Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles

Abstract

Ligand activation of the aryl hydrocarbon (AHR) has profound effects upon the immunological status of the gastrointestinal tract, establishing and maintaining signaling networks, which facilitate host-microbe homeostasis at the mucosal interface. However, the identity of the ligand(s) responsible for such AHR-mediated activation within the gut remains to be firmly established. Here, we combine in vitro ligand binding, quantitative gene expression, protein-DNA interaction and ligand structure activity analyses together with in silico modeling of the AHR ligand binding domain to identify indole, a microbial tryptophan metabolite, as a human-AHR selective agonist. Human AHR, acting as a host indole receptor may exhibit a unique bimolecular (2:1) binding stoichiometry not observed with typical AHR ligands. Such bimolecular indole-mediated activation of the human AHR within the gastrointestinal tract may provide a foundation for inter-kingdom signaling between the enteric microflora and the immune system to promote commensalism within the gut.

Figures

Figure 1. Indole dose-response assessment of AHR-dependent activity.

(A) HepG2 (40/6) cells and (B) Hepa 1.1 cells were treated as indicated for 4 h; cells were lysed, and luciferase activity was measured.

Figure 2. Indole stimulates AHR-target gene expression.

(A) Expression of AHR-responsive CYP1A1, (B) CYP1B1, and (C) AHR within Caco2 cells was determined through qPCR analysis following 4 h of treatment with vehicle, TCDD (10 nM), or indole (IND) at the indicated dose. (D) The mean CYP1A1 enzymatic activity was measured in Caco2 cells following 12 h treatment with DMSO, TCDD (10 nM), or Indole (100 μM) and 3 h incubation with luciferin-CEE reagent. (E) IL6 expression within Caco2 cells was determined by qPCR following 4 h treatment with indole (20 μM) with or without the addition of IL1B (10 ng/mL), AHR dependence was evaluated by 1 h antagonist pretreatment using GNF 351 (200 nM). (F) IL6 secretion by Caco2 cells was determined by ELISA following 24 h treatment with vehicle, TCDD (10 nM) or Indole (100 μM), with or without the addition of IL1B (10 ng/mL). (G) Cyp1a1 gene expression within isolated peritoneal macrophages from C57BL6 and AHR humanized mice were evaluated by qPCR following indicated treatment of 4 h.

Figure 3. Indole is a human specific AHR ligand.

Photoaffinity ligand binding competition assay in which increasing amounts of βNF and indole were added to hAHR or mAHR liver cytosol in combination with a fixed amount of the photoaffinity ligand to evaluate relative competition of indole within the ligand binding pocket of AHR between species. Higher concentrations of competing ligand were not tested as concentrations above 10 μM can yield non-specific competition.

Figure 4. Indole facilitates human specific AHR nuclear localization and DRE binding capacity.

(A) Nuclear translocation of AHR was determined following indicated treatment (1 h) in HepG2 (human) and Hepa1 (mouse) cell lines via western blot analysis. Relative quantification of AHR (normalized to β-actin or Lamin A/C) was determined via Phosphoimager and OptiQuant software, and presented as digitized light units (DLU). (B) In vitro translated hAHR/ARNT gel shift assay displaying treatment capacity to transform hAHR or mAHR to AHR/ARNT/DNA complex.

Figure 5. Ligand specificity of hAHR for microbiota-derived indoles.

HepG2 (40/6) cells were treated as indicated for 4 h; cells were lysed, and luciferase activity was measured.

Figure 6. Methyl-indole isomers exhibit differential capacity to mediate AHR activity.

(A) HepG2 (40/6) cells were treated as indicated for 4 h; cells were lysed, and luciferase activity was measured. (B) Expression of AHR-responsive CYP1A1 and CYP1B1 within Caco2 cells was determined through qPCR analysis following 4 h of treatment with vehicle, indole (IND), 3-methyl indole (3-MI), 2-methyl indole (2-MI), or 1-methyl indole (1-MI) at a concentration of 20 μM. (C) The mean CYP1A1 enzymatic activity was measured in Caco2 cells following 12 h treatment with DMSO, TCDD (10 nM), or indole/methyl indole isomers (100 μM) and 3 h incubation with luciferin-CEE reagent. (D) Synergistic IL6 expression within Caco2 cells was determined by qPCR following 4 h treatment with vehicle, TCDD (10 nM), or indole/methyl indole isomers (20 μM) with or without the addition of IL1B (10 ng/mL). IL6 secretion by Caco2 cells was determined by ELISA following 24 h treatment with vehicle, TCDD (10 nM) or indole/ methyl indole isomers (100 μM) with or without the addition of IL1B (10 ng/mL). (E) In vitro translated AHR/ARNT gel shift assay displaying treatment capacity to transform human AHR to AHR/ARNT/DNA complex.

Figure 7. In silico modeling of AHR ligand binding domain.

Homology modeling of indirubin optimized ligand binding in (A) hAHR and (B) mAHR. The predicted two indole-binding model in (C) hAHR and (D) mAHR ligand binding domain. The predicted two 3-methyl indole-binding models in (E) hAHR and (F) mAHR ligand binding domain. Blue shading indicates the space-filling volume of the ligand binding pocket.