Cholinergic chemosensory cells in the trachea regulate breathing Gabriela Krastevaa,1, Brendan J. Canningb, Petra Hartmanna, Tibor Z. Veresc, Tamara Papadakisa, Christian Mühlfelda, Kirstin Schlieckera, Yvonne N. Tallinid, Armin Braunc, Holger Hacksteine, Nelli Baale, Eberhard Weihef, Burkhard Schützf, Michael Kotlikoffd, Ines Ibanez-Tallong, and Wolfgang Kummera aInstitute of Anatomy and Cell Biology and eInstitute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University, Giessen D-35385, Germany; bJohns Hopkins Asthma and Allergy Center, Baltimore, MD 21224; cFraunhofer Institute for Toxicology and Experimental Medicine, Hannover D-30625, Germany; dDepartment of Biomedical Sciences, College of Veterinary Medicine, Ithaca, NY 14853; fInstitute for Anatomy and Cell Biology, Philipps-University Marburg, D-35037 Marburg, Germany; and gMax-Delbrück-Centre for Molecular Medicine, Berlin D-13092, Germany Edited* by Ewald R. Weibel, University of Bern, Bern, Switzerland, and approved May 2, 2011 (received for review December 23, 2010) In the epithelium of the lower airways, a cell type of unknown two independently generated mouse strains with knockin of eGFP function has been termed “brush cell” because of a distinctive ul- within a BAC spanning the ChAT locus (10, 11). The average trastructural feature, an apical tuft of microvilli. Morphologically number of these cells in a mouse trachea was 6242 ± 989 with similar cells in the nose have been identified as solitary chemosen- approximately twice as many cells located above noncartilagenous sory cells responding to taste stimuli and triggering trigeminal regions (4,065 ± 640 cells) than in epithelial stretches overlaying reflexes. Here we show that brush cells of the mouse trachea ex- cartilage rings (2,177 ± 550 cells) (Fig. S1). Only a few of such cells press the receptors (Tas2R105, Tas2R108), the downstream signal- were located in the main extrapulmonary bronchi, and they oc- α ing molecules ( -gustducin, phospholipase Cβ2) of bitter taste curred only in exceptional cases distal to the lung hilus. Immu- transduction, the synthesis and packaging machinery for acetylcho- nohistochemistry demonstrated expression of villin in these cells, line, and are addressed by vagal sensory nerve fibers carrying nic- a marker protein of brush cells (Fig. 1 A and A′), and ultra- otinic acetylcholine receptors. Tracheal application of an nAChR structural eGFP immunohistochemistry revealed their brush cell agonist caused a reduction in breathing frequency. Similarly, cyclo- morphology (Fig. 1B). The cholinergic nature of the ChAT- heximide, a Tas2R108 agonist, evoked a drop in respiratory rate, eGFP+ brush cells was further documented by their immunore- CELL BIOLOGY being sensitive to nicotinic receptor blockade and epithelium re- activity for the vesicular acetylcholine transporter (VAChT), a fi moval. This identi es brush cells as cholinergic sensors of the chem- membrane protein of cholinergic vesicles (12) (Fig. 1 C and C′) ical composition of the lower airway luminal microenvironment and observation of VAChT-IR in villin-positive cells in wild-type that are directly linked to the regulation of respiration. tracheas (not shown). Although all ChAT-eGFP+ cells were im- munoreactive to villin, a small fraction (17%; 164 cells/4 mice) of – airway sensory innervation | respiratory epithelium | jugular nodose villin-positive cells did not exhibit eGFP (Fig. 1D), even when vi- ganglion | bitter-tasting substances sualization of eGFP was enhanced by eGFP immunolabeling (Fig. 1E), strongly suggesting heterogeneity of tracheal brush cells. he so-called “brush cells” of the respiratory epithelium of the Tlower airways are morphologically defined by an apical tuft of Cholinergic Brush Cells Express a Bitter Taste Transduction System. microvilli containing the structural proteins villin and fimbrin (1). Detection of bitter substances is achieved by a variety of taste Despite some similarities, they are different from solitary neu- receptors of the T2R family that are coupled to the G protein, roendocrine cells of the epithelium that can give rise to small-cell α-gustducin. Their stimulation leads to activation of PLC-β2 and, lung carcinoma (2). The function of brush cells in the lower subsequently, to opening of TRPM5 channels (13–15). We pre- airway epithelium is still unclear. Originally, a resorptive func- viously identified TRPM5 in tracheal brush cells (6). Here, we tion and hence a role in regulation of the viscosity of the mucous detected α-gustducin immunoreactivity and PLC-β2 immunore- had been postulated (3), whereas recent evidence points toward activity in cholinergic (ChAT-eGFP+) brush cells (Fig. 2 A–B′). a chemosensory function (4). Alpha-gustducin and