Polyphenols and Tryptophan Metabolites Activate the Aryl Hydrocarbon Receptor in an in vitro Model of Colonic Fermentation

Scope: Many dietary phytochemicals have been reported to promote gut health. Specific dietary phytochemicals, such as luteolin, as well as specific microbial metabolites of tryptophan are ligands of the aryl hydrocarbon receptor (AhR), which plays a role in immunity and homeostasis of the gut barrier. Here, the fate of luteolin during colonic fermentation and the contribution of tryptophan metabolites to AhR activity in different parts of the colon are investigated. Methods and results: Several polyphenols are screened for AhR activation and oregano, containing the ligand luteolin, is added to batch cultures of human microbiota from the distal colon. Luteolin is rapidly metabolized, with no measurable increase in AhR activity. In the second experiment, using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), not all luteolin is metabolized in the ascending colon, but disappear rapidly in the transverse colon. The greatest AhR activity is due to microbiota-derived metabolites of tryptophan, particularly in the descending colon. Conclusions: Luteolin in food is rapidly metabolized in the transverse colon. Tryptophan metabolism by the microbiota in the colon contributes substantially to the pool of lumen metabolites that can activate the AhR.</p

Coffee induces AHR- and Nrf2-mediated transcription in intestinal epithelial cells

Coffee induces a health-promoting adaptive response of cells in the body. Here, we investigated enterocyte responses to AHR agonists in coffee and measured their transport across a polarized intestinal epithelium. AHR-activating potencies of Turkish, filter, and instant coffee were determined using DR CALUX® bioassay, before and after intestinal metabolization by Caco-2 cells. Furthermore, effects of coffee on induction of AHR- and Nrf2-pathway genes in Caco-2 cells were evaluated by real-time qPCR. Coffee samples showed considerable AHR-activating potencies in DR CALUX® bioassay (up to 79% of positive control activity). After incubation with Caco-2 cells, AHR activity of different coffees was between 35 and 64% of their initial value, suggesting rapid uptake and metabolization by epithelial cells. Expression of AHR-regulated gene CYP1A1 increased up to 41-fold and most Nrf2-pathway genes were up-regulated by coffee. This in vitro study may support the notion that coffee bioactives contribute to antioxidant defense and detoxification processes in vivo

Tryptophan Supplementation Increases the Production of Microbial-Derived AhR Agonists in an in Vitro Simulator of Intestinal Microbial Ecosystem

The aryl hydrocarbon receptor (AhR) plays an important role in intestinal homeostasis, and some microbial metabolites of tryptophan are known AhR agonists. In this study, we assessed the impact of tryptophan supplementation on the formation of tryptophan metabolites, AhR activation, and microbiota composition in the simulator of the human intestinal microbial ecosystem (SHIME). AhR activation, microbial composition, and tryptophan metabolites were compared during high tryptophan supplementation (4 g/L tryptophan), control, and wash-out periods. During tryptophan supplementation, the concentration of several tryptophan metabolites was increased compared to the control and wash-out period, but AhR activation by fermenter supernatant was significantly decreased. This was due to the higher levels of tryptophan, which was found to be an antagonist of AhR signaling. Tryptophan supplementation induced most microbial changes in the transverse colon including increased relative abundance of lactobacillus. We conclude that tryptophan supplementation leads to increased formation of AhR agonists in the colon

Effects of undigested protein-rich ingredients on polarised small intestinal organoid monolayers

Here, we describe the use of monolayers of intestinal epithelial cells derived from intestinal organoids and transcriptomics to investigate the direct effects of dietary protein sources on epithelial function. Mechanically dissociated 3D organoids of mouse duodenum were used to generate a polarized epithelium containing all cell types found in the tissue of origin. The organoid-derived cell monolayers were exposed to 4% (w/v) of 'undigested (non-hydrolysed)-soluble' fraction of protein sources used as feed ingredients [soybean meal (SBM) and casein], or alternative protein sources (spray dried plasma protein, and yellow meal worm), or controls for 6 h prior to RNA isolation and transcriptomics. All protein sources altered expression of unique biological processes in the epithelial cells. Exposure of intestinal organoids to SBM downregulated expression of retinol and retinoid metabolic processes as well as cholesterol and lipid biosynthetic pathways, consistent with the reported hypotriglyceridaemic effect of soy protein in vivo. These findings support the use of intestinal organoids as models to evaluate complex interactions between dietary ingredients and the intestinal epithelium and highlights some unique host effects of alternative protein sources in animal feed and potentially human food. Graphical abstract: Schematic representation of the study. 3-dimensional organoids were generated from mouse duodenum (1). The organoids were subsequently dissociated into single cells (2) and grown as 2-dimensional polarised monolayers (3). Polarized monolayers of organoid cells were exposed to different protein sources [CAS, SBM, SDPP, YMW, or medium control (MC)] for 6 h (4) and further processed for imaging (5) gene expression (6), and biochemical assays (7), to investigate the effects of undigested protein sources on the duodenal epithelium. [Figure not available: See fulltext.]</p

Aryl hydrocarbon receptor ligand production by the gut microbiota is decreased in celiac disease leading to intestinal inflammation

Metabolism of tryptophan by the gut microbiota into derivatives that activate the aryl hydrocarbon receptor (AhR) contributes to intestinal homeostasis. Many chronic inflammatory conditions, including celiac disease involving a loss of tolerance to dietary gluten, are influenced by cues from the gut microbiota. We investigated whether AhR ligand production by the gut microbiota could influence gluten immunopathology in nonobese diabetic (NOD) mice expressing DQ8, a celiac disease susceptibility gene. NOD/DQ8 mice, exposed or not exposed to gluten, were subjected to three interventions directed at enhancing AhR pathway activation. These included a high-tryptophan diet, gavage with Lactobacillus reuteri that produces AhR ligands or treatment with an AhR agonist. We investigated intestinal permeability, gut microbiota composition determined by 16S rRNA gene sequencing, AhR pathway activation in intestinal contents, and small intestinal pathology and inflammatory markers. In NOD/ DQ8 mice, a high-tryptophan diet modulated gut microbiota composition and enhanced AhR ligand production. AhR pathway activation by an enriched tryptophan diet, treatment with the AhR ligand producer L. reuteri, or pharmacological stimulation using 6-formylindolo (3,2-b) carbazole (Ficz) decreased immunopathology in NOD/ DQ8 mice exposed to gluten. We then determined AhR ligand production by the fecal microbiota and AhR activation in patients with active celiac disease compared to nonceliac control individuals. Patients with active celiac disease demonstrated reduced AhR ligand production and lower intestinal AhR pathway activation. These results highlight gut microbiota-dependent modulation of the AhR pathway in celiac disease and suggest a new therapeutic strategy for treating this disorder.</p