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

Intrinsic enteric neurons (iENs) form a crucial neuronal network within the myenteric and submucosal plexus of the gastrointestinal tract, primarily responsible for regulating gut peristalsis. The mechanisms by which iENs sense and integrate dietary and microbial signals to regulate intestinal homeostasis and inflammation remain unclear. Here we showed that environmental sensor aryl hydrocarbon receptor (AHR) was expressed in different iEN subsets in the ileum and colon, and AHR ligands differentially modulated iEN activity in these regions. Genetic perturbation of Ahr in neurons increased iEN activation in the ileum but conversely decreased it in the colon in response to different intestinal pathogens. Furthermore, neuronal AHR deficiency enhanced the clearance of bacterial pathogens, which was associated with increased proliferation and abundance of group 3 innate lymphoid cells in the ileum. Together, our findings demonstrate the region-specific functions of AHR in neurons in response to infections. Overall design: For single-nucleus RNA sequencing of iEN nuclei, Ahr?Actl6b: Sun1-GFP (CKO) and Ahrflox/flox: Sun1-GFP (CTL) mice were infected with Citrobacter rodentium ( 2×109 CFU in 200 µl of PBS) by oral gavage. The ileum and colon muscularis externa were harvested on day 7 after the infection for nucleus isolation and fluorescence-activated cell sorting (FACS). Flow-sorted GFP-positive iEN nuclei were pooled for gene expression library and Feature Barcode library construction via using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10×Genomics). For single-cell RNA sequencing of ILCs and T helper cells, Ahr?Actl6b (CKO) and Ahrflox/flox (CTL) mice were infected with Citrobacter rodentium ( 2×109 CFU in 200 µl of PBS) by oral gavage. The intestinal immune cells in the lamina propria of the ileum were obtained on day 7 after the infection for fluorescence-activated cell sorting (FACS). The flow-sorted ILCs and T helper cells (Viability-CD45+Lin-(CD11c, CD11b, Gr-1, CD19, NK1.1, FceRIa, TCR?/d, CD8a)CD90.2+) were pooled for gene expression library and Feature Barcode library construction via Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10×Genomics).