Intracellular signaling in airway solitary chemosensory (tuft) cells

Project Summary Airway tuft cells (also known as “brush” or “solitary chemosensory” cells) are rare cells found in the nose and trachea. Tuft cells are also found in the lung after injury and/or inflammation or in genetic diseases like primary ciliary dyskinesia. Tuft cells regulate local antimicrobial peptide, acetylcholine (ACh), and IL-25 secretion. IL-25 is an important driver of Th2 inflammatory responses observed in airway disease like chronic rhinosinusitis (CRS) with nasal polyps and asthma. Tuft cell ACh may activate sensory neurons and/or local mucociliary or inflammatory responses. We know little about how to target tuft cells in airway diseases, because we know little about human tuft cell function. Their rarity (≤1 in 100 cells in the nose) makes them difficult to study, though their frequency increases significantly (up to 30% of the epithelium) in nasal polyps. We know that tuft cells express a range of chemosensory G protein-coupled receptors (GPCRs) but know little about how they signal and regulate cell responses. We previously showed that activation of T2R bitter taste GPCRs stimulates Ca2+ -driven secretion of antimicrobial peptides from surrounding epithelial cells. This response is inhibited by activation of cAMP downstream of T1R sweet taste GPCRs, which sense airway surface liquid glucose or sweet bacterial D-amino acids. Other GPCRs (e.g., succinate, cholinergic receptors, adenosine receptors, etc.) exist in tuft cells, but their functions are less clear. Many studies have been done in mice, but our research revealed differences in how mouse vs human tuft cells signal. We need better methods for studying human tuft cell function to complement and extend prior mouse studies. The goal here is to identify the signaling and downstream consequences of nasal tuft cell GPCRs, which may be important therapeutic targets for respiratory infections, either to enhance antibacterial immunity or reduce nasal/lung inflammation. We will utilize a novel genetic labeling strategy to express fluorescent protein biosensors specifically in human tuft cells cultured from residual nasal surgical material. This allows optical imaging of tuft cell function within an intact epithelial monolayer differentiated at air-liquid interface. In Aim 1, we will use these methods to study how tuft cell GPCRs regulate Ca2+, cAMP, and other pathways to fine tune antimicrobial peptide secretion. In Aim 2, we will study how these pathways regulate electrical excitability of airway tuft cells and if/how membrane voltage changes contribute to tuft cell responses. In Aim 3, we will elucidate mechanisms of how tuft cells secrete/release ACh and IL-25 in the context of Th2 iairway disease. These data will clarify the regulation of intracellular signaling of human tuft cells to better understand how to target them in airway diseases like CRS and possibly asthma.