# Butyrate produced by commensal bacteria down-regulates indolamine 2, 3-dioxygenase 1 (IDO-1) expression via a dual mechanism in human intestinal epithelial cells

Camille Martin-Gallausiaux, Pierre Larraufie, Anne Jarry, Fabienne Beguet-Crespel, Ludovica Marinelli, Florence Ledue, Frank Reimann, Herve H. Blottiere, Nicolas Lapaque

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Camille Martin-Gallausiaux, Pierre Larraufie, Anne Jarry, Fabienne Beguet-Crespel, Ludovica Marinelli, et al. Butyrate produced by commensal bacteria down-regulates indolamine 2, 3-dioxygenase 1 (IDO-1) expression via a dual mechanism in human intestinal epithelial cells. Frontiers in Immunology, 2018, 9, 10.3389/fimmu.2018.02838. inserm-01937996

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# Butyrate produced by commensal bacteria down-regulates indolamine 2, 3-dioxygenase 1 (IDO-1) expression via a dual mechanism in human intestinal epithelial cells.

Camille Martin-Gallausiaux¹, ², Pierre Larraufie¹, ³, Anne Jarry⁴, ⁵, Fabienne Béguet-Crespel¹, Ludovica Marinelli¹, ², Florence Ledue¹, Frank Reimann³, Hervé M. BLOTTIERE¹, ⁶, Nicolas Lapaque¹*

¹Institut Micalis, France, ²Sorbonne Universités, France, ³MRC Metabolic Diseases Unit, Metabolic

Research Laboratories, University of Cambridge, United Kingdom, ⁴INSERM U1232 Centre de Recherche en Cancérologie et Immunologie Nantes Angers (CRCINA), France, ⁵University of Nantes, France,

⁶MetaGenoPoliS (MGP), Université Paris-Saclay, France

**Submitted to Journal:**

Frontiers in Immunology

**Specialty Section:**

Mucosal Immunity

**Article type:**

Original Research Article

**Manuscript ID:**

421934

**Received on:**

30 Aug 2018

**Revised on:**

14 Nov 2018

**Frontiers website link:**

www.frontiersin.org
**Conflict of interest statement**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

**Author contribution statement**

Conceived and designed the experiments: CMG, NL; performed most of the experiments: CMG; performed some experiments: PL, AJ, FBC, LM, FL, NL; analysed the data: CMG, AJ, NL; contributed materials: FR; wrote the paper: CMG, NL; edited and revised the manuscript: AJ, PL, FR, HMB.

**Keywords**

Gut Microbiota, ido-1, Intestinal epithelial cell (IEC), Butyrate, commensal bacteria, host-microbiota crosstalk, Immune gene regulation

**Abstract**

Word count: 274

Commensal bacteria are crucial for the development and maintenance of a healthy immune system therefore contributing to the global wellbeing of their host. A wide variety of metabolites produced by commensal bacteria are influencing host health but the characterisation of the multiple molecular mechanisms involved in host-microbiota interactions is still only partially unravelled. The intestinal epithelial cells (IEC) take a central part in the host-microbiota dialogue by inducing the first microbial-derived immune signals. Amongst the numerous effector molecules modulating the immune responses produced by IECs, indoleamine 2,3-dioxygenase-1 (IDO-1) is essential for gut homeostasis. IDO-1 expression is dependent on the microbiota and despite its central role, how the commensal bacteria impacts its expression is still unclear. Therefore, we investigated the impact of individual cultivable commensal bacteria on IDO-1 transcriptional expression and found that the short chain fatty acid (SCFA) butyrate was the main metabolite controlling IDO-1 expression in human primary IECs and IEC cell-lines. This butyrate-driven effect was independent of the G-protein coupled receptors GPR41, GPR43 and GPR109a and of the transcription factors SP1, AP1 and PPARγ for which binding sites were reported in the IDO-1 promoter. We demonstrated for the first time that butyrate represses IDO-1 expression by two distinct mechanisms. Firstly, butyrate decreases STAT1 expression leading to the inhibition of the IFNγ-dependent and phosphoSTAT1-driven transcription of IDO-1. In addition, we described a second mechanism by which butyrate impairs IDO-1 transcription in a STAT1-independent manner that could be attributed to its histone deacetylase (HDAC) inhibitor property.

In conclusion, our results showed that IDO-1 expression is down-regulated by butyrate via a dual mechanism: the reduction of STAT1 level and the HDAC inhibitor property of SCFAs.

**Funding statement**

CMG was the recipient of a fellowship from the Ministère de la Recherche et de l'Éducation Nationale (UPMC-Sorbonne University).

**Ethics statements**

(Authors are required to state the ethical considerations of their study in the manuscript, including for cases where the study was exempt from ethical approval procedures)

Does the study presented in the manuscript involve human or animal subjects: Yes

Please provide the complete ethics statement for your manuscript. Note that the statement will be directly added to the manuscript file for peer-review, and should include the following information:

* Full name of the ethics committee that approved the study

* Consent procedure used for human participants or for animal owners

* Any additional considerations of the study in cases where vulnerable populations were involved, for example minors, persons with disabilities or endangered animal species
This study was carried out in accordance with the recommendations of [name of guidelines], [name of committee]. The protocol was approved by the [name of committee]. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

For statements involving animal subjects, please use:

This study was carried out in accordance with the recommendations of 'name of guidelines, name of committee'. The protocol was approved by the 'name of committee'.

If the study was exempt from one or more of the above requirements, please provide a statement with the reason for the exemption(s).

Ensure that your statement is phrased in a complete way, with clear and concise sentences.

Human gut explains: The tissue fragments were processed accordingly to the French guidelines for research on human tissues, including patients' consent.

Primary colonocytes cultures: Human tissues were obtained from the Human Research Tissue Bank at the Addenbrooke's hospital, Cambridge under the license 09/H0308/24.

In review
1 Butyrate produced by commensal bacteria down-regulates

2 indolamine 2, 3-dioxygenase 1 (IDO-1) expression via a dual

3 mechanism in human intestinal epithelial cells.

4
5 Camille Martin-Gallausiaux¹, ², Pierre Larraufie¹, ³, Anne Jarry⁴, Fabienne Béguet-Crespel¹,

6 Ludovica Marinelli¹, ², Florence Ledue¹, Frank Reimann³, Hervé M. Blottière¹, ⁵ and Nicolas
7 Lapaque¹*.

8
9
10 ¹Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France;

11
¹²Sorbonne Universités, UPMC Univ Paris 06, IFD, Paris, France.

12
³University of Cambridge, Metabolic Research Laboratories and MRC Metabolic Diseases

13 Unit, WT-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK;
14
⁴INSERM UMR 1232, Université de Nantes, Nantes, France;

15
⁵US 1367 MetaGenoPolis, INRA, Université Paris-Saclay, Jouy en Josas, France.

16
17
* Corresponding author: Dr Nicolas Lapaque

18 INRA-MICALIS UMR1319, Bat 442, Domaine de Vilvert 78350, Jouy-en-Josas, France.

19 e-mail: nicolas.lapaque@inra.fr

20
21
Keywords: Gut microbiota, IDO-1, intestinal epithelial cells, butyrate, Immune gene

22 regulation.

23
1
24

Commensal bacteria are crucial for the development and maintenance of a healthy immune system therefore contributing to the global wellbeing of their host. A wide variety of metabolites produced by commensal bacteria are influencing host health but the characterisation of the multiple molecular mechanisms involved in host-microbiota interactions is still only partially unravelled. The intestinal epithelial cells (IEC) take a central part in the host-microbiota dialogue by inducing the first microbial-derived immune signals. Amongst the numerous effector molecules modulating the immune responses produced by IECs, indoleamine 2,3-dioxygenase-1 (IDO-1) is essential for gut homeostasis. IDO-1 expression is dependent on the microbiota and despite its central role, how the commensal bacteria impacts its expression is still unclear. Therefore, we investigated the impact of individual cultivable commensal bacteria on IDO-1 transcriptional expression and found that the short chain fatty acid (SCFA) butyrate was the main metabolite controlling IDO-1 expression in human primary IECs and IEC cell-lines. This butyrate-driven effect was independent of the G-protein coupled receptors GPR41, GPR43 and GPR109a and of the transcription factors SP1, AP1 and PPARγ for which binding sites were reported in the IDO-1 promoter. We demonstrated for the first time that butyrate represses IDO-1 expression by two distinct mechanisms. Firstly, butyrate decreases STAT1 expression leading to the inhibition of the IFNγ-dependent and phosphoSTAT1-driven transcription of IDO-1. In addition, we described a second mechanism by which butyrate impairs IDO-1 transcription in a STAT1-independent manner that could be attributed to its histone deacetylase (HDAC) inhibitor property.

