 0.035 (PAV/AVI), and 1.06 ± 0.02 (AVI/AVI). (C) Fluo-4 traces (means from 10 cultures) during stimulation with 200 μM C4HSL and ATP. (D) Peak Ca2+ responses from C, averaged as described in B. Fluo-4 fluorescence after 5 minutes of C4HSL: 1.29 ± 0.03 (PAV/PAV), 1.14 ± 0.04 (PAV/AVI), and 1.10 ± 0.04 (AVI/AVI). (E and F) Experiments were performed as in C and D using 100 μM C12HSL and 3 cultures from 3 patients/genotype. Fluo-4 fluorescence after 10 minutes of C12HSL stimulation: 1.52 ± 0.08 (PAV/PAV), 1.28 ± 0.04 (PAV/AVI), and 1.14 ± 0.01 (AVI/AVI). (G) Peak Fluo-4 fluorescence (ΔF/F) in hTAS2R38- and Gα16gustducin44-expressing HEK293 cells in response to PTC (114% ± 4% PAV; –4% ± 1% AVI), C4HSL (61% ± 2% PAV; 0.3% ± 0.4% AVI), and C12HSL (73% ± 5% PAV; 3% ± 2% AVI), denatonium (Denat; 1% ± 2% PAV; 3% ± 1% AVI), and salicin (Sal; 1.4% ± 0.3% PAV; 3% ± 1% AVI). *P < 0.05, **P < 0.01, ANOVA with Tukey-Kramer analysis.

Figure 3. Activation of T2R38 by PTC or Pseudomonas AHLs results in NO production.

(A) Traces of DAF-FM fluorescence increased with PTC (1 culture each, ∼100 cells; SEM smaller than symbols; 9–12 cultures each). (B) DAF-FM fluorescence increased after PTC stimulation. 100 μM/5 minutes: 76 ± 13 (PAV/PAV), 20 ± 3 (PAV/AVI), and 17 ± 5 (AVI/AVI); 100 μM/10 minutes; 170 ± 21 (PAV/PAV), 76 ± 17 (PAV/AVI), and 52 ± 13 (AVI/AVI); 1 mM/5 minutes: 176 ± 10 (PAV/PAV), 98 ± 19 (PAV/AVI), and 107 ± 20 (AVI/AVI); 1 mM/10 minutes: 285 ± 18 (PAV/PAV), 123 ± 38 (PAV/AVI;), and 122 ± 5 (AVI/AVI). Increases after SNAP were not different between cultures of different genotypes. Each patient treated as an independent observation; n = 4 patients each. (C) Average traces of DAF-FM with C4HSL (2 cultures each from 4 patients for each genotype). (D) Results from C averaged by patient. Fluorescence changes after 5 minutes were as follows for 10 μM C4HSL: 132 ± 14 (PAV/PAV), 68 ± 6 (PAV/AVI), 50 ± 2 (AVI/AVI); 100 μM C4HSL: 230 ± 30 (PAV/PAV), 130 ± 17 (PAV/AVI), and 69 ± 8 (AVI/AVI). (E) Average traces of DAF-FM with C12HSL (2 cultures each from 4 PAV/PAV patients and 3 PAV/AVI and AVI/AVI patients). (F) Results from E averaged as described in D. Fluorescence changes after 5 minutes were as follows for 10 μM C12HSL: 235 ± 20 (PAV/PAV), 77 ± 13 (PAV/AVI), and 44 ± 9 (AVI/AVI); 100 μM C12HSL: 351 ± 65 (PAV/PAV), 109 ± 25 (PAV/AVI), and 64 ± 16 (AVI/AVI). Increases after SNAP were not significantly different. *P < 0.05, **P < 0.01, ANOVA with Tukey-Kramer analysis.

Figure 4. Pseudomonas conditioned medium (CM) and AHLs stimulate an increase in CBF that requires T2R38 function.

