284 | Nature | Vol 578 | 13 February 2020
Article
Neuronal programming by microbiota
regulates intestinal physiology
Yuuki Obata^1 *, Álvaro Castaño^1 , Stefan Boeing^1 , Ana Carina Bon-Frauches^1 , Candice Fung^2 ,
Todd Fallesen^1 , Mercedes Gomez de Agüero^3 , Bahtiyar Yilmaz^3 , Rita Lopes^1 ,
Almaz Huseynova^1 , Stuart Horswell^1 , Muralidhara Rao Maradana^1 , Werend Boesmans4,5,
Pieter Vanden Berghe^2 , Andrew J. Murray^6 , Brigitta Stockinger1,7, Andrew J. Macpherson3,7
& Vassilis Pachnis1,7*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 crit-
ical 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 specifi-
cally 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 dif-
ferent intestinal segments and microbiota states (Fig. 1a, Extended Data
Fig. 1a–l). First, we compared the transcriptional profiles of myenterichttps://doi.org/10.1038/s41586-020-1975-8
Received: 10 August 2019
Accepted: 9 December 2019
Published online: 5 February 2020
(^1) The Francis Crick Institute, London, UK. (^2) Laboratory of Enteric Neuroscience (LENS), Translational Research in Gastrointestinal Disorders (TARGID), Department of Clinical and Experimental
Medicine, University of Leuven, Leuven, Belgium.^3 Maurice Muller Laboratories (DKF), Universitätsklinik fur Viszerale Chirurgie und Medizin Inselspital, University of Bern, Bern, Switzerland.
(^4) Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, Belgium. (^5) Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University
Medical Center, Maastricht, The Netherlands.^6 Sainsbury Wellcome Centre for Neural Circuits and Behaviour, University College London, London, UK.^7 These authors contributed equally:
Brigitta Stockinger, Andrew J. Macpherson, Vassilis Pachnis. *e-mail: [email protected]; [email protected]