Project description:Neural control of visceral organ function is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence and is often dysregulated in gastrointestinal (GI) disorders. Luminal factors, such as diet and microbiota regulate neurogenic programs of gut motility, 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 representing distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles controlled by the combined effects of host genetic programmes and microbial colonisation. Microbiota-induced expression of AhR in neurons of the distal gastrointestinal tract enables them to respond to the luminal environment and 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 enteric neurons of antibiotic-treated mice partially restores intestinal motility. Taken 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 towards gut homeostasis and health. The enteric nervous system (ENS) encompasses the intrinsic neural networks of the gastrointestinal (GI) tract, which regulate most aspects of intestinal physiology, including peristalsis. In addition to host-specific genetic programmes, microbiota and diet have emerged as critical regulators of gut tissue physiology and changes in the microbial composition of the lumen often accompany GI disorders. We found that gut environmental sensor Aryl hydrocarbon receptor (AhR) is induced in colonic neurons in response to microbiota colonisation and regulates intestinal peristalsis in an AhR ligand-dependent manner. In this experiment, we used RNA sequencing to identify genes regulated in mouse colonic neurons by AhR activation.

Project description:Neural control of visceral organ function is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence and is often dysregulated in gastrointestinal (GI) disorders. Luminal factors, such as diet and microbiota regulate neurogenic programs of gut motility, 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 representing distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles controlled by the combined effects of host genetic programmes and microbial colonisation. Microbiota-induced expression of AhR in neurons of the distal gastrointestinal tract enables them to respond to the luminal environment and 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 enteric neurons of antibiotic-treated mice partially restores intestinal motility. Taken 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 towards gut homeostasis and health. The enteric nervous system (ENS) encompasses the intrinsic neural networks of the gastrointestinal (GI) tract, which regulate most aspects of intestinal physiology, including peristalsis. In addition to host-specific genetic programmes, microbiota and diet have emerged as critical regulators of gut tissue physiology and changes in the microbial composition of the lumen often accompany GI disorders. However the molecular mechanisms by which gut enviromental factors regulate ENS homeostasis remain unknown. In order to address this issue, we used RNA sequencing to identify genes specifically upregulated in mouse colonic neurons in response to microbial colonisation.

Project description:Mapttm1(EGFP)Klt/J mice (Mapt-EGFP; The Jackson Laboratory, Bar Harbor, ME, USA; stock 004779) carry a knock-in of the EGFP coding sequence in the first exon of the microtubule-associated protein tau (Mapt) gene producing a cytoplasmic EGFP fused to the first 31 amino acids of MAPT. EGFP expression marks neurons including enteric neurons regardless of their lineage, closely patterning the expression of neuron-specific beta-tubulin III (TUBB3). Mapt-EGFP ice were backcrossed to C57BL/6J (Jackson Laboratory strain #:000664) for three to five generations at Mayo Clinic. Six male and six female Mapt-EGFP mice (54-98 days of age) underwent surgical laparotomy in 3 groups (surgery #1: 1 male and 1 female, surgery #2: 3 males and 1 female, surgery #3: 2 males and 4 females) under pentobarbital (50mg/kg) anesthesia. The celiac ganglion of each mouse was injected with 3-5 μL of 25 mg/mL Alexa Fluor 647-labeled cholera toxin subunit B (CTB-AF647; Thermo Fisher Scientific, Waltham, MA, USA) with the intention of labeling the cell soma of intestinofugal neurons in the myenteric plexus of the colon. The animals were killed 3-4 days after surgery. The muscularis externa of the colon from each Mapt-EGFP mouse was pooled together between all mice of the same surgery date (2, 4, and 6 mice) and mechanically and enzymatically dissociated into single cells with a two-step process that first enriches for cells within myenteric ganglia (PMCID: PMC8114175). The pooled cells from each group of mice formed one biological replicate and subjected to FACS immediately after dissociation to generate populations of Mapt-EGFP+ neurons with or without the CTB-AF647 tracer and Mapt-EGFP− non-neuronal cells. The frequency of Mapt-EGFP+CTB-AF647+ neurons was approximately 125-fold lower than that of Mapt-EGFP+CTB-AF647− neurons and RNA from these preparations did not pass quality control. Therefore, only data from Mapt-EGFP+CTB-AF647− neurons were analyzed and referred to as Mapt-EGFP+ cells. Total RNA was isolated from Mapt-EGFP+ colonic neurons and Mapt-EGFP− myenteric cells using RNA-Bee (AMSBIO, Cambridge, MA, USA) and purified with RNeasy Mini Kit (Qiagen, Germantown, MD, USA). RNA quality was tested using Agilent Electropherogram (Agilent Technologies, Santa Clara, CA, USA) and hybridized to Affymetrix Mouse Genome 430.2 gene expression microarrays (Thermo Fisher Scientific, Waltham, MA, USA). This study utilized Affymetrix Mouse Genome 430.2 oligonucleotide microarray analysis to charaterize the transcriptome of Mapt-EGFP+ neurons and Mapt-EGFP- non-neuronal myenteric cells isolated from the colon of Mapttm1(EGFP)Klt/J mice.

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Intestinal region-specific functions of AHR in intrinsic enteric neurons during infections

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