(A) Average traces showing CBF increase in PAV/AVI cultures stimulated (stim) with CM or LB as well as ATP. (B) Average traces showing the effects of 3 concentrations of CM on CBF in PAV/PAV taster (9 cultures from 3 patients) and AVI/AVI non-taster cultures (11 cultures from 3 patients). (C) Summary of A and B, treating each patient as an independent observation (n = 3 each; nd, not determined). CBF increased to 1.26 ± 0.6 (PAV/PAV) and 1.08 ± 0.2 (AVI/AVI; P = NS) with 1.56% CM; 1.50 ± 0.03 (PAV/PAV) and 1.18 ± 0.04 (AVI/AVI; P = 0.03) with 3.13% CM; and 1.83 ± 0.11 (PAV/PAV), 1.23 ± 0.09 (PAV/AVI; P < 0.01 vs. PAV/PAV), and 1.32 ± 0.05 (AVI/AVI; P < 0.01 vs. PAV/PAV; P = NS vs. PAV/AVI) with 6.25% CM. Peak CBF after ATP stimulation was not significantly different between cultures of different genotypes. (D) Average traces showing peak CBF in response to 200 μM C4HSL and 100 μM C12HSL in PAV/PAV and AVI/AVI cultures (6 cultures from 3 patients each). (E) Summary of results from D, treating each patient as an independent observation (n = 3–4 patients each). Peak CBF with C4HSL was 1.17 ± 0.02 (PAV/PAV) and 1.02 ± 0.02 (AVI/AVI); peak CBF with C12HSL was 1.25 ± 0.05 (PAV/PAV) and 1.03 ± 0.01 (AVI/AVI). *P < 0.05, **P < 0.01, ANOVA with Tukey-Kramer analysis.

Figure 5. Pseudomonas CM and AHLs stimulate a T2R38-dependent, NO-dependent increase in mucociliary clearance.

(A) Particle streaks before (left) and 3 minutes after (right) stimulation with 6.25% biofilm-CM in PAV/PAV and AVI/AVI cultures from PAV/PAV and AVI/AVI patients. (B) Mean normalized velocity increase was 1.9 ± 0.07 (PAV/PAV), 1.16 ± 0.05 (AVI/AVI), 1.17 ± 0.07 (PAV/PAV + L-NAME), and 1.15 ± 0.07 (PAV/PAV + cPTIO). (C) Results from transport analysis using CPBS (after 3 minute exposure of 50%, 6 hour CPBS) from PAO1, PAO-JP2, and Sad36 strains (4 cultures/genotype/strain). Normalized velocity increases were as follows for PAO-1: 1.67 ± 0.06 (PAV/PAV) vs. 1.13 ± 0.04 (AVI/AVI); PAO-JP2: 1.135 ± 0.04 (PAV/PAV) vs. 1.058 ± 0.03 (AVI/AVI); Sad36: 1.703 ± 0.08 (PAV/PAV) vs. 1.14 ± 0.03 (AVI/AVI). (D) Transport analysis using C4HSL and C12HSL. Increases in velocity upon PBS addition: 1.04 ± 0.02 (PAV/PAV) vs. 1.08 ± 0.2 (AVI/AVI); after C4HSL addition: 1.07 ± 0.02 (0.1 μM; PAV/PAV), 1.12 ± 0.03 (1 μM; PAV/PAV), 1.21 ± 0.04 (10 μM; PAV/PAV), 1.47 ± 0.07 (100 μM; PAV/PAV), and 1.12 ± 0.03 (100 μM; AVI/AVI); after C12HSL addition: 1.09 ± 0.02 (0.1 μM; PAV/PAV), 1.23 ± 0.04 (1 μM; PAV/PAV), 1.48 ± 0.04 (10 μM; PAV/PAV), 1.64 ± 0.04 (100 μM; PAV/PAV), and 1.14 ± 0.03 (100 μM; AVI/AVI). *P < 0.05, **P < 0.01 as denoted by brackets; #P < 0.01 vs. PBS; NS, no significance vs. PBS. All statistical analyses were performed using an ANOVA model with Tukey-Kramer post hoc analysis.

