Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells

The ligand activated transcription factor, aryl hydrocarbon receptor (AhR) emerged as a critical regulator of immune and metabolic processes in the gastrointestinal tract. In the gut, a main source of AhR ligands derives from commensal bacteria. However, many of the reported microbiota-derived ligands have been restricted to indolyl metabolites. Here, by screening commensal bacteria supernatants on an AhR reporter system expressed in human intestinal epithelial cell line (IEC), we found that the short chain fatty acid (SCFA) butyrate induced AhR activity and the transcription of AhR-dependent genes in IECs. We showed that AhR ligand antagonists reduced the effects of butyrate on IEC suggesting that butyrate could act as a ligand of AhR, which was supported by the nuclear translocation of AhR induced by butyrate and in silico structural modelling. In conclusion, our findings suggest that (i) butyrate activates AhR pathway and AhR-dependent genes in human intestinal epithelial cell-lines (ii) butyrate is a potential ligand for AhR which is an original mechanism of gene regulation by SCFA.

Scientific RepoRts | (2019) 9:643 | DOI: 10.1038/s41598-018-37019-2 peptides such as RegIIIγ and the expression of tight junctions molecules in IECs thus reinforcing the barrier and defence functions 13,14 . Interestingly, AhR signalling and known AhR ligands are low in inflammatory bowel diseases (IBD) patients, highlighting the clinical relevance of the AhR pathway in these pathologies 15,16 . AhR activation by ligand administration and AhR knock-down in mouse suppressed and enhanced DSS-induced colitis, respectively suggesting a beneficial effect of AhR activation in IBD 17,18 . An increasing number of bacterial metabolites have been shown to drive the AhR activation, with described protective effects against intestinal inflammation and pathogens colonisations, suggesting a possible role of this signalling pathway in the intestinal homeostasis 7 . This hypothesis has been further supported by a recent study showing that one IBD-associated single nucleotide polymorphism (SNP) within the CARD9 gene, affects microbiota composition thus altering the production of bacterial AhR ligands and consequently intestinal inflammation 15 .
The relevant role of AhR in the maintenance of mucosal homeostasis has been largely focused on immune cells from the lamina propria, and the impact of AhR activation in intestinal epithelial cells (IEC) has only been starting to be unravelled. Emerging evidences highlight that AhR activation impacts also IECs responses contributing to intestinal homeostasis. AhR activation in IECs protects mice from colitis by enhancing IECs barrier functions via the increase of the IL10 receptor expression and the enhancement of tight junctions integrity through the regulation of Notch1 19,20 . Importantly, the excess of AhR ligands in the gut is detrimental to the host. In this context, a recent study in mice model nicely demonstrates that IECs play the role of gatekeeper via the expression of the AhR-regulated gene Cyp1a1 encoding cytochrome P450 1A1 involved in AhR ligands clearance 21,22 . These studies highlighted the importance of AhR ligands in modulating host gut immune homeostasis and prompted us to identify new microbiota-derived activators of the AhR pathway in IECs. We thus tested the bacterial supernatants of over 100 bacterial species of the human microbiota on an AhR reporter system in human intestinal cell lines and found that butyrate-producing bacteria activate the AhR-dependent pathway. A recent work previously highlighted the ability of butyrate to enhance the expression of AhR-dependent genes through its histone deacetylase inhibitor (HDACi) properties 23 . We confirmed the butyrate-activating role at the transcriptional level on AhR-dependent genes in Caco-2 and HT-29 cell lines. Interestingly, other HDACi that could enhance AhR-dependent gene expression were unable to mimic the butyrate dependent-activation of the AhR reporter system, suggesting the existence of a second mechanism. We showed that (i) AhR ligand antagonists impaired the butyrate-induced activation of AhR reporter system, (ii) butyrate enhanced AhR nuclear translocation and (iii) in silico modelling of butyrate interaction with human AhR, highlighting for the first time that butyrate could act as a ligand of AhR.