phospholipase In this respect, tracheal cholinergic brush cells differ from cho- Cβ2 (PLC-β2), elements of the bitter and sweet sensing trans- linergic cells in the taste buds of the circumvallate papillae (16), duction cascade, have been detected in solitary, putative brush which we found to be largely separate from α-gustducin- and cells of the rat trachea (5), and we identified TRPM5, a taste- PLC-β2-immunoreactive taste cells (Fig. 2 C and D). signaling (sweet, bitter, amino acid) transient receptor potential In the absence of specific antibodies against murine T2R sub- cation-channel in rat and mouse tracheal brush cells (6). Mor- types, we investigated their expression by RT-PCR. Of the 35 phologically similar cells in the mouse nasal epithelium express known mouse T2Rs, ligands are identified for T2R105 (cyclohex- α-gustducin (7) and respond to bitter stimuli, and their activation imide) and T2R108 (denatonium) (17, 18). For both, mRNA was evokes respiratory reflexes via afferent neurons of the trigeminal detected in abraded tracheal epithelium, along with α-gustducin + nerves (8, 9). Analyzing a mouse strain expressing enhanced GFP mRNA and PLC-β2mRNA(Fig.2E). ChAT-eGFP cells were (eGFP) under the control of the promoter of the acetylcholine then purified by FACS from freshly dissociated epithelial cells. RT- synthesizing enzyme, choline acetyltransferase (ChAT), solitary ChAT-eGFP+ cells were located in the tracheal epithelium that did not express a marker protein of solitary neuroendocrine cells Author contributions: G.K., B.J.C., and W.K. designed research; G.K., P.H., T.Z.V., T.P., C.M., K.S., and W.K. performed research; Y.N.T., A.B., H.H., N.B., E.W., B.S., M.K., and I.I.-T. (10). This led us to the hypothesis that brush cells of the lower contributed new reagents/analytic tools; G.K., B.J.C., P.H., and W.K. analyzed data; and airway epithelium are solitary chemosensory cells that sense the G.K., B.J.C., and W.K. wrote the paper. chemical composition of the airway lining fluid and transmit this The authors declare no conflict of interest. information to sensory nerve endings via cholinergic transmission. *This Direct Submission article had a prearranged editor. 1To whom correspondence should be addressed. E-mail: Gabriela.Krasteva@anatomie. Results med.uni-giessen.de. fl Tracheal Brush Cells Are Cholinergic. We observed solitary ask- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + shaped or triangular eGFP cells in the tracheal epithelium of 1073/pnas.1019418108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1019418108 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 EGFP) (23). Sensory neurons expressing eGFP fluorescence in this transgenic line indeed carry functional nAChRs: they respond to nicotine with a sharp increase in cytosolic calcium concentration (Fig. S4). Intraepithelial nAChR-positive nerve fibers were cola- beled for CGRP (Fig. 4 B–B″) and seemed to establish close contacts to villin-positive (brush) cells (Fig. 4 A–A″). The sensory vagal origin of such nerve fibers was confirmed by retrograde neuronal labeling using Fast Blue instillation in the airways. Six percent of all retrogradely labeled neurons in the jugular–nodose complex (n = 1,878 Fast Blue+ neurons from four animals) exhibited an nAChR-eGFP+/CGRP+ phenotype (Fig. 4 C–C″), another 2% were nAChR-eGFP+ without additional CGRP immunolabeling, and 9% were solely CGRP immunoreactive. Hence, cholinergic brush cells contact a population of peptidergic (CGRP) cholinoceptive vagal sensory neurons. Mucosal Application of a Bitter Substance Elicits a Cholinergically Driven Respiratory Reflex. The morphological studies summarized above suggest that tracheal cholinergic brush cells detect bitter substances in the airway lining fluid and transmit this in- formation through the release of acetylcholine, which in turn activates nAChRs on adjacent sensory nerve fibers, resulting in action potentials and ultimately respiratory reflexes. To address Fig. 1. Tracheal ChAT-eGFP cells are cholinergic brush cells. (A and A′) this hypothesis directly, a method was established that allows ChAT-eGFP–expressing cells are immunoreactive for villin, a structural pro- monitoring respiratory efforts in a freely breathing mouse while μ tein of microvilli of brush cells. (Scale bar, 20 m.) (B) Ultrastructural pre- perfusing the mucosal surface of the upper cervical trachea with embedding anti-eGFP immunohistochemistry. ChAT-eGFP cell with micovilli fl test substances (Fig. S5). Capsaicin, a known activator of afferent tuft (mt) typical for brush cells, anked by secretory cells (SC). (Scale bar, fi 1 μm.) (C and C′) Epifluorescence. ChAT-eGFP–expressing cell is immunore- C- bers (24, 25) and here used as a test substance