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In conclusion, our results showed that IDO-1 expression is down-regulated by butyrate via a dual mechanism: the reduction of STAT1 level and the HDAC inhibitor property of SCFAs.
## 48 Introduction

The gut microbiome is a microbial ecosystem that exerts diverse functions often associated with beneficial physiological effects for its host. Among these essential functions, the intestinal microbiome provides an extended repertoire of molecules that influences the host health notably *via* the development and the maturation of its immune system (Sekirov et al., 2010; Postler and Ghosh, 2017). The molecular bases of the host-microbiota interactions are only just beginning to be unravelled and are mediated by a wide variety of metabolites produced by commensal bacteria (Blacher et al., 2017; Postler and Ghosh, 2017). Many bacteria-derived metabolites originate from dietary sources. Among them, an important role has been attributed to the metabolites derived from the bacterial fermentation of dietary fibres, namely the short chain fatty acids (SCFAs) linking host nutrition to immune development and functions (Blacher et al., 2017; Postler and Ghosh, 2017). Human cells respond to SCFAs through a signalling activation cascade involving specific G-protein coupled receptors (GPR41, GPR43 and GPR109a) and through an epigenetic regulation of gene expression by the inhibition of lysine or histone deacetylases (HDACs) (Hinnebusch et al., 2002; Schilderink et al., 2013; Fellows et al., 2018).

Numerous studies suggest that the close intimacy between the mucosal microbial populations and the host intestinal cells is central for the fine regulation of the host physiology. Indeed, intestinal epithelial cells (IEC) provide a crucial physical barrier against harmful pathogens and are also key players in the initiation and maintenance of mucosal immune responses (Kagnoff, 2014). Accordingly, indigenous members of the microbiota have dramatic and specific impacts on the host immune system through their intimate interactions with the host epithelium (Gaboriau-Routhiau et al., 2009; Ivanov et al., 2009; Atarashi et al., 2011; Schilderink et al., 2013; Atarashi et al., 2015).
Indoleamine 2,3-dioxygenase-1 (IDO-1) is an enzyme that catalyses the oxidation of the indole moiety of the essential amino acid tryptophan leading to production of N-formylkynurenine and its derivatives. In the last decades, a growing number of studies showed the importance of IDO-1 in various pathologies, including, autoimmune diseases, allergy and cancer (Platten et al., 2005; Munn and Mellor, 2007). Despite the fact that IDO-1 expression was largely thought to be protective, several recent studies suggest a detrimental role of IDO-1 expression in obesity, atherosclerosis, vascular inflammation and aneurysm (Metghalchi et al., 2015; Laurans et al., 2018; Metghalchi et al., 2018). These results suggest that IDO-1 plays a far more complex role in health and fine-tuning of its expression and activity might occur in healthy individuals. Mechanisms inducing IDO-1 expression during inflammation have already been described and include IFNγ and type-I IFN. However, natural factors inhibiting IDO-1 expression have not been reported yet.

The gut, along with the skin, is a major site of IDO-1 activity at steady state. IDO-1 expression in human healthy IECs is poorly described but has been reported in several studies to be increased in IBD (Barcelo-Batllori et al., 2002; Ferdinande et al., 2008; Cherayil, 2009; Zhou et al., 2012). In the murine gut, its expression is dependent on the microbiota (Rhee et al., 2005; Atarashi et al., 2011). These observations prompted us to investigate the impact of individual cultivable commensal bacteria on IDO-1 transcriptional expression. In the current study, we screened over 401 bacterial supernatants on an IDO-1 reporter system and found that butyrate was the main inhibitor of IDO-1 expression in human primary IECs and cell lines. The IDO-1 down-regulation was independent of GPR41, GPR43 and GPR109a, three known G-protein coupled receptors for SCFAs and of SP1, AP-1 and PPARγ, three transcription factors targeted by butyrate and for which binding sites were reported in the IDO-1 promoter. Our results showed that butyrate regulated IDO-1 expression via a dual mechanism. First, butyrate decreased STAT1 expression leading to the inhibition of the IFNγ-
dependent phosphorylation of STAT1 and consequently the STAT1-driven transcriptional activity of *IDO-1*. In addition, we described a second mechanism by which butyrate impaired *IDO-1* transcription in a STAT1 independent manner that could be attributed to the HDAC inhibitory property of SCFAs.

In review
102

**Materials and methods**

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**IDO-1 expression in human normal colon at the protein and mRNA levels**

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Macroscopically and microscopically unaffected human normal colon was obtained from 10 patients undergoing surgery for colon cancer, at least at 10 cm downstream the tumour [7 men, 3 women; mean age 62 years; left (7) or right colon (3)]. The tissue fragments were processed accordingly to the French guidelines for research on human tissues, including patients' consent. IDO-1 immunostaining was performed using a monoclonal antibody (clone 4D2, Serotec) and a standard streptavidin-biotin-peroxidase technique after antigen retrieval in citrate buffer pH6. Diaminobenzidine was used as a chromogen and nuclei were counterstained with hematoxylin. IDO-1 mRNA levels were assessed on preparations of isolated IECs after EDTA treatment and on whole mucosa microdissected from the normal colon as previously described (Jarry et al., 2008). Samples were prepared by beads-beating mechanical lysis using Fastprep (MP Biomedicals) and centrifuged at 8,000g for 10 min at 4°C prior RNA extraction and RT-PCR analysis.

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**Cell Culture of human intestinal cell lines and primary colonocytes**

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The human epithelial cell lines HT-29 and Caco-2 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and grown as described (Martin-Gallausiaux et al., 2018). Four human primary colonic cell culture from three different donors were performed as described (Habib et al., 2013). Briefly, PBS-washed colonic tissues were digested with 0.5mg/ml of collagenase type XI. The crypts were plated onto Matrigel coated plates and cultured for 24h in DMEM 24mM glucose supplemented with 10% FCS, 2mM L-Glutamine, 50 U/mL penicillin, 50 U/mL streptomycin and Y-27632 (Tocris). The day after plating, media was rinsed with fresh media and replaced with culture media with or without
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butyrate 2mM. Human tissues were obtained from the Human Research Tissue Bank at the
Addenbrooke's hospital, Cambridge under the license 09/H0308/24.

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Luciferase Reporter and cell viability Assays

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A 1.6-Kb section of the human *IDO-1* promoter was cloned using KpnI and NheI restrictions
sites (Primers used were Fw: AAAGGTACCGGGTAGGATAGATTTAGTGAG; Rv:
AAAAAGCTAGCCATTCTTGTAGTCTGCTCC) into the pGL4.14 (Promega) luciferase
plasmid and used to establish the stable HT-29 *IDO-1* reporter cell-line after antibiotic
selection (hygromycin, 600 µg/mL, InvivoGen) and validated with IFNγ (100U/mL,
Peprotech) and IL1β (10ng/mL, Peprotech). For each experiment, HT-29-*IDO-1* reporter cells
were seeded at 3x10⁴ cells per well in 96-well plates 24h prior to incubation with bacterial
supernatants or reagents. The cells were stimulated for 24h with 10 µL of bacterial
supernatants in a total culture volume of 100 µL per well (i.e., 10% vol/vol) prior to the
luciferase assay. The luciferase activity was quantified as relative luminescence units using a
microplate reader (Tecan) and the Neolite Luminescence Reporter Assay (Perkin-Elmer)
according to the manufacturers' instructions. The *IDO-1* activity was normalized to the
controls, i.e., the un-stimulated cells or cells in presence of non-inoculated bacteria culture
medium. Experiments were performed in triplicates for at least three biological independent
assays. Cell viability was monitored by MTS measurement using the CellTiter 96 Aqueous
One solution (Promega) according to the manufacturer's recommendations.

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Culture of commensal bacteria, preparation of supernatants and SCFAs concentration
assessment.

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135 human intestinal commensal bacterial strains which include 111 different species from
the in-house INRA-Micalis collection or from DSMZ were grown. Bacterial cultures and

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supernatants were performed as described (Martin-Gallausiaux et al., 2018). Screened species
and strains, corresponding growth media, optical densities (OD), short chain fatty acids
(SCFAs) concentrations are listed in Supplementary Table 1. Concentrations of SCFAs
produced by cultured bacteria were measured by HPLC and gas chromatography as described
(Bourriaud et al., 2005).