Results
Metabolites derived from commensal bacteria enhanced AhR activity. In the gut, Aryl hydrocarbon receptor (AhR) ligands derive from diverse origins that include the intestinal microbiota as one of the main sources. To decipher which bacteria from human gut activate the AhR pathway, we performed a screening of commensal bacteria on a human intestinal epithelial cell line (HT-29-AhR) stably expressing an AhR-dependant reporter system. The AhR reporter system contains three copies of the DNA-binding domain motif recognized by AhR (xenobiotic-responsive element; XRE) driving the transcription of the luciferase reporter gene. Since AhR activators produced by bacteria are secreted in the intestinal lumen, we performed the screening of bacterial culture supernatants 8 . The screening included 132 bacterial strains, belonging to the major phyla of the human intestinal microbiota (Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Verrucomicrobia) ( Supplementary Fig. S1, Supplementary Table S1) and grown under appropriate bacterial growth conditions (Supplementary Table S2). When possible, we cultured the bacteria in different media, to avoid biased results due to the rich composition of culture media, at least for almost all the bacteria activating the AhR reporter system. In our experimental set-up, AhR activation was detected in HT-29 cells challenged with some supernatants derived from Proteobacteria, Firmicutes, Fusobacteria and few Actinobacteria (Fig. 1).

Butyrate activates AhR pathway in intestinal epithelial cell lines. Despite the huge literature on
AhR ligands, only few are commensal-derived molecules. Amongst them, indole and other tryptophan derivatives are produced by a wide range of bacteria including Lactobacillus (Firmicutes) and Proteobacteria 14,24 . In our screening we confirmed the AhR activation induced by some lactobacilli as well as by members of the Proteobacteria, probably due to the production of indole derivatives. Interestingly, among the bacteria not reported or not predicted to be indole producers, we identified some members of genera Ruminoclostridium and Roseburia, belonging to the Firmicutes phylum. These genera, together with other AhR-activating bacteria predicted to produce indole (Clostridium and Lachnoclostridium) share the ability to degrade diet-derived fibres, leading to the production of short-chain fatty acids (SCFAs) 25 . We thus hypothesized that SCFAs concentration in the supernatants of these bacteria could explain the activation of the AhR pathway as previously reported 23 . We therefore quantified the concentration of SCFAs in some bacterial supernatants (Supplementary Table S1 and Fig. 2A). A principal component analysis (PCA) of the complete dataset (SCFA concentrations and AhR activity) revealed that AhR activity and butyrate concentration were major contributors of total variability (Fig. 2B). Further Spearman correlation analysis showed a strong positive correlation between AhR activation and the production of butyrate quantified in commensal bacteria supernatants (rho = 0.4966, Fig. 2C).
To reinforce experimentally the observed correlations, we tested pure SCFAs at different physiological concentrations found in the intestine (ranging from 0.125 mM to 8 mM) on HT-29-AhR reporter cells (Fig. 2D,F) 26,27 . Among the tested SCFAs, we confirmed that butyrate was able to induce AhR activation in a dose-dependent manner, in HT-29-AhR cell line as well as in another intestinal reporter cell line, Caco2-AhR (Fig. 2D,E). Propionate, another abundant SCFA produced by bacteria, was also able to activate AhR in a dose-dependent manner in HT-29-AhR cells at a concentration starting from 1 mM (Fig. 2F). Interestingly, we also observed that valerate and the branched chain fatty acids iso-valerate, both described to influence epithelial physiology, were Scientific RepoRts | (2019) 9:643 | DOI:10.1038/s41598-018-37019-2 also able to activate AhR pathway in HT-29-AhR cell line at similar concentrations 28,29 Iso-butyrate activated AhR only at 8 mM a concentration rarely reached in the intestine while acetate, the most abundant SCFA produced by commensal bacteria showed activation at high concentration (20 mM, Fig. 2F and Supplementary Fig. S2). Apart for acetate, the SCFA concentrations inducing AhR activity were consistent with the final SCFAs concentrations on bacterial supernatants measured in the screen thus reinforcing our hypothesis (Supplementary Table S1). Moreover, we showed by qRT-PCR that AhR-regulated genes, CYP1A1, AHR and AHRR, were up-regulated by butyrate both in HT-29 and Caco2 cells confirming that this SCFA activated AhR pathway (Fig. 3A,B). We confirmed the butyrate-driven up-regulation of AHR at the protein level by Western-Blot analysis in HT-29 (Fig. 3C).
