Oral tryptophan activates duodenal aryl hydrocarbon receptor in healthy subjects: a crossover randomized controlled trial.

Tryptophan is an essential amino acid transformed by host and gut microbial enzymes into metabolites that regulate mucosal homeostasis through aryl hydrocarbon receptor (AhR) activation. Alteration of tryptophan metabolism has been associated with chronic inflammation; however, whether tryptophan supplementation affects the metabolite repertoire and AhR activation under physiological conditions in humans is unknown. We performed a randomized, double blind, placebo-controlled, crossover study in 20 healthy volunteers. Subjects on a low tryptophan background diet were randomly assigned to a 3-wk l-tryptophan supplementation (3 g/day) or placebo, and after a 2-wk washout switched to opposite interventions. We assessed gastrointestinal and psychological symptoms by validated questionnaires, AhR activation by cell reporter assay, tryptophan metabolites by liquid chromatography and high-resolution mass spectrometry, cytokine production in isolated monocytes by ELISA, and microbiota profile by 16S rRNA Illumina technique. Oral tryptophan supplementation was well tolerated, with no changes in gastrointestinal or psychological scores. Compared with placebo, tryptophan increased AhR activation capacity by duodenal contents, but not by feces. This was paralleled by higher urinary and plasma kynurenine metabolites and indoles. Tryptophan had a modest impact on fecal microbiome profiles and no significant effect on cytokine production. At the doses used in this study, oral tryptophan supplementation in humans induces microbial indole and host kynurenine metabolic pathways in the small intestine, known to be immunomodulatory. The results should prompt tryptophan intervention strategies in inflammatory conditions of the small intestine where the AhR pathway is impaired.NEW & NOTEWORTHY We demonstrate that in healthy subjects, orally administered tryptophan activates microbial indole and host kynurenine pathways in the small intestine, the primary metabolic site for dietary components, and the richest source of immune cells along the gut. This study provides novel insights in how to optimally activate immunomodulatory AhR pathways and indole metabolism in the small intestine, serving as basis for future therapeutic trials using l-tryptophan supplementation in chronic inflammatory conditions affecting the small intestine.

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.

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.

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

ABSTRACT: 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.

Aryl hydrocarbon Receptor activation during in vitro and in vivo digestion of raw and cooked broccoli (brassica oleracea var. Italica).

Broccoli is rich in glucosinolates, which can be converted upon chewing and processing into Aryl hydrocarbon Receptor (AhR) ligands. Activation of AhR plays an important role in overall gut homeostasis but the role of broccoli processing on the generation of AhR ligands is still largely unknown. In this study, the effects of temperature, cooking method (steaming versus boiling), gastric pH and further digestion of broccoli on AhR activation were investigated in vitro and in ileostomy subjects. For the in vitro study, raw, steamed (t = 3 min and t = 6 min) and boiled (t = 3 min and t = 6 min) broccoli were digested in vitro with different gastric pH. In the in vivo ileostomy study, 8 subjects received a broccoli soup or a broccoli soup plus an exogenous myrosinase source. AhR activation was measured in both in vitro and in vivo samples by using HepG2-Lucia™ AhR reporter cells. Cooking broccoli reduced the AhR activation measured after gastric digestion in vitro, but no effect of gastric pH was found. Indole AhR ligands were not detected or detected at very low levels both after intestinal in vitro digestion and in the ileostomy patient samples, which resulted in no AhR activation. This suggests that the evaluation of the relevance of glucosinolates for AhR modulation in the gut cannot prescind from the way broccoli is processed, and that broccoli consumption does not necessarily produce substantial amounts of AhR ligands in the large intestine.

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.

Identification of Commensal Species Positively Correlated with Early Stress Responses to a Compromised Mucus Barrier.

BACKGROUND: Our aims were (1) to correlate changes in the microbiota to intestinal gene expression before and during the development of colitis in Muc2 mice and (2) to investigate whether the heterozygote Muc2 mouse would reveal host markers of gut barrier stress. METHODS: Colon histology, transcriptomics, and microbiota profiling of faecal samples was performed on wild type, Muc2, and Muc2 mice at 2, 4, and 8 weeks of age. RESULTS: Muc2 mice develop colitis in proximal colon after weaning, resulting in inflammatory and adaptive immune responses, and expression of genes associated with human inflammatory bowel disease. Muc2 mice do not develop colitis, but produce a thinner mucus layer. The transcriptome of Muc2 mice revealed differential expression of genes participating in mucosal stress responses and exacerbation of a transient inflammatory state around the time of weaning. Young wild type and Muc2 mice have a more constrained group of bacteria as compared with the Muc2 mice, but at 8 weeks the microbiota composition is more similar in all mice. At all ages, microbiota composition discriminated the groups of mice according to their genotype. Specific bacterial clusters correlated with altered gene expression responses to stress and bacteria, before colitis development, including colitogenic members of the genus Bacteroides. CONCLUSIONS: The abundance of Bacteroides pathobionts increased before histological signs of pathology suggesting they may play a role in triggering the development of colitis. The Muc2 mouse produces a thinner mucus layer and can be used to study mucus barrier stress in the absence of colitis.