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## Reagents and cytokines

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## Plasmids and transfection

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Human GPR43 and GPR109a were cloned after EcoRI and XhoI digestion in pCMV-eGFP-N1 vector. Oligonucleotides used for amplification of GPR43 were *aaaactcgagatgctgccggactggaa* and *aaaagaattcctactctgtagtgaagtccga*. Oligonucleotides used for amplification of GPR109a were *aaaactcgagatgaatcggcaccatctgcaggat* and
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aaaagaattcttaaggagaggtgggcccaga. HT-29 cells were seeded at 3.10⁴ density per well in 96-

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well plates and transiently transfected with Lipofectamine 2000 (Thermofischer). 24h after

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transfection, incubation with reagents was done for an additional 24h prior luciferase activity

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measurement.

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## siRNA assays

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HT29 cells were seeded at 4.10⁵ cells per well in a 6 wells plates on day 1 and siRNA were

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transfected with DharmaFect I at final concentrations of 1 and 25nM on day 2 and 3,

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following the manufacturer's instructions (Dharmacom). Incubation with drugs was done on

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day 6 and IDO-1 activity was assessed on day 7. siRNA SMARTpool ON-TARGETplus

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STAT1 siRNA (L-003543-00-0005) and ON-TARGETplus Non-targeting Pool (D-001810-

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10-05) were from Dharmacon.

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## Real-Time PCR

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Real-Time PCR were performed as described (Martin-Gallausiaux et al., 2018). qPCRs were

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carried out using a StepOnePlus Real-Time PCR System (ThermoFischer Scientific) with

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Taqman gene expression assay probes : GAPDH Hs02758991_g1, IDO-1 Hs00984148_m1,

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GPR43 Hs00271142_s1, GPR41 Hs02519193_g1, GPR109a Hs02341584_s1, RBP1

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Hs01011512_g1, Actinbeta Hs99999903_m1, STAT1 s01013996_m1, B2M Hs99999907_m1.

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GAPDH, Actin, RBP1 and B2M were used for internal normalisation. Samples were tested in

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experimental duplicates and at least in biological triplicates. For primary cells treated with

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butyrate and control, cDNAs were pre-amplified (10 cycles) using the TaqMan PreAmp

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Master Mix Kit following the manufacturer's recommendations.
193 **Western blot analysis**

194 HT-29 cells were seeded at densities of 5x10⁵ cells per well in 24-well-plates for 24h prior
195 stimulation. Cells were washed twice and lysed in buffer (1% NP40, 150mM NaCl, 50mM
196 Tris-HCL pH8, 5mM EDTA, 1 x Complete Protease Inhibitor Cocktail (Roche), 1X x Phos
197 STOP phosphatase Inhibitor Cocktail (Roche). Nucleus were eliminated by centrifugation for
198 10 minutes 4°C at 17500g. Protein extracts were run in SDS-PAGE gels and transferred onto
199 PVDF membranes. Membranes were blocked overnight in TBS 0.1% tween 4% skim milk or
200 BSA (Sigma). Primary antibodies were incubated overnight at 4°C (STAT1 1:1000 (D1K9Y),
201 STAT1-phospho TYR 701 1:1000 (58D6), STAT3 1:1000 (124H6), Lamin A/C 1:2000
202 (4C11) all from Cell signalling; Actin 1:2000 (AC-40) from Sigma, GAPDH 1:2000 from
203 Santa Cruz). Secondary (Goat anti-Rabbit IgG HRP (P0448) and Goat anti-mousse HRP
204 (P0447) from Dako) antibodies were successively added for 1h before detection with the
205 Clarity Western ECL Substrate using the Chemidoc MP System (Bio-Rad). Quantifications
206 were performed using the image Lab software (Bio-Rad). Proteins levels were internally
207 normalised with GAPDH or Actin before comparison with experimental controls.

208 **Cytoplasmic and nuclear proteins extraction**

209 HT-29 cells were seeded at densities of 5x10⁵ cells/well in 24-well-plates for 24h prior
210 stimulation with butyrate. Cytoplasmic and nuclear protein extracts were prepared using the
211 NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's
212 instructions (ThermoScientific). Lamin A/C and GAPDH were used as nuclear and
213 cytoplasmic protein loading controls respectively.
214 Promoter analysis

215 *In silico* analysis of the promoter sequence upstream of the transcription start of *IDO-1* was
216 performed using Genomatix MatInspector software (core similarity=1; matrix similarity
217 >0.8).

218 Statistical Analysis

219 Data were analysed using R and RStudio software. Function for PCA analysis: prcomp.
220 Correlation matrix was done with Hmisc package. Graphics were produced with ggplot2
221 package and Prism GraphPad software. Statistical analysis was done with Student two-sided
222 test or Wilcoxon rank test.

223
225 **IDO-1 is expressed in epithelial cells of the human normal colonic mucosa**

226 IDO-1 expression is well documented in dendritic cells (DC) and macrophages (Matteoli et al., 2010). However its expression in intestinal epithelial cells (IECs) has been scarcely studied in 227 human. We assessed IDO-1 expression both by immunohistochemistry on paraffin sections of 228 normal human colonic mucosa (n=10) and at the mRNA level. In 8 cases, IDO-1 was 229 expressed by IECs with either strong homogeneous staining of more than 80% IECs all along 230 the colonic crypts (perinuclear and/or membrane staining in enterocytes and goblet cells; 231 Figure 1A, left panel) or heterogeneous staining of IECs (10-20% of IECs; Figure 1A, right 232 panel). In 2 samples, IDO-1 was barely detectable in IECs. IDO-1 was also expressed in the 233 lamina propria, in some mononuclear cells and endothelial cells (Figure 1A). IDO-1 234 expression was then confirmed by RT-PCR on RNA extracted from preparations of isolated 235 human IECs from normal colon. As shown in Figure 1B, isolated human IECs expressed an 236 IDO-1 level comparable with the expression level from the entire colonic mucosa suggesting 237 that IECs were an important source of IDO-1 mRNAs in the colon. 238

239 **Metabolites derived from commensal bacteria modulate IDO-1 expression**

240 In the gut, IDO-1 expression is dependent on the microbiota since colonisation of mice with 241 commensal bacteria induced high levels of IDO-1 in IECs (Rhee et al., 2005; Atarashi et al., 242 2011). In an attempt to decipher which commensal bacteria influence IDO-1 expression, we 243 performed a screening with an IDO-1 reporter system expressed in the human epithelial cell 244 line HT-29. As recently reported in animal studies and in functional metagenomic studies, 245 bioactive compounds produced by commensal bacteria are likely to be small-secreted 246 molecules, we thus tested the bacterial supernatants of 135 members of the human microbiota
that include 60% of species close to the human core microbiome on an *IDO-1* reporter system (Supplementary Table 1) (Qin et al., 2010; Cohen et al., 2015; Blacher et al., 2017; Postler and Ghosh, 2017). In this set-up, only few bacterial supernatants were activating *IDO-1* expression in HT-29 cells, including some *Lactobacillaceae* (Supplementary Figure 1). Interestingly, a global and dramatic down-regulation of *IDO-1* was observed in HT-29 challenged with supernatants of Firmicutes and Fusobacteria, while Actinobacteria, Bacteroidetes, Proteobacteria and Verrucomicrobia barely modulated *IDO-1* expression (Figure 2A, Supplementary Figure 1).

