Brainstem Dbh+ Neurons Control Chronic Allergen-Induced Airway Hyperreactivity

Exaggerated airway constriction triggered by exposure to irritants such as allergen, also called hyperreactivity, is a hallmark of asthma and can be life-threatening. Aside from immune cells, vagal sensory neurons are important for airway hyperreactivity 1-4. However, the identity and signature of the downstream nodes of this adaptive circuit remains poorly understood. Here we show that Dbh+ neurons in the nucleus of the solitary tract (nTS) of the brainstem, and downstream neurons in the nucleus ambiguus (NA), are both necessary and sufficient for chronic allergen-induced airway hyperreactivity. We found that repeated exposures of mice to inhaled allergen activates nTS neurons in a mast cell-, interleukin 4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA-seq followed by RNAscope quantification of the nTS at baseline and following allergen challenges reveals that a Dbh+ population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted, while chemogenetic activation promoted hyperreactivity. Viral tracing indicates that Dbh+ nTS neurons, capable of producing norepinephrine, project to the NA, and NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that then directly drive airway constriction. Focusing on transmitters, delivery of norepinephrine antagonists to the NA blunted allergen-induced hyperreactivity. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. The knowledge opens the possibility of targeting neural modulation as an approach to control refractory allergen-induced airway constriction. Overall design: We dissected the GFP+ NA region from 7 Chat-cre; CAG-Sun1/sfGFP mice brainstem under the fluorescence microscope. Mice were euthanized using CO2 inhalation. Brains were acutely harvested. Dissected NA were put in liquid N2 immediately and stored at -80°C or proceeded directly to nuclei isolation. NA nuclei were processed into cDNA libraries using Chromium Single Cell 3' v3 kit (10XGenomics). Sequencing was carried out on NovaSeq (Illumina) platform. CellRanger package (version 3.0.2) was used to align the raw reads onto the mouse reference genome (GRCm38) and generate the feature-barcode matrix. R package Seurat (version 4.0) was used to perform data quality control, normalization, principal components analysis and uniform manifold approximation and projection (UMAP method).