Neuronal programming by microbiota regulates intestinal physiology

Abstract

Neural control of the function of visceral organs is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence, and is often dysregulated in gastrointestinal disorders 1. Luminal factors, such as diet and microbiota, regulate neurogenic programs of gut motility 2-5 , but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor aryl hydrocarbon receptor (AHR) functions as a biosensor in intestinal neural circuits, linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons that represent distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles that are controlled by the combined effects of host genetic programs and microbial colonization. Microbiota-induced expression of AHR in neurons of the distal gastrointestinal tract enables these neurons to respond to the luminal environment and to induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in the enteric neurons of mice treated with antibiotics partially restores intestinal motility. Together, our experiments identify AHR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits to maintain gut homeostasis and health. The enteric nervous system (ENS) encompasses the intrinsic neural networks of the gastrointestinal tract, which regulate most aspects of intestinal physiology (including peristalsis) 6,7. In addition to host-specific genetic programs, microbiota and diet have emerged as critical regulators of the physiology of gut tissue 2,8 and changes in the microbial composition of the lumen often accompany gastrointestinal disorders 4. Thus, depletion of the microbiota causes a reduced excit-ability of enteric neurons, changes in motility programs (such as the neurogenic colonic migrating motor complexes 5,9,10) and prolonged intestinal transit time (ITT) 11,12. However, conventionalization of adult germ-free mice reduces the deficit in ITT 11 and restores neuronal excit-ability 13 , which suggests that intestinal neural circuits are endowed with molecular mechanisms that monitor the state of the gut lumen and adjust neuronal activity and motility accordingly. Despite considerable recent progress 2 in describing the effects of the microbiota and diet on gastrointestinal physiology, the molecular mechanisms by which the luminal environment regulates ENS activity and intestinal peristalsis remain unknown. We hypothesized that molecular mechanisms that link the micro-biota to intestinal motor behaviour are likely to be encoded by genetic programs that operate predominantly in neural circuits of the colon, the intestinal segment with the heaviest load of microorganisms 14. We therefore used RNA sequencing to identify genes that are specifically upregulated in enteric neurons of the mouse colon in response to microbial colonization. Because our pilot experiments indicated that the current protocols for tissue dissociation and the recovery of intact ENS cells often resulted in considerable cellular damage and non-specific transcriptional changes, we developed a strategy that uses an adeno-associated virus (AAV) for labelling followed by the isolation and RNA sequencing of enteric neuron nuclei (nRNA-seq) that represent different intestinal segments and microbiota states (Fig. 1a, Extended Data Fig. 1a-l). First, we compared the transcriptional profiles of myenteric