IL-22-STAT3 pathway plays a key role in the maintenance of ileal homeostasis in mice lacking secreted mucus barrier.

BACKGROUND: Muc2-deficient mice show no signs of ileal pathology but the mechanisms remained unknown. METHODS: Wild-type (WT), Muc2, and Muc2 mice were killed at 2, 4, and 8 weeks of age. Total RNA from ileum was used for full genome transcriptome analysis and qPCR. Microbiota composition was determined using a mouse intestinal chip (MITChip). Morphological and immunohistological studies were performed on segments of ileum. RESULTS: The ileum was colonized by more diverse microbiota in young (week 4) WT than in Muc2 mice, and composition was influenced by genotype. Weaning was associated with major changes in the transcriptome of all mice, and the highest number of differentially expressed genes compared with adults, reflecting temporal changes in microbiota. Although the spatial compartmentalization of bacteria was compromised in Muc2 mice, gene set enrichment analysis revealed a downregulation of Toll-like receptor, immune, and chemokine signaling pathways compared to WT mice. The predicted effects of enhanced IL-22 signaling were identified in the Muc2 transcriptome as the upregulation of epithelial cell proliferation altered expression of mitosis and cell-cycle control pathways. This is consistent with increased villus length and number of Ki67 epithelial cells in Muc2 mice. Additionally, expression of the network of IL-22 regulated defense genes, including Fut2, Reg3ß, Reg3γ, Relmb, and the Defensin Defb46 were increased in Muc2 mice. CONCLUSIONS: These findings highlight a role for the IL-22-STAT3 pathway in maintaining ileal homeostasis when the mucus barrier is compromised and its potential as a target for novel therapeutic strategies in inflammatory bowel disease.

Mucin Muc2 deficiency and weaning influences the expression of the innate defense genes Reg3ß, Reg3γ and angiogenin-4.

BACKGROUND: Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2(-/-)) on the expression of regenerating islet-derived protein 3 beta (Reg3ß), regenerating islet-derived protein 3 gamma (Reg3γ), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. METHODS: Intestinal tissues of Muc2(-/-) and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3ß, Reg3γ, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. RESULTS: Reg3ß and Reg3γ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3ß, Reg3γ, and Ang4 differed between WT and Muc2(-/-) mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3ß and Reg3γ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2(-/-) mice at P28, where and when expression levels of Reg3ß, Reg3γ, and Ang4 were the lowest. CONCLUSIONS: Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3ß and Reg3γ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2(-/-) mice. The inverse correlation between colitis development and Reg3ß, Reg3γ, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammation.

Intestinally secreted C-type lectin Reg3b attenuates salmonellosis but not listeriosis in mice.

The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b(-/-)) mice and wild-type (WT) mice were orally infected with gram-negative Salmonella enteritidis or gram-positive Listeria monocytogenes. At day 2 after oral Listeria infection and at day 4 after oral Salmonella infection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition, ex vivo binding of ileal mucosal Reg3b to Listeria and Salmonella was investigated. Whereas recovery of Salmonella or Listeria from feces of Reg3b(-/-) mice did not differ from that from feces of WT mice, significantly higher numbers of viable Salmonella, but not Listeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b(-/-) mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the gram-negative bacterium S. enteritidis in mice but not against the gram-positive bacterium L. monocytogenes.

The role of innate signaling in the homeostasis of tolerance and immunity in the intestine.

In the intestine innate recognition of microbes is achieved through pattern recognition receptor (PRR) families expressed in immune cells and different cell lineages of the intestinal epithelium. Toll-like receptor (TLR) and nucleotide-binding and oligomerization domain-like receptor (NLR) families are emerging as key mediators of immunity through their role as maturation factors of immune cells and triggers for the production of cytokines and chemokines and antimicrobial factors. At the mucosal surface chronic activation of the immune system is avoided through the epithelial production of a glycocalyx, steady-state production of antimicrobial factors as well as the selective expression and localization of PRRs. Additionally, the polarization of epithelial TLR signaling and suppression of NF-kappaB activation by luminal commensals appears to contribute to the homeostasis of tolerance and immunity. Several studies have demonstrated that TLR signaling in epithelial cells contributes to a range of homeostatic mechanisms including proliferation, wound healing, epithelial integrity, and regulation of mucosal immune functions. The intestinal epithelium appears to have uniquely evolved to maintain mucosal tolerance and immunity, and future efforts to further understand the molecular mechanisms of intestinal homeostasis may have a major impact on human health.