Butyrate acts through HDAC inhibition to enhance aryl hydrocarbon receptor activation by gut microbiota-derived ligands

Abstract

Aryl hydrocarbon receptor (AhR) is a critical player in the crosstalk between the gut microbiota and its host. However, factors regulating AhR within the gut, which is a complex metabolomic environment, are poorly understood. This study investigates the effect of a combination of metabolites on the activation mechanism of AhR. AhR activity was evaluated using both a luciferase reporter system and mRNA levels of AhR target genes on human cell lines and human colonic explants. AhR activation was studied by radioligand-binding assay, nuclear translocation of AhR by immuofluorescence and protein co-immunoprecipitation of AhR with ARNT. Indirect activation of AhR was evaluated using several tests and inhibitors. The promoter of the target gene CYP1A1 was studied both by chromatin immunoprecipitation and by using an histone deacetylase HDAC inhibitor (iHDAC). Short-chain fatty acids, and butyrate in particular, enhance AhR activity mediated by endogenous tryptophan metabolites without binding to the receptor. This effect was confirmed in human intestinal explants and did not rely on activation of receptors targeted by SCFAs, inhibition of AhR degradation or clearance of its ligands. Butyrate acted directly on AhR target gene promoter to reshape chromatin through iHDAC activity. Our findings revealed that butyrate is not an AhR ligand but acts as iHDAC leading to an increase recruitment of AhR to the target gene promoter in the presence of tryptophan-derived AhR agonists. These data contribute to a novel understanding of the complex regulation of AhR activation by gut microbiota-derived metabolites.

Keywords: AhR; CYP1A1; FICZ; HDAC; Microbiota; SCFAs; butyrate; metabolites; tryptophan.

Figure 1.

Several gut microbiota-dependent metabolites activate AhR. (a) HepG2^luc were treated in triplicate for 24 hours with differents doses of metabolites (1 µM – 10 mM). Results were normalized on the basis of negative luciferase activity of the control (unstimulated cells, dotted line) and cytotoxicity measurement and doses with the highest AhR activity without cell mortality are shown. Data are representative of two independent experiments. (b) HT29^luc were treated in triplicate for 24 hours with the metabolites having the highest AhR activity on HepG2^luc. Doses with the highest AhR activity without cell mortality are shown. (c) AhR activity in HepG2^luc without (white) or with 10% FBS (gray) after 24 hours. Cells were treated in triplicate and data are presented as mean ± SEM of two independent experiments. (d) Heat map representation of AhR activation in HepG2^luc treated in triplicate with Trp metabolites and SCFAs (1 µM – 1 mM) with or without 10% FBS. Means RLU obtained in two independent experiments were represented and relative color graduation was attributed using Morpheus software (https://software.broadinstitute.org/morpheus). (e) AhR activity obtained in HepG2^luc treated in triplicate with butyrate (1 and 10 mM) without (white) or with (gray) 10% FBS. Data are presented as mean ± SEM of two independent experiments. *p < .05; **p < .01; ***p < .001; ****p < .0001.

Figure 2.

Butyrate and AhR ligands synergize to activate the AhR pathway. (a-b) AhR activity in HepG2^luc treated in triplicate with butyrate (10 mM) in combination with FICZ(10 and 100 ng/ml)(a) or IAA (0.1 and 1 mM) (b) in FBS-free media. Data are presented as mean ± SEM of three independent experiments. (c-d) RT-qPCR analysis of CYP1A1 mRNA levels in HepG2 (c) and HT29 (d) treated with butyrate and/or FICZ for 8 and 6 hours respectively. (e-f) RT-qPCR analysis of AHRR mRNA levels in HepG2 (e) and HT29 (f) treated with butyrate and/or FICZ for 8 and 6 hours respectively. Data are presented as mean ± SEM of two independent experiments. (g-h) RT-qPCR analysis of CYP1A1 mRNA levels in colonic explants treated with butyrate (0.1 and 0.01 mM) in combination with FICZ 10 ng/ml (g) or 100 ng/ml (h) for 24 hours. Data are presented as mean ± SEM and each dot represent a patient. *p < .05, **p < .01, ***p < .001, ****p < .0001.

Figure 3.

Butyrate is not an AhR ligand. (a-b) Ligand binding to AhR was evaluated for SCFAs (a) and the combination of butyrate with FICZ (b). Data are presented as mean ± SEM of three independent experiments. (c) Nuclear translocation of AhR in LS180 cells treated with vehicle (DMSO), butyrate, FICZ and FICZ combined with butyrate. (d) Semi-quantitative estimation of nuclear translocation was expressed as a percentage of intensity of cytoplasm fluorescence. (e) Formation of AhR-ARNT heterodimers in LS180 treated for 90 min with TCDD, (10 nM), butyrate (10 mM), FICZ (10 nM) and combination of butyrate with FICZ. * p < .05, **p < .01, ***p < .001.

Figure 4.

Butyrate acts through its iHDAC activity to activate AhR in synergie with AhR ligands. (a) AhR activity in HepG2^luc treated in triplicate with butyrate (1 mM), FICZ (10 ng/ml), TSA (100 nM) or the combination of FICZ with butyrate or TSA in a FBS-free media. Data are presented as mean ± SEM of three independent experiments. (b) AhR binding to the promoter region of the CYP1A1 gene detected by ChIP-qPCR in HepG2 treated with DMSO, FICZ and butyrate at several time points. (c) Synergistic recruitment of AhR to the CYP1A1 promoter mediated by butyrate in combination with FICZ. Only experiments with an enrichment greater than or equal to 1.5 for FICZ were retained. Data are presented as mean ± SEM and each dot represent an independent experiment. (d) RT-qPCR analysis of CYP1A1 mRNA levels in HepG2 treated with butyrate (1 mM), FICZ (10 ng/ml), C646 (10 µM) or the combination of FICZ with butyrate or C646 for 8 hours. Data are presented as mean ± SEM of two independent experiment. **p < .01; ***p < .001; ****p < .0001.