Regional encoding of enteric nervous system responses to microbiota and type 2 inflammation.

Enteric neurons are essential regulators of intestinal physiology, yet their responses to varying microbial and immune environments along the intestinal tract and or during challenges remain poorly understood. In this study, we regionally profiled enteric neurons across gnotobiotic, allergic, and parasite-infected mice. Timing and complexity of microbial perturbations and type 2 inflammation result in motor neuron state shifts and alter multiple functionally distinct sensory neurons, including interleukin-13– and leukotriene-responsive Nmu-hi cells and Grp-hi neurons, which expand in germ-free colonic tissue and interact with Grpr+ interstitial cells of Cajal. Leveraging adeno-associated virus–based Perturb-seq, we identified Edf1 and Mitf as controllers of motor neuron state transition and gastrointestinal transit time, directly linking enteric neuron states to physiology. Editor's summary: Enteric neurons of the intestine play a major role in regulating intestinal physiology. Tan et al. characterized the effect of microbiota and inflammation on the enteric nervous system in mice. The authors integrated single-cell transcriptomics across three intestinal regions with in vivo functional perturbation to uncover how microbial cues and immune signals reshape inhibitory motor neuron states and sensory neuron plasticity. Using single-cell RNA sequencing and genetic perturbations, they identified regulators of motor neuron states and intestinal gastric time. In addition to providing biological insights into the response of the enteric nervous system to various perturbations, the results are a valuable resource that will serve as an important benchmark for many future discoveries. —Mattia Maroso INTRODUCTION: The enteric nervous system (ENS) is a branch of the autonomous nervous system composed of neurons located across two plexi of the intestinal tract. The ENS can function semiautonomously to regulate digestion and peristalsis, through a circuit involving five main neuronal types: (i) sensory neurons activate (ii) excitatory and (iii) inhibitory motor neurons through (iv) ascending and descending interneurons, which concomitantly activate (v) secretomotor and vasodilator neurons to regulate epithelial secretion and absorption. Accumulating evidence suggests that, in addition to working through this core circuit, the ENS plays a key role in responses to bacteria, allergens, and parasites through extensive cross-talk with the immune system. RATIONALE: The gastrointestinal tract is constantly exposed to environmental changes driven by the microbiota, pathogens, and immunity, yet our understanding of the ENS's adaptation to these fluctuations remains limited, in part because of difficulties in isolating and profiling enteric neurons. To address this gap, we generated gnotobiotic mouse reporters and colonized them with distinct microbiomes, and in parallel, studied the ENS of animals exposed to type 2 inflammation, including food allergy and Heligmosomoides polygyrus infection. In each of these settings, we deeply profiled the ENS across distinct segments of the intestine—duodenum, jejunum, ileum, and colon—to define its response to perturbation. RESULTS: Plate-based single-nucleus RNA profiling of the ENS captured a total of 7640 enteric neurons with more than 6000 median genes per cell. This atlas highlights two main axes of diversity across enteric neurons. First, we observed a functional specification of sensory neurons across distinct cell types characterized by distinct neuropeptides and receptors, including neuromedin U (Nmu)–expressing neurons that respond to type 2 cytokines and leukotrienes. Second, we observed transcriptomic gradients across both subsets of motor neurons (inhibitory and excitatory), likely encompassing distinct cell states that can be modulated by environmental challenges. Notably, these changes were highly coordinated across distinct perturbations, with changes in the frequency of Nmu+neurons and a shift of inhibitory motor neurons between Bglap and Dcc/Aldh1a3 populations occurring in animals that were allergic, parasite-infected, and born germ-free. To define the programs that contribute to neuronal adaptation, we next used adeno-associated virus (AAV)–PHP.S viral vectors—which efficiently target all peripheral neurons—to delete genes of interest in Cas9-expressing animals. We first delivered pools of guides and performed single-nucleus RNA sequencing of infected neurons to identify genes associated with changes in neuronal cell states. Lastly, we deleted candidate regulators in individual animals and validated two genes, Edf1 and Mitf, as regulators of both inhibitory motor neuron cell state and total gastrointestinal transit time. More specifically, deletion of Edf1 resulted in an expansion of BGLAP+ neurons among infected cells and increased transit time, whereas deletion of Mitf expanded DCC+ neurons and shortened transit time. CONCLUSION: Our studies provide a detailed transcriptomic atlas of the ENS during distinct environmental challenges and reveal a set of coordinated responses to perturbations. This work directly links ENS cell states to changes in intestinal physiology and provides a blueprint for future functional studies of the ENS in health and disease. Enteric neurons across sites and perturbations.: Enteric neuron single-nucleus RNA sequencing (snRNA-seq) was performed on the indicated segments and models, resulting in a single-cell (sc) atlas of 7640 deeply profiled neurons (bottom left). Nmu-hi sensory neurons and inhibitory motor neuron states were highly responsive to perturbations (top right), and AAV-based gene deletions demonstrated a role for Mitf and Edf1 in modulating inhibitory motor neurons and gastrointestinal transit time (bottom right). PSN, putative sensory neurons; PEMN, putative excitatory motor neurons; PIMN, putative inhibitory motor neurons; PIN, putative interneurons; PSVN, putative secretomotor/vasodilator neurons. [ABSTRACT FROM AUTHOR]