The Role of T2R38 in Sinonasal Innate Immunity
One class of receptors emerging as important components of airway innate immunity is the T2R family of bitter taste receptors. In type II cells of the tongue, T2Rs protect against the ingestion of harmful compounds, including toxic bacterial and plant products. Extraoral expression of T2Rs is found in a variety of organs, including the brain, gut, pancreas, and bladder. T2Rs were also recently identified within the motile cilia of cells of the human bronchial and sinonasal epithelium. The physiological roles and ligands for extraoral T2Rs are largely unknown. As many extraoral T2Rs never come into direct contact with ingested food, many known T2R agonists (i.e. bitter products from edible plants) are probably not directly relevant to extraoral function. Instead, it has been hypothesized that extraoral T2Rs detect bitter products from pathogenic bacteria or fungi. A role for T2Rs in immunity might explain their widespread distribution throughout the body. Initial support for this came from studies of solitary chemosensory cells (SCCs) in the mouse nose. SCCs express T2Rs and respond to acyl-homoserine lactones (AHLs), which are quorum-sensing molecules from gram-negative bacteria, including the airway pathogen Pseudomonas aeruginosa. Many lactones are bitter, suggesting that AHLs are relevant ligands for some extraoral T2Rs.
The hypothesis that T2Rs play a role in immunity may have important clinical consequences. The TAS2R genes encoding T2Rs have many naturally occurring polymorphisms underlying individual taste preferences for bitter foods and beverages, including vegetables, coffee, scotch, and beer. We initially hypothesized that, if T2Rs function in the human airway to detect bacteria and regulate immunity, it is possible that genetic variation in TAS2R genes contributes to susceptibility to respiratory infection and/or CRS.
Sinonasal-ciliated epithelial cells express the bitter taste receptor T2R38, localized within the motile cilia (Fig. 2a). Sinonasal T2R38 function was studied in human tissue explants, as well as air–liquid interface cultures (ALI) of primary sinonasal cells. ALI cultures recapitulate a polarized respiratory epithelium with well differentiated ciliated and goblet cells. When human ciliated epithelial cells were stimulated with the T2R38-specific bitter agonist phenylthiocarbamide (PTC; also known as phenylthiourea), they exhibited low-level calcium responses that activated nitric oxide synthase-mediated production of intracellular nitric oxide. Pharmacological inhibition revealed that T2R38 signaling required two important components of the canonical taste pathway characterized in taste cells, namely the transient receptor potential melastatin isoform 5 (TRPM5) ion channel and the phospholipase C isoform beta 2 (PLCβ2), which has now been experimentally confirmed using ALIs derived from nasal septum of wild-type (Wt), TRPM5 knockout, and PLCβ2 knockout mice.
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Figure 2.
T2R38 in sinonasal innate immunity. (a) Immunofluorescence confocal micrograph of the apical section of a fixed human sinonasal tissue explant stained using antibodies directed against β-tubulin IV (β-tubIV, with green fluorescent secondary antibody; top panel), a cilia protein, and T2R38 (with red fluorescent secondary antibody; bottom panel), as described in [12]. Scale bar is 20μm. (b) T2R38-activation by bacterial quorum-sensing molecules stimulates calcium-mediated nitric oxide production, which increases ciliary beat frequency and directly kills bacteria. AHL, acyl-homoserine lactone; Ca, calcium; CBF, ciliary beat frequency; NO, nitric oxide; NOS, nitric oxide synthase; PKG, protein kinase G.
A major result of the nitric oxide produced during T2R38 activation is increased MCC. Nitric oxide activates guanylyl cyclase, which produces cyclic-guanidine monophosphate and activates protein kinase G, which directly phosphorylates ciliary axoneme proteins to increase beating. However, nitric oxide production is also an important airway defense mechanism independently of MCC. Nitric oxide is a highly reactive radical that can diffuse inside bacteria cells. It produces reactive S-nitrosothiols and peroxynitrites that can damage bacterial DNA, membrane lipids, and enzymes. High levels of nitric oxide synthase are expressed in the cilia and microvilli of the sinonasal epithelium, and thus the sinuses are thought to be a major source of airway nitric oxide. The nitric oxide produced by sinonasal-ciliated epithelial cells in vitro was found to diffuse into the airway surface liquid and have direct bactericidal effects against P. aeruginosa, strongly suggesting that this nitric oxide may have direct antibacterial effects in vivo.
An important piece of evidence supporting the role of T2R38 as a bona-fide contributor to airway immunity was the identification of physiological bacterial ligands that activate T2R38 in vitro. The two major P. aeruginosa AHLs, N-butyryl-L-homoserine lactone and N-3-oxo-dodecanoyl-L-homoserine lactone, activate T2R38 both in sinonasal cells and in a heterologous expression system in vitro. This was confirmed using a P. aeruginosa strain mutated for the enzymes that synthesize AHLs (strain PAO-JP2; ΔlasI, ΔrhlI;). Sinonasal cells produced robust amounts of nitric oxide in a T2R38-dependent manner when stimulated with physiologically relevant concentrations of AHLs or with dilute conditioned medium from Wt, but not mutant P. aeruginosa. Because many gram-negative bacteria secrete AHLs, these data suggest that T2R38 in airway cilia is a sentinel to detect invading gram-negative bacteria and mount nitric oxide-dependent defense responses. Figure 2b depicts a diagram of this pathway.
Human T2R38 functionality is affected dramatically by several well studied polymorphisms in TAS2R38. Two polymorphisms are common in Caucasians: one encodes a functional T2R38 and the other encodes a nonfunctional T2R38, resulting from differences in amino acids at positions 49, 262, and 296. The functional T2R38 contains Pro (P), Ala (A), and Val (V) (the PAV variant) residues, respectively, at these positions. The nonfunctional T2R38 contains Ala (A), Val (V), and Ile (I) (the AVI variant) at these positions, respectively. Loss of the V at position 296 disrupts AVI receptor activation. These polymorphisms have well studied taste phenotypes. Homozygous AVI/AVI individuals (~30% frequency in Caucasians) are nontasters for T2R38-specific agonists, such as PTC; AVI/AVI individuals perceive PTC as either not or weakly bitter. PAV/PAV individuals (~20% frequency in Caucasians) are supertasters for PTC, tasting PTC as intensely bitter. AVI/PAV heterozygotes have varying intermediate levels of taste, correlating with differences of the relative expression levels of AVI and PAV alleles.
The effects of TAS2R38 polymorphisms were studied using primary ALIs derived from genotyped patients. When ALIs were stimulated in vitro with PTC, AHLs, or conditioned medium from P. aeruginosa, the antibacterial nitric oxide responses correlated with TAS2R38 polymorphisms. Epithelial cells from PAV/PAV supertasters exhibited markedly enhanced nitric oxide production, MCC, and bacterial killing compared with AVI/AVI or AVI/PAV cells. Cells derived from AVI/AVI nontasters or AVI/PAV heterozygotes had blunted nitric oxide responses that were not effective at killing P. aeruginosa in vitro. This strongly suggested that PAV/PAV individuals might be less susceptible to gram-negative infection, which has now been further studied in a clinical setting.