## Butyrate down-regulates *IDO-1* expression in epithelial cells

Among the Firmicutes, the most active genera on *IDO-1* expression were *Clostridium*, *Lachnoclostridium*, *Ruminoclostridium* and *Roseburia* (Supplementary Figure 1). All these genera in addition to the *Fusobacterium* genus share a common active role in the diet-derived fibre degradation leading to the production of short-chain fatty acids (SCFAs) by anaerobic fermentation (Vital et al., 2014). We thus hypothesized that the down-regulated pattern of *IDO-1* expression could be explained by the SCFA concentration in the bacterial supernatants. We therefore quantified the concentrations of acetate, propionate, butyrate, isobutyrate, valerate and isovalerate by GC-MS or HPLC in some bacterial supernatants (Supplementary Table 1). Principal component (PCA) and correlation analyses on SCFAs concentrations and *IDO-1* activity showed a negative correlation between butyrate concentration and *IDO-1* expression (Figure 2B and Supplementary Figure 2A). Specific impact of butyrate on *IDO-1* was confirmed by a pairwise spearman correlation (Figure 2C). Analysis with acetate concentrations showed no correlation with *IDO-1* expression (Figure 2D).
We validated experimentally the observed correlations by testing the effect of a range of physiological intestinal concentration of SCFAs on *IDO-1* reporter system. Acetate which is the more abundant SCFA produced by gut bacteria had no impact on *IDO-1* expression. Butyrate and to a lesser extent propionate, isobutyrate, isovalerate and valerate down-regulated *IDO-1* (Figure 3A and Supplementary Figure 2B). Indeed, as shown in Figure 3A, a significant *IDO-1* down-regulation was observed at a concentration as low as 0.5 mM for butyrate and propionate. These concentrations were consistent with the final SCFA concentrations in bacterial supernatants used in the screen thus supporting their involvement in *IDO-1* down-regulation (Supplementary Table 1). Butyrate and propionate are found in the human gut lumen at around 20mM (Cummings et al., 1987). Moreover, we showed that butyrate and propionate also inhibited Interferon γ (IFNγ)-induced *IDO-1* expression in a dose-dependent manner in our reporter system (Figure 3B and Supplementary Figure 2C). This result was confirmed at the mRNA level by RT-PCR in IFNγ-treated HT-29 cells with a total abolishment of *IDO-1* expression by butyrate and propionate while acetate had no significant impact (Figure 3C). In addition, the inhibitory impact of butyrate and propionate on *IDO-1* expression was observed in an *IDO-1* reporter system expressed in another IEC line, Caco-2 (Figure 3D and Supplementary Figure 2D). More importantly, we showed that this phenotype is not restricted to cell-lines as *IDO-1* mRNA level was also significantly down-regulated by butyrate in human primary colonocytes culture, compared to non-treated cells (Figure 3E).

## Butyrate inhibits IFNγ-induced IDO-1 expression by STAT1 down-regulation

Several mechanisms of *IDO-1* induction have been reported. A classical cascade involves IFNγ-dependent phosphorylation of Signal transducer and activator of transcription 1 (STAT1) promoting *IDO-1* expression (Chon et al., 1996). Previous studies have
demonstrated the inhibition of IFNγ-dependent phosphorylation of STAT1 by butyrate, in a nasopharyngeal carcinoma model (Jiang et al., 2010; He et al., 2013). We thus assayed by immunoblot analysis the impact of a 24h-treatment of butyrate on the IFNγ-induced phosphorylation of STAT1 in HT-29 cells. In line with other studies, we observed less Tyr 701 phosphorylated form of STAT1 in cells pre-treated with butyrate (Figure 4A-B).
Interestingly, in contrast to previous studies, we observed that this phenotype was directly correlated to a down-regulation of the protein level of STAT1 itself mediated by butyrate as both total STAT1 and phosphorylated STAT1 levels were similarly diminished (Figure 4A-C). The butyrate-driven STAT1 down-regulation was observed on both IFNγ stimulated and non-stimulated cells (Figure 4A, 4C and Supplementary Figure 3A). Interestingly, we did not monitor any inhibition of STAT1 gene expression by RT-PCR at 6 and 24h post incubation with butyrate (Supplementary Figure 3B) suggesting post-transcriptional modifications of STAT1. To further determine whether STAT1 was translocated in the nucleus by butyrate treatment, nuclear STAT1 protein level was assessed by immunoblotting in butyrate-treated and control HT-29 cells. As shown in figure 4D, we did not detect accumulation of nuclear STAT1 in butyrate-treated cells. In summary, these findings demonstrated that butyrate strongly reduced STAT1 protein level which is a mechanism contributing to the inhibition of IFNγ-induced IDO-1 in human intestinal epithelial cells.

**Butyrate inhibits IDO-1 expression independently of STAT1 and STAT3.**

To further decipher the mechanism of butyrate-driven IDO-1 regulation observed in cells untreated with IFNγ, we studied STAT1 involvement in the IDO-1 down-regulation observed in unstimulated IECs (Figure 3). The pivotal role of STAT1 was assayed using siRNA down-regulation (Supplementary Figure 4A). We observed no impact on butyrate-dependent inhibition of IDO-1 in absence of STAT1 signalling. These results suggested that
butyrate did not impact on basal STAT1-dependent signalling and that STAT1-independent mechanism may also be involved in IDO-1 down-regulation (Figure 5A).

Two alternative pathways for IDO-1 induction have been reported, involving STAT3 and aryl hydroxycarbon receptor (AHR) on one hand and an NFκB-dependent pathway on the other hand (Litzenburger et al., 2014; Yu et al., 2014; Li et al., 2016). We showed that blocking STAT3 phosphorylation (Cucurbitacin I) or activating AHR pathway (TCDD) did not induce IDO-1 or prevent butyrate inhibition in our model supporting that the STAT3/AHR pathway was not involved in this process (Figure 5B-C). Moreover, immunoblotting assays on STAT3 level revealed, that in contrast to STAT1, STAT3 was not decreased following butyrate incubation for 24h in HT-29 (Supplementary Figure 4B). In addition, we ruled out NFκB activation as NFκB inhibitor BAY 11-7082 did not impact on butyrate-driven IDO-1 down-regulation, as positive control NFκB activation was induced by TNFα (Figure 5D). Altogether, these results suggested that butyrate down-regulated IDO-1 independently of STAT1, STAT3, AHR and NFκB.

**Butyrate-mediated impact on IDO-1 is independent of the SCFAs receptors GPR41, GPR43 and GPR109a.**

Our data suggest that butyrate down-regulates IDO-1 expression in a STAT1 and STAT3-independent manner and, thus, might involve an additional mechanism. SCFAs impact human cells through two main mechanisms: inhibition of histone and lysine deacetylases (K/HDAC) and activation of specific G-protein coupled receptors (GPR41, GPR109a: both Gα/i coupled receptors and GPR43: Gα/i and Gαq coupled receptor) (Hinnebusch et al., 2002; Tolhurst et al., 2012; Schilderink et al., 2013). We confirmed that the three G-protein coupled receptors are expressed in HT-29 and Caco-2 cells (Supplementary Figure 5A-B). To test the potential role of these receptors, we first used
342 selective agonists of GPR41 (1-MCPC and AR420626), GPR43 (Tiglic acid and 4-CMTB)
343 and GPR109a (Niacin and MK1903). If the butyrate-driven down-regulation of *IDO-1*
344 expression were mediated by the GPR-dependent signalling pathways, we should expect that
345 activation of these receptors would inhibit *IDO-1* expression. Interestingly, none of these
346 agonists, alone or in combination, impacted *IDO-1* expression (Figure 6A and Supplementary
347 Figure 5C). To further confirm this observation, we used inhibitors of the Gαi and the Gαq
348 pathways: the pertussis toxin (Ptx) and phospholipase Cβ inhibitor (U73122) respectively. As
349 shown in Figure 6B, none of these inhibitors impacted on the butyrate-dependent *IDO-1*
350 down-regulation. Moreover, over-expression of GPR43 and GPR109a in HT-29 did not
351 impact the butyrate-dependent inhibition of *IDO-1* expression (Supplementary Figure 6).
352 Altogether these results suggest that the SCFAs receptors GPR41, GPR43 and GPR109a were
353 not involved in the observed butyrate-driven inhibition of *IDO-1* expression.

354 **Butyrate down-regulates IDO-1 expression via its HDAC inhibitory property in a AP-1,**
355 **PPARγ and SP1-independent manner**

356 SCFAs, and butyrate in particular, are potent modulators of protein acetylation targeting
357 histones and transcription factors. Indeed, SCFAs impact human cells through their ability to
358 inhibit lysine and histone deacetylases (HDAC) and are thus considered as members of the
359 HDAC inhibitor (HDACi) family (Schilderink et al., 2013; Koh et al., 2016). As part of the
360 aliphatic family of HDACi, butyrate targets HDAC class I (HDAC 1, 2, 3, 8) and IIa (HDAC
361 4, 5, 7, 9) (Gallinari et al., 2007). To assess if butyrate impacts *IDO-1* expression through its
362 HDACi property, we tested three HDACi targeting a wide range of HDAC. Two belonging to
363 the hydroxamic acids family, structurally and metabolically unrelated to SCFAs: trichostatin
364 A (TSA), Vorinostat (SAHA) and one belonging to the fatty acid family: sodium valproate
365 (VAP) (Gallinari et al., 2007). The effect of butyrate on *IDO-1* expression was mimicked by
366 the three HDACi tested suggesting that the *IDO-1* down-regulation observed with butyrate
367 might be a consequence of its HDAC inhibitory properties (Figure 7A).