AhR activation by butyrate is independent of the SCFA receptors GPR41, GPR43, GPR109a and the SCFAs transporter MCT-1. Butyrate, like other SCFAs, activates eukaryotic cells through two main mechanisms: activation of specific G-protein coupled receptors (GPCR: GPR41, GPR43, GPR109a) and inhibition of histone deacetylases (HDAC) [30][31][32][33][34] . Considering that the three G-protein coupled receptors are expressed in HT-29 and Caco-2 cells (Martin-Gallausiaux et al. in press), we firstly tested if the butyrate-induced AhR activation could be mediated by GPRs signalling by stimulating HT-29-AhR and Caco2-AhR cells with known GPRs agonists, targeting GPR41, GPR43 and GPR109a. For each GPR, two agonists were tested (GPR41: 4-CMTB and Tiglic acid; GPR43: AR420626 and 1-MCPC; GPR109a: Niacine and MK1903). Interestingly, none of the tested agonists induced AhR activation in luciferase reporter system suggesting that these GPRs are not involved in the butyrate-induced activation in HT-29-AhR and Caco-2-AhR cell lines (Fig. 4A,B). GPR41, GPR109a are both Gαi coupled receptors whereas GPR43 is a Gαi and Gαq coupled receptor. To further confirm our observation, we used an inhibitor of the Gαi pathway inhibitor: the pertussis toxin (Ptx) in HT-29-AhR (Fig. 4C). No impact on the butyrate-induced AhR activation was detected in cells when the Gα i subunit was inhibited, further confirming that these GPRs were not involved in the observed AhR activation.
Butyrate uptake by the monocarboxylate transporter MCT-1 has been described as crucial for the GPR-independent regulation of a wide range of genes by butyrate 35 . To assess if MCT-1 participated in the activation of AhR by butyrate, a well-described inhibitor of MCT-1 transporter, pCMB, was tested on HT-29-AhR cells (Fig. 4D). The inhibition of MCT-1 transporter did not affect the ability of butyrate to activate AhR reporter system, suggesting that MCT-1 was not involved in this process.
Activation of the AhR signalling pathway by butyrate is independent of its role as inhibitor of HDAC. SCFAs, via their ability to inhibit lysine and histone deacetylases (HDAC), are potent modulators of histones and transcription factors acetylation that are well-documented regulatory mechanisms of gene transcription [30][31][32] . A recent study showed that SCFAs and other HDAC inhibitors (HDACi) enhanced the expression of AHR, AHRR and CYP1A1 via the increased level of histone acetylation of AhR-dependent genes 23 . These results prompted us to investigate the direct role of HDACi in the activation of AhR signalling pathway. To assess if butyrate impacted AhR pathway through its HDACi property, we tested three HDACi targeting a wide range of HDAC, trichostatin A (TSA), Vorinostat (SAHA) belonging to the hydroxamic acids family, structurally and metabolically unrelated to SCFAs and sodium valproate (VAP) belonging to the fatty acid family 36 . Interestingly, TSA, SAHA and VAP did not reproduce the butyrate-induced AhR activation in both HT-29-AhR and Caco2-AhR reporter system, suggesting that an additional mechanism leading to butyrate activation of the AhR pathway might exist (Fig. 5A,B). However, we could reproduce the activation of CYP1A1 by TSA as observed by Jin et al. (Fig. 5C) 23 . Altogether, these results suggested that HDACi property of SCFAs was not involved in the butyrate-dependent activation of the AhR signalling pathway as monitored with reporter systems although HDAC inhibition might participate in the regulation of AhR-induced genes.
