Endothelial sensing of AHR ligands regulates intestinal homeostasis

Endothelial cells line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance and coordinate angiogenesis and lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric endothelial cells actively sense and integrate such signals is currently unknown. Here we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for endothelial cell sensing of dietary metabolites in adult mice and human primary endothelial cells. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic endothelial cells. Analyses of AHR-mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR deficiency in adult mice resulted in dysregulated inflammatory responses and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human endothelial cells, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the effect of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting endothelial cell quiescence and vascular normalcy.


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No statistical methods were used to estimate sample sizes before the study.We based our sample numbers on a combination of preliminary data from pilot studies, previous experience with in vivo and in vitro systems (Schiering et al. Nature 2017, Metidji et al. Immunity 2018), and standards in the field.
Data exclusions scRNAseq thresholding involved excluding cells with abnormally high or low transcripts, and based on high mitochondrial gene content as is standard practice for downstream analysis of this sequencing data.We also excluded doublets, and removed contaminant clusters to improve stringency, as described in the methods.For each BEC and LEC RNAseq, 1 control and 1 experimental repeat were excluded from the original n=4/group based on low Cyp1a1 activation and highly divergent transcriptome (experimental samples), and highly divergent transcriptome only (control samples).

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Blinding was not used in this study as the researchers must predetermine genotype before the study in order to obtain equal group sizes, minimize animal usage, achieve appropriate randomization and prevent infection-based artifacts (see above).