368 Regulation of gene transcription by butyrate involved a wide range of transcription
369 factors. To delineate whether transcription factors targeted by butyrate could impact *IDO-1*
370 expression, we analysed the human *IDO-1* promoter sequence. Analysis revealed binding
371 sites for several transcription factors implicated in butyrate-regulated gene expression, namely
372 Specificity Protein-1 (SP1) binding GC-rich boxes, as well as AP1 and PPARγ responsive
373 elements (Supplementary Table 2) (Nakano et al., 1997; Davie, 2003; Nepelska et al., 2012;
374 Alex et al., 2013). To delineate if butyrate affects *IDO-1* expression *via* SP1, we treated
375 stimulated cells with mithramycin A that binds to GC-rich DNA sequences, thereby inhibiting
376 SP1-dependent gene modulation (Blume et al., 1991). As shown in Figure 7A, incubation of
377 butyrate or HDACi-stimulated cells with mithramycin did not impact on the *IDO-1* down-
378 regulation, suggesting that SP1 was not involved in this process. As butyrate is a major
379 activator of PPARγ-dependent gene activation, we also investigated its role in *IDO-1* down-
380 regulation (Alex et al., 2013). Two specific PPARγ activators, pioglitazone and rosiglitazone,
381 did not affect *IDO-1* expression, suggesting that the PPARγ responsive elements in *IDO-1*
382 promoter might not be functional (Figure 7B). We further tested whether PPARγ was
383 involved in the butyrate-dependent inhibition of *IDO-1* by using a specific PPARγ inhibitor
384 (GW9662). The PPARγ inhibitor GW9662 did not impact on the butyrate-induced *IDO-1*
385 down-regulation, confirming that the transcription factor PPARγ was not involved in this
386 process (Figure 7B). Finally, the implication of AP1 motifs, present in *IDO-1* promoter was
387 tested using an AP1 chemical inhibitor (SR11302). Pre-treatment with AP1 inhibitor did not
388 significantly prevent the inhibition of *IDO-1* mediated by butyrate, suggesting that AP1 was
389 not involved either (Figure 7C). Altogether, our findings suggest that butyrate down-regulates
390 *IDO-1*-expression by a second mechanism involving its iHDAC property, independently of

391 the butyrate-targeted transcription factors AP1, PPARγ and SP1.

392

393

In review
394

Discussion

395

The immune system is traditionally viewed as a highly elaborated defence system developed to fight intruders, especially rapidly evolving pathogens such as bacteria. However, accumulating studies highlight a widespread cooperation established between hosts and bacteria during millions of years that have shaped their own development (Smith et al., 2007).

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Intestinal commensal bacteria are crucial for the development and maintenance of a healthy immune system locally and have a homeostatic role beyond the gut, therefore contributing to the global wellbeing of their host. The particular abundance and combination of commensal bacteria may have dramatic and specific impacts on the host immune system through their intimate interaction with the host epithelium. Accordingly, the intestinal epithelial cells (IEC) play a central role in the dialogue established between the host and the microbiota by providing an active physical segregation of commensal bacteria and by initiating the first microbial-dependent signals. Indeed, IECs express receptors recognising microbial motifs that activate downstream signalling cascades thus promoting the production of bactericidal peptides and the recruitment and activation of innate and adaptive immune cells notably by the production of effector proteins and enzymes (Atarashi et al., 2011; Kagnoff, 2014).

Amongst the effector molecules modulating the immune responses produced by IECs, indoleamine 2,3-dioxygenase-1 (IDO-1) has an important role in the gut homeostasis (Cherayil, 2009; Ciorba, 2013). However, whether human IECs express IDO-1 and how bacteria control IDO-1 expression in IECs is still unclear. Here, we show that human normal colonic IECs express IDO-1 at the mRNA and protein level and that epithelial IDO-1 is modulated by short chain fatty acids (SCFAs), more specifically by butyrate. Indeed, we demonstrate herein that physiological concentrations of butyrate down-regulate IDO-1 expression in HT-29 and Caco-2 reporter systems, but also at the mRNAs level in both the HT-29 cell line and in human primary colonic epithelial cells (Cummings et al., 1987).
In the context of IFNγ stimulation, STAT1 is an essential mediator of IDO-1 expression (Chon et al., 1996). Our results indicate that butyrate-treated IECs showed reduced STAT1 phosphorylation on the tyrosine 701, as described in other models (Jiang et al., 2010; He et al., 2013). However, our results indicate that the reduced amount of phosphorylated STAT1 observed with butyrate is a consequence of a butyrate-driven STAT1 protein level reduction. STAT1 diminution was not a result of an increase of nuclear translocation and we did not observe any transcriptional inhibition of STAT1 expression, suggesting a post-transcriptional modification of STAT1. Many post-translational modifications of STAT1 such as SUMOylation and ubiquitination have been identified leading to STAT1 degradation and consequently modifying STAT1 protein levels in cells (Tanaka et al., 2005; Yuan et al., 2012; Maarifi et al., 2015). Interestingly, butyrate has been described as a global enhancer of protein ubiquitination (Jiang et al., 2010). We thus believe that combination of post-translational modifications of STAT1 might occur explaining its down-regulation by butyrate. The precise mechanism, and cellular actor, notably the implication of HDAC inhibition or GPRs implicated in STAT1 down-regulation need to be investigated further.

In addition to the butyrate-dependent down-regulation of STAT1 that impaired IFNγ-induced IDO-1 expression, we demonstrated that STAT1 is dispensable for the basal IDO-1 repression induced by butyrate suggesting that this SCFA repressed IDO-1 expression by a second distinct mechanism. To decipher this STAT1-independent mechanism, we investigated the implication of butyrate specific G-protein coupled receptors (GPR41, GPR109a and GPR43). However, by using agonists of these receptors and G protein subunit inhibitors, we showed that this mechanism was not implicated in the inhibition of IDO-1 mediated by butyrate. SCFAs impact the host biological responses by the direct regulation of gene transcription by their properties of lysine deacetylase inhibitors that consequently favour acetylation of histones and transcription factors (Hinnebusch et al., 2002; Schilderink et al.,
2013). We showed that three HDAC inhibitors targeting a wide range of HDAC mimicked the effect of butyrate on *IDO-1* expression in un-stimulated cells suggesting that the *IDO-1* down-regulation observed was likely linked to the HDAC inhibitory properties of SCFAs. As regulation of gene transcription by HDACi involved many transcription factors, we reported, by analysing the sequence of the *IDO-1* promoter, the presence of responsive elements of three transcription factors potentially targeted by butyrate: SP1, AP1 and PPARγ (Nakano et al., 1997; Davie, 2003; Nepelska et al., 2012; Alex et al., 2013). However, by using specific inhibitors and agonists, we demonstrated that these three transcription factors were not involved in the STAT1-independent butyrate-driven inhibition of *IDO-1* expression.

Despite being limited to human cell-lines and primary IECs, our results highlighted a role of butyrate in *IDO-1* expression. However *in vivo* studies are required to confirm these in vitro results and to precise the downstream effects of modulation of *IDO-1* in the colon. What would be the impact of *IDO-1* inhibition on human health is still an open question, as, depending on the disease context, its expression has positive or negative outcomes (Fallarino et al., 2012; Metghalchi et al., 2015; Laurans et al., 2018; Metghalchi et al., 2018). IDO-1 is highly expressed in human tumour cells and consequently creates an immunosuppressive microenvironment that has been associated with poor prognosis notably in colorectal cancer (Cherayil, 2009; Ciorba, 2013). IDO-1 expression is high in inflammatory bowel diseases notably in IECs and has often been positively associated with the severity of gastrointestinal diseases and inflammatory-induced colon tumorigenesis, with no causal implication (Barcelo-Batllori et al., 2002; Ferdinande et al., 2008; Cherayil, 2009; Ciorba, 2013). However IDO-1<sup>-/-</sup> mice do not present any spontaneous colitis and its role in induced colitis models varies between studies according to the inducing agent and mouse strain used and probably the microbiota composition (Gurtner et al., 2003; Ciorba et al., 2010; Takamatsu et al., 2013; Shon et al., 2015). IDO-1 regulates immune responses *via* the so-called “metabolic immune
regulation” that suppresses the Th1 and Th17 differentiation and enhances the *de novo* differentiation of anti-inflammatory regulatory T cells (Fallarino et al., 2012). A recent study suggests that the role of IDO-1 in the regulation of the immune response is more complex as it repressed the production of IL10, a major anti-inflammatory cytokine (Metghalchi et al., 2015). In line with this, recent studies suggest that IDO-1 expression have a detrimental role in aneurysm, atherosclerosis and obesity (Metghalchi et al., 2015; Laurans et al., 2018; Metghalchi et al., 2018). Moreover, Laurans *et al.* demonstrate that IDO-1 activity enhanced chronic inflammation and intestinal permeability that consequently impacts on obesity outcomes (Laurans et al., 2018). In addition, IDO-1 has been described as a main regulator of the intestinal B cell responses to commensal bacteria that drives microbiota composition and indirectly the microbiota-dependent barrier responses (Harrington et al., 2008; Zelante et al., 2013). These studies demonstrate that intestinal IDO-1 expression might also shape gut microbiota with potent impact on host health. Altogether, these studies suggest that the role of IDO-1 in influencing gut inflammation is far more complex than expected, and might depend on the cell types expressing it. *IDO-1* down-regulation by microbiota-derived butyrate in IECs, as demonstrated here, could be crucial for the fine-tuning of *IDO-1* expression in healthy conditions and for the initiation of appropriate immune responses depending on the context: chronic inflammation, cancer, obesity or infections.