Butyrate acts as an AhR ligand. We showed that the two main mechanisms described for butyrate to activate transcription factors, HDACi and the activation pathway via GPRs, are not directly involved in the butyrate-induced activation of AhR signalling pathway monitored by reporter cell assay. Two groups described an original mechanism of gene regulation where butyrate acts as a ligand of the transcription factor PPARγ 37,38 . As AhR is a ligand binding transcription factors, we hypothesized that butyrate could act as an AhR ligand similarly to what was described for PPARγ. To demonstrate this hypothesis, we incubated butyrate-stimulated HT-29-AhR and Caco-2-AhR reporter cell-lines with two well-characterized antagonists of the AhR-ligand binding (CH-223191 and GNF-351) and one HSP90 inhibitor, described to avoid the downstream AhR-XRE binding, by blocking the release of HSP90 chaperon from the AhR complex (epigallocatechine-3-gallate, EGCG) [39][40][41][42] . The three tested inhibitors significantly decreased the butyrate-induced activation of AhR signalling pathway in both HT-29 and Caco-2 reporter cell lines (Fig. 6A,B). In addition, we confirmed in HT-29 cells that CH-223191 and GNF-351 were able to block CYP1A1 expression induced by butyrate (Fig. 6C). The pivotal role of AhR in CYP1A1 upregulation by butyrate was assayed using siRNA. As shown in Fig. 6D, we observed a diminished CYP1A1 activation by butyrate and TCDD in cells treated with AhR siRNA compared to control siRNA. These results suggested that the activation of AhR signalling pathway by butyrate was due to a ligand-dependent AhR-XRE interaction, indicating a possible role of butyrate as a direct AhR ligand.
Relative gene expression (fold) It is well established that ligand binding triggers the accessibility of the nuclear localisation signal on the AhR N-terminus that consequently initiates the AhR nuclear translocation event 43 . Thus, additional evidence for the human AhR agonist potential of butyrate was obtained by performing nuclear translocation assay. Sub-cellular localisation of AhR in HT-29 cells incubated with butyrate (1, 3 and 6 h) and TCDD (1 h) was assessed by immunoblotting assay. We showed an accumulation of AhR proteins in the nucleus upon treatment with butyrate starting at 3 h and with a peak at 6 h, consistent with the action of an AhR agonist such as TCDD (Fig. 7).
Altogether, our results suggested that butyrate activation of the AhR reporter system relied on AhR translocation to the nucleus and consequent DNA-binding and that butyrate might act as a ligand of AhR.
In silico modelling of butyrate interaction with AhR. Numerous AhR modulators, binding in the large central pocket of the AhR PAS-B domain, have been extensively studied using a combination of homology modelling and docking stimulations (for review see 44 ). To support our results suggesting that butyrate acts as an AhR ligand, we performed a molecular docking simulation of the butyrate/AhR binding process. We first generated a homology model of human AhR ligand binding domain (AhR PAS-B) using HIF-2α PAS-B domain as template similarly to Bisson et al. 45,46 . The AhR PAS-B homology domain was obtained with RaptorX modelling software and the binding cavity was defined using HOLLOW and Carver softwares (Fig. 8A). Then, the model was minimized to avoid clashes between side chains. From the best scoring solutions cluster using HADDOCK, we observed different orientations of the butyrate inside the AhR PAS-B cavity. Based on four different docking experiments, two main orientations were found for butyrate in the pocket of human AhR PAS-B domain. Interactions between butyrate and AhR are dominated by polar contacts with the side chains of Q383 and S365, for the first orientation (Fig. 8B) and with side chains of Q383 and H291, for the second one (Fig. 8C). Interestingly, by comparing our results with published docking analyses of other ligands on human AhR PAS-B, the first proposed orientation for butyrate docking shared the same interacting side chains (Q383 and S365) with the docking of FICZ, proposed by Bisson et al. 45 . Overall these molecular docking analyses are coherent with our findings of butyrate as a direct modulator of AhR by ligand binding.

