Commensal microbiota-dependent tryptophan catabolism within the gastrointestinal tract is known to exert profound effects upon host physiology, including the maintenance of epithelial barrier and immune function. A number of abundant microbiota-derived tryptophan metabolites exhibit activation potential for the aryl hydrocarbon receptor (AHR). Gene expression facilitated by AHR activation through the presence of dietary or microbiota-generated metabolites can influence gastrointestinal homeostasis and confer protection from intestinal challenges. Utilizing untargeted mass spectrometry-based metabolomics profiling, combined with AHR activity screening assays, we identify four previously unrecognized tryptophan metabolites, present in mouse cecal contents and human stool, with the capacity to activate AHR. Using GC/MS and LC/MS platforms, quantification of these novel AHR activators, along with previously established AHR-activating tryptophan metabolites, was achieved, providing a relative order of abundance. Using physiologically relevant concentrations and quantitative gene expression analyses, the relative efficacy of these tryptophan metabolites with regard to mouse or human AHR activation potential is examined. These data reveal indole, 2-oxindole, indole-3-acetic acid and kynurenic acid as the dominant AHR activators in mouse cecal contents and human stool from participants on a controlled diet. Here we provide the first documentation of the relative abundance and AHR activation potential of a panel of microbiota-derived tryptophan metabolites. Furthermore, these data reveal the human AHR to be more sensitive, at physiologically relevant concentrations, to tryptophan metabolite activation than mouse AHR. Additionally, correlation analyses indicate a relationship linking major tryptophan metabolite abundance with AHR activity, suggesting these cecal/fecal metabolites represent biomarkers of intestinal AHR activity.

Urolithins (e.g., UroA and B) are gut microbiota-derived metabolites of the natural polyphenol ellagic acid. Urolithins are associated with various health benefits, including attenuation of inflammatory signaling, anti-cancer effects and repression of lipid accumulation. The molecular mechanisms underlying the beneficial effects of urolithins remain unclear. We hypothesize that some of the human health benefits of urolithins are mediated through the aryl hydrocarbon receptor (AHR). Utilizing a cell-based reporter system, we tested urolithins for the capacity to modulate AHR activity. Cytochrome P450 1A1 (CYP1A1) mRNA levels were assessed by real-time quantitative polymerase chain reaction. Competitive ligand binding assays were performed to determine whether UroA is a direct ligand for the AHR. Subcellular AHR protein levels were examined utilizing immunoblotting analysis. AHR expression was repressed in Caco-2 cells by siRNA transfection to investigate AHR-dependency. UroA and B were able to antagonize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AHR-mediated transcriptional activity. Furthermore, UroA and B attenuated TCDD-mediated stimulation of CYP1A1 mRNA levels. In addition, competitive ligand binding assays characterized UroA as a direct AHR ligand. Consistent with other AHR antagonists, UroA failed to induce AHR retention in the nucleus. AHR is necessary for UroA-mediated attenuation of cytokine-induced interleukin 6 (IL6) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression in Caco-2 cells. Here we identified UroA as the first dietary-derived human selective AHR antagonist produced by the gut microbiota through multi-step metabolism. Furthermore, previously reported anti-inflammatory activity of UroA may at least in part be mediated through AHR.

Increasingly, the aryl hydrocarbon receptor (AHR) is being recognized as a sensor for endogenous and pseudo-endogenous metabolites, and in particular microbiota and host generated tryptophan metabolites. One proposed explanation for this is the role of the AHR in innate immune signaling within barrier tissues in response to the presence of microorganisms. A number of cytokine/chemokine genes exhibit a combinatorial increase in transcription upon toll-like receptors and AHR activation, supporting this concept. The AHR also plays a role in the enhanced differentiation of intestinal and dermal epithelium leading to improved barrier function. Importantly, from an evolutionary perspective many of these tryptophan metabolites exhibit greater activation potential for the human AHR when compared to the rodent AHR. These observations underscore the importance of the AHR in barrier tissues and may lead to pharmacologic therapeutic intervention.

Persistent organic pollutants (POPs) are important environmental chemicals and continued study of their mechanism of action remains a high priority. POPs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and polychlorinated biphenyls (PCBs), are widespread environmental contaminants that are agonists for the aryl hydrocarbon receptor (AHR). Activation of the AHR modulates the gut microbiome community structure and function, host immunity, and the host metabolome. In the current study, male C57BL6/J mice were exposed, via the diet, to 5 µg/kg body weight (BW) TCDF or 24 µg/kg BW of TCDF every day for 5 days. The functional and structural changes imparted by TCDF exposure to the gut microbiome and host metabolome were explored via 16S rRNA gene amplicon sequencing, metabolomics, and bacterial metatranscriptomics. Significant changes included increases in lipopolysaccharide (LPS) biosynthesis gene expression after exposure to 24 µg/kg BW of TCDF. Increases in LPS biosynthesis were confirmed with metabolomics and LPS assays using serum obtained from TCDF-treated mice. Significant increases in gene expression within aspartate and glutamate metabolism were noted after exposure to 24 µg/kg BW of TCDF. Together, these results suggest that after exposure to 24 µg/kg BW of TCDF, the gut microbiome increases the production of LPS and glutamate to promote localized gut inflammation, potentially using glutamate as a stress response.

The intestinal epithelial layer is susceptible to damage by chemical, physiological and mechanical stress. While it is essential to maintain the integrity of epithelium, the biochemical pathways that contribute to the barrier function have not been completely investigated. Here we demonstrate an aryl hydrocarbon receptor (AHR)‐dependent mechanism facilitating the production of the antimicrobial peptide AMP regenerating islet‐derived protein 3 gamma (REG3G), which is essential for intestinal homeostasis. Genetic ablation of AHR in mice impairs pSTAT3‐mediated REG3G expression and increases bacterial numbers of Segmented filamentous bacteria ( SFB) and Akkermansia muciniphila in the small intestine. Studies with tissue‐specific conditional knockout mice revealed that the presence of AHR in the epithelial cells of the small intestine is not required for the production of REG3G through the phosphorylated STAT3‐mediated pathway. However, immune‐cell‐specific AHR activity is necessary for normal expression of REG3G in all regions of the small intestine. A diet rich in broccoli, capable of inducing AHR activity, increases REG3G production when compared to a semi‐purified diet that is devoid of ligands that can potentially activate the AHR, thus highlighting the importance of AHR in antimicrobial function. Overall, these data suggest that homeostatic antimicrobial REG3G production is increased by an AHR pathway intrinsic to the immune cells in the small intestine.