Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

Abstract

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl-homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl-homoserine lactones serve as quorum-sensing molecules for gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Fig. 1.

SCCs in the nose are innervated by trigeminal sensory nerves. (A) Midline section through the head of a mouse showing the nasal airway passages. The area of respiratory epithelium indicated by the blue rectangle contains a high density of SCCs. (B and C) Double-label images show the SCC cells (exhibiting green TrpM5-driven GFP fluorescence) richly innervated by pain fibers of the trigeminal nerve, which contain the neuropeptides substance P (red fluorescence in B and B′′) and CGRP (red fluorescence in C and C′′). (D) Area marked by blue rectangle in A in a TrpM5–GFP mouse showing green fluorescent SCCs in the nasal epithelium.

Fig. 2.

Ca²⁺ responses in SCCs show responsiveness to AHLs. More than 50% (16/31) of TrpM5–GFP cells and 75% (36/47) of Gα-gustducin-GFP-–labeled cells respond to 10–20 mM denatonium (Den) and to the bacterially produced AHLs but not to the control plasmid (pViet). (A and B) Responses are eliminated by treatment with the PLC inhibitor U73122, indicating that these Ca²⁺ responses are mediated by a G-protein–coupled receptor system. (C) Only GFP-labeled cells respond to denatonium (Den) and AHL molecules (C6, 3-oxo-C6-HSL; C12, 3-oxo-C12-HSL); neither GFP-labeled nor unlabeled epithelium cells show Ca²⁺ responses to stimulation by capsaicin (Cap). (D) Dose–response curves relating the magnitude of the Ca²⁺ response to the concentration of the applied synthetic AHL. (E) Cell responses (Mean ± SEM) to denatonium (Den) and 3-oxo-C12-HSL (C12) applied in the presence of the control (noneffective) inhibitor (U73343) and the active PLC inhibitor U73122 normalized to the response with no inhibitor (normalized response = 1.0). The PLC blocker U73122 significantly (** denotes P < 0.001, one-sample t test with Bonferroni correction, n = 7 cells) reduces the Ca²⁺ response of isolated TrpM5–GFP SCCs to both denatonium (Den) and C12 synthetic AHL whereas the control drug has no effect (P > 0.05). (F) Structure of the principal AHL compounds produced by each of the bacterial genes used. See Fig. S1 for a complete analysis of the bacterial products.

Fig. 3.

Respiratory changes evoked by AHL stimulation in wild type (blue) and TrpM5–KO (red) mice. Gα-gustducin–KO mice show a similar lack of responsiveness. (A–D) Graphs (Upper) and respiratory records (Lower) showing changes in relative breath duration following stimulation [stimulus application indicated by a caret (“_^”)] in wild-type (WT) and KO strains. Values >1 indicate a slowing in breathing with higher numbers indicating a longer pause. The lack of respiratory response to denatonium, LasI, or EsaI products in the KO mice implicates the SCCs in the detection of these substances. The continued responsiveness of the KO mice to capsaicin (D) confirms that the trigeminal nerve is directly responsive to capsaicin and does not require functioning of the SCCs. (E) Summary graph (mean ± SEM) comparing relative breath duration following stimulation with irritants and control solutions in WT, Gα-gustducin–KO, and TrpM5–KO animals. Application of denatonium, LasI, or EsaI products and the synthetic HSLs each resulted in a highly significant (two-tailed t test: ***P < 0.001; *P < 0.05) pause in respiration in the WT but not in the KO mice whereas application of capsaicin and acetic acid elicits responses in WT as well as in KO lines. Application of 1% methanol (vehicle control) or extract from bacteria carrying the control pViet plasmid showed no such responses.

Fig. 4.

Summary diagram of the SCC trigeminal system. The SCCs synapse on the same trigeminal nerve fibers that provide sensory innervation to the surrounding epithelium in the form of free nerve endings. The nerve fibers express several chemosensitive ion channels, including TRPs and ASICs. These nerve endings terminate below the level of the epithelial tight junctions (26) and therefore can respond only to stimuli capable of crossing this barrier. The SCCs extend above the barrier of tight junctions and so have access to all stimuli in the overlying mucus layer. Our results show that certain stimuli, including denatonium and AHLs, must act via the SCCs to activate the trigeminal nerve.