Discussion
AhR is a ligand-activated transcription factor that is crucial for intestinal homeostasis by repressing inflammation and by maintaining the epithelial barrier in the gastrointestinal tract 3,47 . The gastrointestinal tract is a rich source of AhR ligands, which have been shown to induce AhR dependent responses and to protect the gut upon infection or induced colitis. Despite the huge literature on the structurally pleiotropic nature of AhR ligands,  only few are commensal-derived molecules. Amongst them, indoles and other tryptophan derivatives are produced by a variety of bacteria including some Lactobacilli (Firmicutes) and Proteobacteria 14,24 . Interestingly, non-commensal bacteria have been reported to produce non-indole AhR ligands such as the phenazine derivative from Mycobacteria and the 1,4-dihydroxy-2-naphthoic acid from the probiotic Propionibacterium freudenreichii 13,48 . Given the abundance and extensive metabolic capacity of gut microbiome, it is thus likely that metabolites apart from tryptophan derivatives are present in high concentration to stimulate AhR pathway. By screening commensal bacteria supernatants, we identified butyrate as a potent activator of AhR pathway and AhR-dependent genes. Butyrate is a short chain fatty acid (SCFA) that derived from bacterial fermentation of dietary fibers. Butyrate, via its role as inhibitor of lysine/histone deacetylase (K/HDACi), influences expression of a large variety of host genes in the colon, including some encoding for immune proteins 49 . Previous studies described butyrate as a regulator of AhR-dependent genes thought its role as histone deacetylase inhibitors (HDACi) 23,50 . However, these studies assessed the butyrate effect through induction of AhR transcriptional targets and not directly on the AhR receptor. Our results are in accordance with these studies as we showed that HDACi and butyrate upregulate CYP1A1 expression. However, we demonstrated that butyrate does not impact AhR-dependent gene expression solely by its HDACi properties. Indeed by using an AhR reporter system in HT-29 and Caco2 cells, we showed that butyrate, and not HDAC inhibitors, activates AhR signalling pathway.
To decipher the mechanism of butyrate-dependent AhR activation, we investigated the implication of butyrate specific G-protein coupled receptors (GPR41, GPR109a and GPR43) and main transporter, monocarboxylate transporter 1 (MCT1) [51][52][53] . By using agonists and inhibitors, we showed that AhR activation by butyrate was not mediated by those GPRs or MCT1. Many studies evidenced that the predominant biological activities of the AHR are through ligand binding. Despite the AhR association with xenobiotic compounds, structurally diverse metabolites from the diet, bacteria or produced by the host have been reported as capable of binding to human AhR 6,54 . Our experimental results suggest that butyrate acts as an AhR ligand leading to the activation of the AhR pathway. Moreover, structural modelling of the binding of butyrate to human AhR PAS-B supports this hypothesis as we observed two main orientations of butyrate in the ligand-binding cavity of AhR. Interestingly, one of these orientations showed polar contact of butyrate with the same side chains of PAS-B reported for the binding of FICZ to human AhR supporting our hypothesis that butyrate is a ligand of AhR 45 . Confirmation of these results by direct amino acids point mutations in the ligand-binding cavity of human AHR similarly to previous publication on mice Ahr would have support our hypothesis 55 . This is an original mechanism of butyrate-dependent modulation of host gene expression, which has only been reported for another ligand-dependent transcription factor, PPARγ. Indeed, two groups reported that PPARγ-dependent genes regulation by butyrate is independent of its HDACi properties and is mediated through a direct binding of the SFCA to this transcription factor 37,38 . The strong impact of butyrate on mucosal immune homeostasis has been largely documented in mice and in vitro models. In human, studies have shown lower concentration of SCFA and butyrate-producing bacteria (i.e. Faecalibaterium and Clostridium) in the gut of inflammatory bowel diseases (IBD) patients suggesting a relevant role of butyrate in intestinal health [56][57][58] . Butyrate and other SCFAs elicit most of their biological activities by binding to GPRs and by acting as HDAC inhibitors 49 . Our results demonstrate an additional mechanism where butyrate binds to AhR thus activating its signalling pathway and in synergy with its HDACi property leads to the expression of AhR-dependent genes. These results might be generalized to other SCFAs as most of them activated the AhR signalling pathway (Fig. 2 and Supplementary Fig. S2) and in silico modelling of propionate interaction with AhR showed an orientation in the pocket of human AhR PAS-B domain similar to butyrate involving H291 and Q383 ( Supplementary Fig. S3). However additional experiments are needed to validate this point. Interestingly, SCFAs such as butyrate exhibit overlapping activities with AhR ligands on intestinal homeostasis and in IBD 59 . AhR ligands regulate epithelial IL-10 receptor α subunit (IL-10RA) expression that damped colitis by promoting epithelial wound healing 60 . Similarly, butyrate promotes epithelial barrier formation through IL-10RA induction on IECs 61 . Hence, it is possible that AhR mediates the butyrate-induced IL-10RA up-regulation. Considering the high quantities of SCFAs in the gut, it is likely that butyrate possibly in synergy with other bacterial-derived AhR ligands have a role in the physiological functions of AhR. Nevertheless, the validation of our results in human primary IECs or colonic organoids is a future challenge to further decipher the mechanisms of butyrate impact on AhR signalling pathway, as well as other ligand-dependent transcription factors.