Here, we describe an important role for the SCFA butyrate in the regulation of *IDO-1* expression in IECs. Contrary to DCs where IDO-1 functions in diverse processes in health and disease have been well documented, its role in IECs is still debated. We demonstrated here for the first time that butyrate represses *IDO-1* expression by two distinct mechanisms. First, butyrate treatment was able to reduce STAT1-dependent induction of *IDO-1*. In addition, we show that this reduction is correlated with the butyrate-driven decrease in STAT1 level. Second, butyrate regulation of *IDO-1* expression is independent of the IFNγ-
494 signalling pathway and involves the HDAC inhibitory property of butyrate. As SCFAs are
495 crucial for human physiology and health, our results strongly suggest that controlling IDO-1
496 expression in IECs under steady state conditions can be part of the global mechanism of
497 SCFAs to maintain immune homeostasis in the gut.

In review
498 Acknowledgements

The authors are grateful to Drs Véronique Douard (INRA UMR1319) and Marion Espeli (INSERM UMR-S996) for helpful discussions and critical comments on the manuscript. The authors are grateful to members of the team for helpful discussions. The authors are grateful to Agnès David (INRA, UMR 1280 PHAN, Nantes, France) for SCFA analysis. This work was supported by the Institut National de la Recherche Agronomique (INRA), by grants funded by EU-FP7 METACARDIS (HEALTH-F4-2012-305312), by the ANR FunMetagen (ANR-11-BSV6-0013). CMG was the recipient of a fellowship from the Ministère de la Recherche et de l'Éducation Nationale (UPMC-Sorbonne University). The Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre. Work in Cambridge was funded by Wellcome (106262/Z/14/Z, 106 263/Z/14/Z) and the MRC (MRC MC UU 12012/3).

510

511 Authors Contribution

Conceived and designed the experiments: CMG, NL; performed most of the experiments: CMG; performed some experiments: PL, AJ, FBC, LM, FL, NL; analysed the data: CMG, AJ, NL; contributed materials: FR; wrote the paper: CMG, NL; edited and revised the manuscript: AJ, PL, FR, HMB.

516

517 Disclosures:

The authors disclose no conflict of interest.

519

520
521 References

522 Alex, S., Lange, K., Amolo, T., Grinstead, J.S., Haakonsson, A.K., Szalowska, E., et al.
523 (2013). Short-chain fatty acids stimulate angiopoietin-like 4 synthesis in human colon
524 adenocarcinoma cells by activating peroxisome proliferator-activated receptor gamma.
525 *Mol Cell Biol* 33(7), 1303-1316. doi: 10.1128/MCB.00858-12.

526 Atarashi, K., Tanoue, T., Ando, M., Kamada, N., Nagano, Y., Narushima, S., et al. (2015).
527 Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells. *Cell*
528 163(2), 367-380. doi: 10.1016/j.cell.2015.08.058.

529 Atarashi, K., Tanoue, T., Shima, T., Imaoka, A., Kuwahara, T., Momose, Y., et al. (2011).
530 Induction of colonic regulatory T cells by indigenous Clostridium species. *Science*
531 331(6015), 337-341.

532 Barcelo-Batllori, S., Andre, M., Servis, C., Levy, N., Takikawa, O., Michetti, P., et al. (2002).
533 Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells:
534 implications for inflammatory bowel diseases. *Proteomics* 2(5), 551-560. doi:
535 10.1002/1615-9861(200205)2:5<551::AID-PROT551>3.0.CO;2-O.

536 Blacher, E., Levy, M., Tatirovsky, E., and Elinav, E. (2017). Microbiome-Modulated
537 Metabolites at the Interface of Host Immunity. *J Immunol* 198(2), 572-580. doi:
538 10.4049/jimmunol.1601247.

539 Blume, S.W., Snyder, R.C., Ray, R., Thomas, S., Koller, C.A., and Miller, D.M. (1991).
540 Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of
541 the dihydrofolate reductase gene in vitro and in vivo. *J Clin Invest* 88(5), 1613-1621.
542 doi: 10.1172/JCI115474.

543 Bourriaud, C., Robins, R.J., Martin, L., Kozlowski, F., Tenailleau, E., Cherbut, C., et al.
544 (2005). Lactate is mainly fermented to butyrate by human intestinal microfloras but
545 inter-individual variation is evident. *J Appl Microbiol* 99(1), 201-212. doi:
546 10.1111/j.1365-2672.2005.02605.x.

547 Cherayil, B.J. (2009). Indoleamine 2,3-dioxygenase in intestinal immunity and inflammation.
548 *Inflamm Bowel Dis* 15(9), 1391-1396. doi: 10.1002/ibd.20910.

549 Chon, S.Y., Hassanain, H.H., and Gupta, S.L. (1996). Cooperative role of interferon
550 regulatory factor 1 and p91 (STAT1) response elements in interferon-gamma-
551 inducible expression of human indoleamine 2,3-dioxygenase gene. *J Biol Chem*
552 271(29), 17247-17252.

553 Ciorba, M.A. (2013). Indoleamine 2,3 dioxygenase in intestinal disease. *Curr Opin
554 Gastroenterol* 29(2), 146-152. doi: 10.1097/MOG.0b013e32835c9cb3.

555 Ciorba, M.A., Bettonville, E.E., McDonald, K.G., Metz, R., Prendergast, G.C., Newberry,
556 R.D., et al. (2010). Induction of IDO-1 by immunostimulatory DNA limits severity of
557 experimental colitis. *J Immunol* 184(7), 3907-3916. doi: 10.4049/jimmunol.0900291.

558 Cohen, L.J., Kang, H.S., Chu, J., Huang, Y.H., Gordon, E.A., Reddy, B.V., et al. (2015).
559 Functional metagenomic discovery of bacterial effectors in the human microbiome
560 and isolation of commendamide, a GPCR G2A/132 agonist. *Proc Natl Acad Sci U S A*
561 112(35), E4825-4834. doi: 10.1073/pnas.1508737112.

562 Cummings, J.H., Pomare, E.W., Branch, W.J., Naylor, C.P., and Macfarlane, G.T. (1987).
563 Short chain fatty acids in human large intestine, portal, hepatic and venous blood. *Gut*
564 28(10), 1221-1227.

565 Davie, J.R. (2003). Inhibition of histone deacetylase activity by butyrate. *J Nutr* 133(7 Suppl),
566 2485S-2493S.

567 Fallarino, F., Grohmann, U., and Puccetti, P. (2012). Indoleamine 2,3-dioxygenase: from
catalyst to signaling function. *Eur J Immunol* 42(8), 1932-1937. doi:
568 10.1002/eji.201242572.
569
570 Fellows, R., Denizot, J., Stellato, C., Cuomo, A., Jain, P., Stoyanova, E., et al. (2018).
571 Microbiota derived short chain fatty acids promote histone crotonylation in the colon
572 through histone deacetylases. *Nat Commun* 9(1), 105. doi: 10.1038/s41467-017-02651-5.

574 Ferdinande, L., Demetter, P., Perez-Novo, C., Waeytens, A., Taildeman, J., Rottiers, I., et al.
575 (2008). Inflamed intestinal mucosa features a specific epithelial expression pattern of
576 indoleamine 2,3-dioxygenase. *Int J Immunopathol Pharmacol* 21(2), 289-295. doi:
577 10.1177/039463200802100205.

578 Gaboriau-Routhiau, V., Rakotobe, S., Lecuyer, E., Mulder, I., Lan, A., Bridonneau, C., et al.
579 (2009). The key role of segmented filamentous bacteria in the coordinated maturation
580 of gut helper T cell responses. *Immunity* 31(4), 677-689. doi:
581 10.1016/j.immuni.2009.08.020.

