Abstract
Background
Intestinal immune homeostasis is maintained by the interplay between microbiota and the mucosal immune system. Changes in gut microbiota have been associated with chronic intestinal conditions, such as inflammatory bowel disease (IBD). The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by dietary and environmental stimuli to control immune responses in the gut and homeostatic mechanisms at mucosal surfaces. In IBD, AhR expression is downregulated. Major agonists of AhR in the gut include microbial tryptophan metabolites such as indole derivatives, which are decreased in IBD patients. The mechanisms involved in tryptophan metabolism by bacteria and their implications in AhR activation and thus IBD pathogenesis are not well understood.
Aims
To investigate whether tryptophan metabolism by intestinal bacteria participates in AhR activation and IBD pathogenesis.
Methods
Microbiota profiles (16S rRNA Illumina) and activation of AhR (luciferase reporter assay) were determined in fecal samples from IBD patients (n=10) and healthy volunteers (n=10). Germ-free C57BL/6 mice were colonized with fecal slurries of 2 healthy subjects and 4 IBD patients (n=4 mice/donor) by oral gavage (humanized-mice). All mice were fed irradiated tryptophan diets with 0.1% or 1% tryptophan content for 14 days. Simultaneously, SPF Mucin 2 (Muc2) deficient mice (C57BL/6 background), which develop colitis spontaneously, were fed tryptophan diets (0.1%, 0.3% and 1% content). Activation of AhR was measured in feces using an AhR luciferase reporter assay. Inflammation was determined by immunohistochemistry and the characterization of immune infiltrate in colon cross-sections. Bacteria from human and mouse fecal samples were isolated and screened for their ability to produce indoles using biochemical reagents. Positive bacteria were identified by colony PCR and 16S rRNA Sanger sequencing.
Results
IBD patients had an altered fecal microbiota with a lower capacity to activate AhR compared to healthy subjects. Colonization of mice with microbiota from healthy subjects induced greater activation of AhR compared to mice colonized with microbiota from patients with IBD. Furthermore, increasing dietary tryptophan composition rescued the capacity to activate AhR. In Muc2 deficient mice, dietary tryptophan treatment enhanced AhR activation capacity and reduced infiltration of innate immune cells before the onset of colitis. Several AhR agonist producing bacterial species were identified and will be used in future experiments.
Conclusions
Activation of AhR is dependent on the gut microbiota and disease status of the donor. Dietary intervention with tryptophan enhances AhR activation capacity and may be a potential therapeutic avenue in IBD individuals with intestinal dysbiosis.
Funding Agencies
Farncombe Family Digestive Health Research Institute, Biocodex Microbiota Foundation