In conclusion, we show that butyrate stimulates AhR-dependent genes through a direct AhR activation and probably in complement to its HDACi property in human intestinal cell. Our results suggest that butyrate acts as a ligand of AhR which is, to our knowledge, an original mechanism only been reported for another ligand-binding transcription factor PPARγ 38 .

Culture of commensal bacteria, preparation of supernatants and SCFA concentration assessment. 132 human intestinal commensal bacterial strains (106 different species) from the in-house
INRA-Micalis collection or from DSMZ were grown. Anaerobic culture conditions were done accordingly to the Hungate method 62 . Screened strains, corresponding growth media, optical densities (OD600), SCFA concentrations are listed in Appendix Table S1 and composition of home-made growth media is listed in Supplementary  Table S2. Bacterial cultures were cultured to reach the maximum OD. Bacterial supernatants were harvest after centrifugation at 5,000 × g for 10 min and filtered on a 0.22 μm PES filters and stored at −80 °C. Quality controls were performed using Gram staining method, aerobic growth test and fresh observation on microscope. Non-inoculated bacteria culture medium served as control. Concentrations of SCFAs produced by cultured bacteria were measured by HPLC and gas chromatography as described by 63,64 . Real-Time PCR. Cell lines were seeded in 12-well culture plates at densities of 0.5 × 10 6 cells per well. The cells were seeded in FBS-supplemented media then, after 24 h, the media was replaced with a non-FBS-supplemented and cells incubated during 24 h before stimulation. After stimulation time of 6 h, total RNA was extracted using RNeasy mini-Kit (Qiagen) according to manufacturer's recommendations. cDNA was synthesized from 2 μg of RNA using the High-Capacity cDNA Archive Kit (Applied Biosystems). qPCRs were carried out using a StepOne (Applied Biosystems) thermal cycler in a reaction volume of 20 μL with Taqman gene expression assay probes: AHR: Hs00169233_m1; AHRR: Hs01005075_m1; CYP1A1: Hs01054796_g1; β-ACTIN: Hs99999903_m1. AHR, CYP1A1 and AHRR expression relative to control expression was determined by the 2 −ΔΔCt method using β-actin as control gene. Data are means ± SEM of at least three distinct experiments, performed in triplicate.
Cytoplasmic and nuclear protein extraction. HT-29 cells were seeded at densities of 0.5 × 10 6 cells per well in 12-well-plates. 24 h after seeding the media was replaced with a non-FBS-supplemented RPMI and cells incubated during 24 h prior to stimulation. When nuclear extracts were not needed, cells were washed twice and lysed in buffer (1% NP40, 150 mM NaCl, 50 mM Tris-HCL pH8, 5 mM EDTA, 1x Complete Protease Inhibitor Cocktail (Roche)). Nucleus were eliminated by centrifugation for 10 minutes 4 °C at 17500 g. For compartments separation, nuclear and cytoplasmic extracts were prepared with NE-PER Nuclear and Cytoplasmic  H4A3). Secondary mouse horseradish peroxidase-coupled antibody (DAKO) was successively incubated at room temperature for 2 h before detection with the Clarity Western ECL Substrate using the Chemidoc MP System (Bio-Rad). Quantifications were performed using the image Lab software (Bio-Rad). AhR nuclear protein levels were normalised to Lamin A/C protein levels. Lamp1 and GAPDH were used as purification controls for the cytoplasmic proteins.
Modelling of butyrate binding to AhR. For modelling the structure of the complex between AhR and butyrate or propionate, HADDOCK software was used 65, 66 . HADDOCK is a highly successful modelling approach that makes use of structural knowledge when available to drive the docking procedure. In this case the crystal structure of the heterodimeric HIF-2α:ARNT complex (PDB code 4ZP4) 67