582 Gallinari, P., Di Marco, S., Jones, P., Pallaoro, M., and Steinkuhler, C. (2007). HDACs, histone deacetylation and gene transcription: from molecular biology to cancer therapeutics. *Cell Res* 17(3), 195-211. doi: 10.1038/sj.cr.7310149.

585 Gurtner, G.J., Newberry, R.D., Schloemann, S.R., McDonald, K.G., and Stenson, W.F.
586 (2003). Inhibition of indoleamine 2,3-dioxygenase augments trinitrobenzene sulfonic
587 acid colitis in mice. *Gastroenterology* 125(6), 1762-1773.

588 Habib, A.M., Richards, P., Rogers, G.J., Reimann, F., and Gribble, F.M. (2013). Co-localisation and secretion of glucagon-like peptide 1 and peptide YY from primary cultured human L cells. *Diabetologia* 56(6), 1413-1416. doi: 10.1007/s00125-013-2887-z.

592 Harrington, L., Srikanth, C.V., Antony, R., Rhee, S.J., Mellor, A.L., Shi, H.N., et al. (2008).
593 Deficiency of indoleamine 2,3-dioxygenase enhances commensal-induced antibody
594 responses and protects against Citrobacter rodentium-induced colitis. *Infect Immun*
595 76(7), 3045-3053. doi: 10.1128/IAI.00193-08.

596 He, Y.W., Wang, H.S., Zeng, J., Fang, X., Chen, H.Y., Du, J., et al. (2013). Sodium butyrate
597 inhibits interferon-gamma induced indoleamine 2,3-dioxygenase expression via
598 STAT1 in nasopharyngeal carcinoma cells. *Life Sci* 93(15), 509-515. doi:
599 10.1016/j.lfs.2013.07.028.

600 Hinnebusch, B.F., Meng, S., Wu, J.T., Archer, S.Y., and Hodin, R.A. (2002). The effects of
601 short-chain fatty acids on human colon cancer cell phenotype are associated with
602 histone hyperacetylation. *J Nutr* 132(5), 1012-1017.

603 Ivanov, II, Atarashi, K., Manel, N., Brodie, E.L., Shima, T., Karaoz, U., et al. (2009).
604 Induction of intestinal Th17 cells by segmented filamentous bacteria. *Cell* 139(3),
605 485-498.

606 Jarry, A., Bossard, C., Bou-Hanna, C., Masson, D., Espaze, E., Denis, M.G., et al. (2008).
607 Mucosal IL-10 and TGF-beta play crucial roles in preventing LPS-driven, IFN-
608 gamma-mediated epithelial damage in human colon explants. *J Clin Invest* 118(3),
609 1132-1142. doi: 10.1172/JCI32140.

610 Jiang, G.M., He, Y.W., Fang, R., Zhang, G., Zeng, J., Yi, Y.M., et al. (2010). Sodium butyrate down-regulation of indoleamine 2, 3-dioxygenase at the transcriptional and post-transcriptional levels. *Int J Biochem Cell Biol* 42(11), 1840-1846. doi: 10.1016/j.biocel.2010.07.020.

614 Kagnoff, M.F. (2014). The intestinal epithelium is an integral component of a
615 communications network. *J Clin Invest* 124(7), 2841-2843. doi: 10.1172/JCI75225.

616 Koh, A., De Vadder, F., Kovatcheva-Datchary, P., and Backhed, F. (2016). From Dietary
617 Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites. *Cell*
618 165(6), 1332-1345. doi: 10.1016/j.cell.2016.05.041.
619 Laurans, L., Venteclef, N., Haddad, Y., Chajadine, M., Alzaid, F., Metghalchi, S., et al.
620 (2018). Genetic deficiency of indoleamine 2,3-dioxygenase promotes gut microbiota-
621 mediated metabolic health. *Nat Med*. doi: 10.1038/s41591-018-0060-4.

622 Li, Q., Harden, J.L., Anderson, C.D., and Egilmez, N.K. (2016). Tolerogenic Phenotype of
623 IFN-gamma-Induced IDO+ Dendritic Cells Is Maintained via an Autocrine IDO-
624 Kynurenine/AhR-IDO Loop. *J Immunol* 197(3), 962-970. doi:
625 10.4049/jimmunol.1502615.

626 Litzenburger, U.M., Opitz, C.A., Sahm, F., Rauschenbach, K.J., Trump, S., Winter, M., et al.
627 (2014). Constitutive IDO expression in human cancer is sustained by an autocrine
628 signaling loop involving IL-6, STAT3 and the AHR. *Oncotarget* 5(4), 1038-1051. doi:
629 10.18632/oncotarget.1637.

630 Maarifi, G., Maroui, M.A., Dutrieux, J., Dianoux, L., Nisole, S., and Chelbi-Alix, M.K.
631 (2015). Small Ubiquitin-like Modifier Alters IFN Response. *J Immunol* 195(5), 2312-
632 2324. doi: 10.4049/jimmunol.1500035.

633 Martin-Gallausiaux, C., Beguet-Crespel, F., Marinelli, L., Jamet, A., Ledue, F., Blottiere,
634 H.M., et al. (2018). Butyrate produced by gut commensal bacteria activates TGF-beta1
635 expression through the transcription factor SP1 in human intestinal epithelial cells. *Sci
636 Rep* 8, 9742 doi: DOI:10.1038/s41598-018-28048-y.

637 Matteoli, G., Mazzini, E., Iliev, I.D., Mileti, E., Fallarino, F., Puccetti, P., et al. (2010). Gut
638 CD103+ dendritic cells express indoleamine 2,3-dioxygenase which influences T
639 regulatory/T effector cell balance and oral tolerance induction. *Gut* 59(5), 595-604.

640 Metghalchi, S., Ponnuswamy, P., Simon, T., Haddad, Y., Laurans, L., Clement, M., et al.
641 (2015). Indoleamine 2,3-Dioxygenase Fine-Tunes Immune Homeostasis in
642 Atherosclerosis and Colitis through Repression of Interleukin-10 Production. *Cell
643 Metab* 22(3), 460-471. doi: 10.1016/j.cmet.2015.07.004.

644 Metghalchi, S., Vandestienne, M., Haddad, Y., Esposito, B., Dairou, J., Tedgui, A., et al.
645 (2018). Indoleamine 2 3-dioxygenase knockout limits angiotensin II-induced
646 aneurysm in low density lipoprotein receptor-deficient mice fed with high fat diet.
647 *PLoS One* 13(3), e0193737. doi: 10.1371/journal.pone.0193737.

648 Munn, D.H., and Mellor, A.L. (2007). Indoleamine 2,3-dioxygenase and tumor-induced
tolerance. *J Clin Invest* 117(5), 1147-1154.

650 Nakano, K., Mizuno, T., Sowa, Y., Orita, T., Yoshino, T., Okuyama, Y., et al. (1997).
651 Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative
652 human colon cancer cell line. *J Biol Chem* 272(35), 22199-22206.

653 Nepelska, M., Cultrone, A., Beguet-Crespel, F., Le Roux, K., Dore, J., Arulampalam, V., et
654 al. (2012). Butyrate produced by commensal bacteria potentiates phorbol esters
655 induced AP-1 response in human intestinal epithelial cells. *PLoS One* 7(12), e52869.
656 doi: 10.1371/journal.pone.0052869.

657 Platten, M., Ho, P.P., Youssef, S., Fontoura, P., Garren, H., Hur, E.M., et al. (2005).
658 Treatment of autoimmune neuroinflammation with a synthetic tryptophan metabolite.
659 *Science* 310(5749), 850-855. doi: 10.1126/science.1117634.

660 Postler, T.S., and Ghosh, S. (2017). Understanding the Holobiont: How Microbial Metabolites
661 Affect Human Health and Shape the Immune System. *Cell Metab* 26(1), 110-130. doi:
662 10.1016/j.cmet.2017.05.008.

663 Qin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K.S., Manichanh, C., et al. (2010). A
664 human gut microbial gene catalogue established by metagenomic sequencing. *Nature*
665 464(7285), 59-65.

666 Rhee, S.J., Walker, W.A., and Cherayil, B.J. (2005). Developmentally regulated intestinal
667 expression of IFN-gamma and its target genes and the age-specific response to enteric
668 Salmonella infection. *J Immunol* 175(2), 1127-1136.
669 Schilderink, R., Verseijden, C., and de Jonge, W.J. (2013). Dietary inhibitors of histone deacetylases in intestinal immunity and homeostasis. *Front Immunol* 4, 226. doi: 10.3389/fimmu.2013.00226.

