Introduction: Solitary cholinergic chemosensory cells (SCCC) are rare tracheal epithelial cells, which are considered as a part of the innate immune system sensing invading pathogens within the airways. They express a wide range of GPCR including taste receptors and their signaling machinery (TRPM5, PLCβ2 and ITP3R3) and the acetylcholine (ACh) producing enzyme ChAT. We previously identified bacterial formylated signal peptides as activators of SCCC, which subsequently release ACh and thereby alter ciliary activity. The receptor recognizing these formylated peptides, however, remained unknown. In the intestine it was shown that tuft cells, the counterpart of tracheal SCCC, can be activated by the microbial metabolite succinate. We here investigated if tracheal SCCC are also equipped with the succinate recognizing receptor SucnR1 and if succinate triggers innate defense mechanism through activation of tracheal SCCC.
Material and Methods: Expression of the succinate receptor SucnR1 was analyzed in isolated tracheal epithelial cells by RT-PCR and by analyzing existing single cell sequencing data sets. Particle transport speed (PTS) and ciliary beat frequency (CBF) were examined in response to metabolites. Ussing chamber experiments were performed with explanted tracheas to investigate ion transport processes across the tracheal epithelium.
Results: SucnR1 was exclusively expressed by a subset of SCCC within the tracheal epithelium, and there was no epithelial Sucnr1 expression in mice lacking SCCC (Pou2f3-/-). Succinate, but neither butyrate nor acetate, increased CBF and, consequently, PTS in a dose-dependent manner. This effect required Sucnr1 (lost in SucnR1-deficient mice) and SCCC (lost in Pou2f3- and Trpm5-deficient mice). In mice with SCCC-specific deletion of ChAT (ChATfl-Avilcre) and in the presence of the muscarinic antagonist atropine, the effect of succinate was reduced by 71 and 87%, respectively. In mice lacking muscarinic receptor M3, which is expressed by ciliated cells, the succinate effect was also abrogated by 77%. In Ussing chamber experiments, succinate induced a sharp increase in ion flux across the tracheal epithelium, which was reduced in SucnR1-, Pou2f3- and Trpm5-deficient mice by 81, 82 and 76%, respectively. The succinate-induced increase could be reduced by blocking cholinergic signaling (atropine + mecamylamine, 4-DAMP) and by the general chloride channel inhibitor NPPB (85, 94 and 98%, respectively). Furthermore, the gap junction blocker Gap27 reduced both, the succinate-induced increase in PTS and ion secretion (79 and 74%, respectively).
Conclusion: Succinate activates SCCC by binding to SucnR1, thereby triggering release of ACh. This increases ciliary activity of neighboring ciliated cells most likely via gap junctions and induces secretion of chloride ions into the periciliary fluid. Secretion of chloride ions is required for sufficient production of periciliary fluid. Hence, these effects are synergistic in terms of mucociliary clearance. Interestingly, succinate is produced in high quantities by epithelial cells during infection with Pseudomonas aeruginosa, a common pathogen especially in cystic fibrosis patients. Thus, the present data extend the molecular spectrum surveilled by tracheal SCCC beyond microbial factors to host-derived factors.