Figure 6. Human sinonasal ALI cultures exhibit T2R38-dependent apical NO diffusion.

(A) Left: NO metabolites were quantified from ASL (1 stimulated and 1 unstimulated culture used from 4 PAV/PAV and 3 AVI/AVI patients each). Right: Calibration using known NaNO3 standards. (B) Left: Fluorescence ratios of DAF-2 and Texas Red Dextran (TRD) were used to measure NO secretion. There was no change in ratios for unstimulated PAV/PAV or AVI/AVI cultures (solid bars; 0.65 ± 0.19 [PAV/PAV] and 0.39 ± 0.07 [AVI/AVI] at 5 minutes, 0.50 ± 0.11 [PAV/PAV] and 0.50 ± 0.10 [AVI/AVI] at 30 minutes, and 0.45 ± 0.10 [PAV/PAV] and 0.41 ± 0.06 [AVI/AVI] at 60 minutes). In contrast, PAV/PAV and AVI/AVI cultures had marked differences after PTC stimulation (0.46 ± 0.10 [PAV/PAV] and 0.54 ± 0.1 [AVI/AVI] at 5 minutes, 1.55 ± 0.17 [PAV/PAV] and 0.71 ± 0.1 [AVI/AVI] at 30 minutes, and 2.52 ± 0.76 [PAV/PAV] and 0.90 ± 0.3 [AVI/AVI] at 60 minutes). Right: Addition of 0, 5, 50, 250, and 500 μM DETA NONOate resulted in a linear increase in the DAF-2/TRD ratio. (C) Exposure to WT, but not AHL-deficient, Pseudomonas induced T2R38-dependent NO secretion. DAF-2/Texas Red ratios after exposure to PAO1 were 0.5 ± 0.1 (PAV/PAV) and 0.5 ± 0.1 (AVI/AVI) at 5 minutes, 2.1 ± 0.3 (PAV/PAV) and 0.6 ± 0.1 (AVI/AVI) at 60 minutes, and 3.5 ± 0.4 (PAV/PAV) and 0.7 ± 0.2 (AVI/AVI) at 120 minutes. Ratios after exposure to PAO-JP2 were 0.5 ± 0.1 (PAV/PAV) and 0.5 ± 0.1 (AVI/AVI) at 5 minutes, 1.0 ± 0.2 (PAV/PAV) and 0.9 ± 0.1 (AVI/AVI) at 60 minutes, and 0.8 ± 0.3 (PAV/PAV) and 0.7 ± 0.3 (AVI/AVI) at 120 minutes. *P < 0.05, ANOVA with Tukey-Kramer analysis.

Figure 7. T2R38 is required for maximal epithelial killing of P. aeruginosa.

(A) PAO1 removed from cultures after 2 hours of exposure showed increased propidium iodide fluorescence (indicating bacterial cell permeability) and decreased SYTO 9 fluorescence in bacteria exposed to PAV/PAV cultures vs. PAV/AVI and AVI/AVI cultures. Original magnification, ×60. (B) Left: Percentage of green (viable) PAO1 after exposure to saline (no epithelial cells; negative control; 97% ± 2%), colistin (no epithelial cells; positive control; 4% ± 2%), PAV/PAV cultures (14% ± 1%), PAV/AVI cultures (80% ± 5%), AVI/AVI cultures (70% ± 5%), PAV/PAV cultures plus L-NAME (90% ± 2%), PAV/PAV cultures plus cPTIO (93% ± 2%), and washed PAV/PAV cultures (45% ± 3%). Right: Percentage of viable PAO-JP2 from separate experiments after exposure to PAV/PAV and AVI/AVI cultures (79% ± 3% and 87% ± 3%, respectively) as well as PAV/PAV and AVI/AVI cultures plus 10 μM each C4HSL and C12HSL (7% ± 2% and 84% ± 3%, respectively). *P < 0.05, **P < 0.01, ANOVA with Bonferroni analysis.