Mixture Effects of Tryptophan Intestinal Microbial Metabolites on Aryl Hydrocarbon Receptor Activity

Abstract

Aryl hydrocarbon receptor (AHR) plays pivotal roles in intestinal physiology and pathophysiology. Intestinal AHR is activated by numerous dietary, endogenous, and microbial ligands. Whereas the effects of individual compounds on AHR are mostly known, the effects of real physiological mixtures occurring in the intestine have not been studied. Using reporter gene assays and RT-PCR, we evaluated the combinatorial effects (3520 combinations) of 11 microbial catabolites of tryptophan (MICTs) on AHR. We robustly (n = 30) determined the potencies and relative efficacies of single MICTs. Synergistic effects of MICT binary mixtures were observed between low- or medium-efficacy agonists, in particular for combinations of indole-3-propionate and indole-3-lactate. Combinations comprising highly efficacious agonists such as indole-3-pyruvate displayed rather antagonist effects, caused by saturation of the assay response. These synergistic effects were confirmed by RT-PCR as CYP1A1 mRNA expression. We also tested mimic multicomponent and binary mixtures of MICTs, prepared based on the metabolomic analyses of human feces and colonoscopy aspirates, respectively. In this case, AHR responsiveness did not correlate with type of diet or health status, and the indole concentrations in the mixtures were determinative of gross AHR activity. Future systematic research on the synergistic activation of AHR by microbial metabolites and other ligands is needed.

Keywords: aryl hydrocarbon receptor; indole derivatives; microbiome; mimic mixtures; tryptophan metabolites.

Figure 1

The effect of tryptophan metabolites on the cell viability and the activity of nuclear receptors. Cells were treated for 24 h with the tested compounds at concentrations ranging from 1 nM to 200 µM (indole from 10 µM to 10 mM). (A) Cell viability in LS174T and AZ-AHR cells. The MTT, NR, and LDH assays were performed as described in the experimental section. Data are the means from at least three consecutive cell passages and are expressed as a percentage of the viability (MTT) or cytotoxicity (NR, LDH) of the control cells. (B) The activity of VDR, GR, PPARγ, and AR receptors in stably transfected reporter cell lines. A luciferase assay was performed as described in the experimental section. Vehicle DMSO (0.1% v/v) was used as a negative control, and VD3 (75 nM), DEX (100 nM), 15d-PGDJ (40 µM), or DHT (100 nM) as positive controls, respectively. Data are the means from at least three consecutive cell passages and the effects of tested compounds are expressed as a % of induction by the positive control.

Figure 2

High-throughput screening of AHR transcriptional activity by tryptophan microbial catabolites (MICT). AZ-AHR cells were incubated for 4 h with vehicle (DMSO; 0.1% v/v), TCDD (5 nM), or increasing concentrations of the tested compounds. Following the treatments, cells were lysed, and luciferase activity was measured. Panel (A): The column scatter plot represents the values of the half-maximal effective concentrations (potency; EC₅₀) and relative efficacies (a ratio of luciferase activity by MICT in highest concentration/luciferase activity by TCDD). Panel (B): Dose–response assessment of AHR-dependent activity. Data are expressed as the fold induction of luciferase activity over control cells and are the mean ± SD from approximately 30 consecutive cell passages. The fold induction of TCDD was 200 ± 105 (n = 165). All values are statistically significant (p < 0.01) in comparison to vehicle-treated cells.

Figure 3

Heat-map of combination index (CI) values from high-throughput screening of the MICT binary mixtures at the AHR receptor. AZ-AHR gene reporter cells were incubated for 4 h with vehicle (DMSO; 0.1% v/v), TCDD as a positive control, and increasing concentrations of MICTs alone or as binary mixtures, one with each other in the entire concentration range. Following the treatments, cells were lysed, and luciferase activity was measured as described in the experimental section. The experiments were performed in technical duplicates and three biological replicates at least. The effect of MICTs in binary mixtures on the AHR transcriptional activity was analyzed by calculations based on the Chou-Talalay method and using the CalcuSyn software. The effects of the combined treatments are represented as a combination index (CI) and are color coded (green indicates high synergy and blue indicates no synergy).

Figure 4

The induction of CYP1A1 by selected MICTs and their binary combinations. (A) Type I synergism, where the efficacy of the binary mixture is higher than the effect of each compound alone; (B) Type II synergism, where the efficacy of the binary mixture is higher than the sum of the efficacies of two single compounds. AZ-AHR cells were incubated with vehicle (DMSO; 0.2% v/v), TCDD (20 nM), or MICTs alone or in binary combinations for 4 h. The levels of CYP1A1 mRNA were determined by qRT-PCR and are expressed as a percent of induction by TCDD. Each dot represents one independent experiment. The experiments were performed in technical triplicates and three biological replicates at least.

Figure 5

Effect of mimic mixtures of MICTs on AHR activity. LS174T-AHR-luc cells were incubated for 4 h with vehicle (DMSO; 0.2% v/v), TCDD (20 nM), and MICTs alone or in the mimic mixture. The concentration of each compound equals the observed mean quantified in human fecal samples [30]. The effects of tested compounds are expressed as a percent of induction of luciferase activity by TCDD over control cells. The experiments were performed in technical quadruplicates and three biological replicates. The error bars represent the mean ± SD, * p < 0.1, ** p < 0.01.

Figure 6

AHR activation by mimic binary mixtures of IND and IPA. LS174T-AHR-luc cells were incubated for 4 h with vehicle (DMSO; 0.2% v/v), TCDD (20 nM), and the tested compounds alone or in the mixture. Mimic binary mixtures were prepared according to the ratio of IND and IPA that equals the concentration detected in individual aspirates of colonoscopy patients. The experiments were performed in technical quadruplicates and three biological replicates.