IADR Abstract Archives
The Role of Bitter Taste Receptors in Oral Innate Immunity
Objectives: Early childhood caries (ECC) has become a major public health concern posing a significant socioeconomic burden worldwide. The causes of ECC are multifactorial and host immunity has been shown to play a major role in its development. Streptococcus mutans a key etiological pathogen has been shown to secrete Competence Stimulating Peptides (CSPs) during caries pathogenesis. However the mechanism by which these CSPs influence host immunity is not well understood. In this study we propose that bitter taste receptors (T2Rs) interact with S. mutants CSPs and generate immune responses.
Objectives: [A] Pharmacological characterization of CSPs with select T2Rs using heterologous HEK293T expression system. [B] Characterize the expression and function of select T2Rs in Gingival Epithelial Cells (GECs).
Methods: Ca2+ assay: Intracellular calcium levels were measured using Ca2+ binding dye (Fluo NW) after addition of CSPs and/or bitter agonists Diphenhydramine (DPH), Quinine and Dexthromethorphan (DXM) to HEK293T stably expressing different T2Rs. Real-time PCR was carried out for T2R3, T2R4, T2R7, T2R14, T2R49 and GAPDH expression in GECs.
Results: [A] In GECs, CSP-1 and CSP-2 were able to mobilize intracellular calcium at 20 μM (*p<0.05) and 50 μM (**p<0.01) concentrations. Bitter agonists induced calcium responses in a concentration dependent manner and the EC50 values are as follows: DPH (440 ± 20 μM), Quinine (465 ± 19 μM), DXM (511 ± 7 μM). Cells treated with PLCβ inhibitor (U-73122) reduced T2R-agonist induced calcium responses compared to controls (***p<0.001). In T2R expressing stable cell lines, we observed a concentration dependent calcium mobilization upon CSP treatment. The EC50 values for CSP-1 treated cells were as follows T2R4 (4.2 ± 1 μM), T2R7 (4.3 ±1 μM), T2R49 (2.7 ± 1.2 μM), CSP-2 treated cells T2R14 (3.0 ± 1.8 μM) and CSP-12261 treated cells T2R7 (7.2 ± 2.3 μM), T2R49 (6.2 ± 1.9 μM). [B] qPCR analysis revealed moderate expression of T2R4 and T2R14.
Conclusions: CSPs are identified as potent T2R agonists. These results suggest that T2Rs are involved in innate immunity and may be protective in function. Future studies will involve quantification of anti-microbial peptides (AMPs) produced by T2R activation and how T2Rs are involved in oral innate immunity.
Objectives: [A] Pharmacological characterization of CSPs with select T2Rs using heterologous HEK293T expression system. [B] Characterize the expression and function of select T2Rs in Gingival Epithelial Cells (GECs).
Methods: Ca2+ assay: Intracellular calcium levels were measured using Ca2+ binding dye (Fluo NW) after addition of CSPs and/or bitter agonists Diphenhydramine (DPH), Quinine and Dexthromethorphan (DXM) to HEK293T stably expressing different T2Rs. Real-time PCR was carried out for T2R3, T2R4, T2R7, T2R14, T2R49 and GAPDH expression in GECs.
Results: [A] In GECs, CSP-1 and CSP-2 were able to mobilize intracellular calcium at 20 μM (*p<0.05) and 50 μM (**p<0.01) concentrations. Bitter agonists induced calcium responses in a concentration dependent manner and the EC50 values are as follows: DPH (440 ± 20 μM), Quinine (465 ± 19 μM), DXM (511 ± 7 μM). Cells treated with PLCβ inhibitor (U-73122) reduced T2R-agonist induced calcium responses compared to controls (***p<0.001). In T2R expressing stable cell lines, we observed a concentration dependent calcium mobilization upon CSP treatment. The EC50 values for CSP-1 treated cells were as follows T2R4 (4.2 ± 1 μM), T2R7 (4.3 ±1 μM), T2R49 (2.7 ± 1.2 μM), CSP-2 treated cells T2R14 (3.0 ± 1.8 μM) and CSP-12261 treated cells T2R7 (7.2 ± 2.3 μM), T2R49 (6.2 ± 1.9 μM). [B] qPCR analysis revealed moderate expression of T2R4 and T2R14.
Conclusions: CSPs are identified as potent T2R agonists. These results suggest that T2Rs are involved in innate immunity and may be protective in function. Future studies will involve quantification of anti-microbial peptides (AMPs) produced by T2R activation and how T2Rs are involved in oral innate immunity.