672 Sekirov, I., Russell, S.L., Antunes, L.C., and Finlay, B.B. (2010). Gut microbiota in health and disease. *Physiol Rev* 90(3), 859-904. doi: 10.1152/physrev.00045.2009.

674 Shon, W.J., Lee, Y.K., Shin, J.H., Choi, E.Y., and Shin, D.M. (2015). Severity of DSS-induced colitis is reduced in Ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks. *Sci Rep* 5, 17305. doi: 10.1038/srep17305.

677 Smith, K., McCoy, K.D., and Macpherson, A.J. (2007). Use of axenic animals in studying the adaptation of mammals to their commensal intestinal microbiota. *Semin Immunol* 19(2), 59-69. doi: 10.1016/j.smim.2006.10.002.

680 Takamatsu, M., Hirata, A., Ohtaki, H., Hoshi, M., Hatano, Y., Tomita, H., et al. (2013). IDO1 plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate-induced colitis in mice. *J Immunol* 191(6), 3057-3064. doi: 10.4049/jimmunol.1203306.

683 Tanaka, T., Soriano, M.A., and Grusby, M.J. (2005). SLIM is a nuclear ubiquitin E3 ligase that negatively regulates STAT signaling. *Immunity* 22(6), 729-736. doi: 10.1016/j.immuni.2005.04.008.

686 Tolhurst, G., Heffron, H., Lam, Y.S., Parker, H.E., Habib, A.M., Diakogiannaki, E., et al. (2012). Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2. *Diabetes* 61(2), 364-371. doi: 10.2337/db11-1019.

689 Vital, M., Howe, A.C., and Tiedje, J.M. (2014). Revealing the bacterial butyrate synthesis pathways by analyzing (meta)genomic data. *MBio* 5(2), e00889. doi: 10.1128/mBio.00889-14.

692 Yu, J., Wang, Y., Yan, F., Zhang, P., Li, H., Zhao, H., et al. (2014). Noncanonical NF-kappaB activation mediates STAT3-stimulated IDO upregulation in myeloid-derived suppressor cells in breast cancer. *J Immunol* 193(5), 2574-2586. doi: 10.4049/jimmunol.1400833.

696 Yuan, C., Qi, J., Zhao, X., and Gao, C. (2012). Smurfl protein negatively regulates interferon-gamma signaling through promoting STAT1 protein ubiquitination and degradation. *J Biol Chem* 287(21), 17006-17015. doi: 10.1074/jbc.M112.341198.

699 Zelante, T., Iannitti, R.G., Cunha, C., De Luca, A., Giovannini, G., Pieraccini, G., et al. (2013). Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. *Immunity* 39(2), 372-385. doi: 10.1016/j.immuni.2013.08.003.

703 Zhou, L., Chen, H., Wen, Q., and Zhang, Y. (2012). Indoleamine 2,3-dioxygenase expression in human inflammatory bowel disease. *Eur J Gastroenterol Hepatol* 24(6), 695-701. doi: 10.1097/MEG.0b013e328351c1c2.

707
708 **Figure legends**

709 **Figure 1:**

710 IDO-1 expression in human colonic epithelial cells. (A) Human normal colonic mucosa was
711 stained for IDO-1. Representative immunohistochemical staining of IDO-1 showed that IDO-
712 1 (brown) is expressed in epithelial cells (left panel: strong perinuclear and/or membrane
713 staining of about 80% of the IECs ; right panel: heterogeneous staining of few IECs (arrows))
714 and in few *lamina propria* mononuclear cells (arrowheads) and endothelial cells (asterisk)
715 (original magnification x200). (B). *IDO-1* gene expression was determined by RT-PCR on
716 RNA extracted from preparations of isolated human colonic epithelial cells (IECs) and of
717 whole mucosa microdissected from normal colon. Results were normalised to β-2
718 microglobulin (B2M) and expressed as 2-ΔCt relative value (median ± quartiles) of 4 patients
719 (1-2 samples/patient).

720 **Figure 2:**

721 Correlation between bacterial metabolites production and IDO-1 gene expression. (A) Effect
722 of bacterial supernatants on IDO-1 reporter system organised by phylum. Culture supernatants
723 of a wide range of cultivable commensal bacteria were applied on the HT-29-IDO-1 reporter
724 system (10% vol/vol) for 24h. IDO-1 expression was measured by luciferase activity and
725 expressed as fold increase towards its control: non inoculated growth medium used for each
726 culture. IDO-1 expression profiles upper and lower the dash lines were considered as
727 significantly changed. (B) PCA analysis showing the correlation between the SCFAs
728 concentrations produced by the commensal bacteria and IDO-1 expression. (C).
729 Representation of IDO-1 expression correlated to butyrate concentration in bacterial cultures
classified by rank value. Actinobacteria in blue, Bacteroidetes in yellow, Firmicutes in grey, Fusobacteria in red and Verrucomicrobia in light blue.

**Figure 3:**

Impact of SCFAs on *IDO-1* expression. (A), HT-29-*IDO-1* reporter cells were incubated with a range of concentration of acetate, butyrate and propionate (0.5; 1; 2; 4; 8 mM) for 24h. *IDO-1* expression was measured by luciferase activity and expressed as the mean ± SD fold change towards un-stimulated cells (N>3). (B), HT-29-*IDO-1* reporter cells were incubated with IFNγ (100U/ml) and a range of concentration of butyrate (0.5-8mM). *IDO-1* expression was measured by luciferase activity and expressed as the median ± quartiles of fold change towards un-stimulated cells (N>3). (C) *IDO-1* gene expression on HT-29 exposed for 6h to IFNγ (100U/ml) +/- butyrate (2mM), propionate (4mM) or acetate (8mM) was determined by RT-PCR. Results were normalised to *GAPDH* and expressed as 2-ΔΔCt relative to control mean value; ND: not detected (N=3). (D) Caco2-*IDO-1* reporter cells were incubated with a range of concentration of acetate, propionate and butyrate (0.5; 1; 2; 4; 8 mM). *IDO-1* expression was measured by luciferase activity and expressed as the mean ± SD fold change towards un-stimulated cells (N>3). (E) *IDO-1* expression level on human colonic epithelial cells treated for 24h with butyrate compared to non-treated cells from the same patient was determined by RT-PCR. Results are normalised to *RPS17* and expressed as 2-ΔΔCt relative to control, median ± quartiles (N=4). P value: *P*<0.05, **P*<0.005, ***P*<0.001.

**Figure 4:**

Inhibition of IFNγ-induced *IDO-1* expression by butyrate is correlated with a decrease of STAT1 protein level. (A-C) HT-29 cells were cultured 24h with butyrate (But 2mM) prior IFNγ (100U/ml) stimulation for 15 (line 3 with butyrate and 5 without butyrate) or 30 min
(line 4 with butyrate and 6 without butyrate). The protein level of p-STAT1 Tyr701, STAT1 and Actin were determined by western-blot on total protein extracted. Densitometric quantifications of total P-STAT1 and STAT1 proteins, from 3 independent experiments, were normalised to Actin and expressed as fold change compared to IFN stimulated cells (B) and unstimulated cell (C) respectively of 3 independent experiments. Data are represented as median ± quartiles. (D) HT-29 cells were incubated 24h with medium or butyrate (But 2mM) prior cytoplasmic and nuclear extractions. The protein levels of STAT1, Laminin A/C and GAPDH were assessed in each fraction by western-blot. P value: *P<0.05, **P<0.005, ***P<0.001.

<caption>762 **Figure 5:**</caption>

Butyrate inhibition of *IDO-1* promoter activity is STAT1 and STAT3 independent. (A) HT-29-*IDO-1* cells were transfected with STAT1 siRNA or control siRNA and incubated with butyrate (But 2mM) or IFNγ (100U/ml) for 24h before measuring IDO-1 level. (B) HT-29-*IDO-1* cells were incubated for 2h with the STAT3 phosphorylation inhibitor (Cucurbitacin I, 1μM) prior to butyrate (But 2mM) treatment for total incubation time of 24h (N=4). (C) HT-29-*IDO-1* cells were incubated with AHR ligand (TCDD 10nM) +/- butyrate (But 2mM) for 24h. Data represented 2 independent experiments (D) HT-29-*IDO-1* cells were incubated for 1h with the NκFB inhibitor, Bay117082 (Bay 40μM) prior stimulation with butyrate (But 2mM) or TNFα (10ng/ml) for 24h (N=3). *IDO-1* expression was measured by luciferase activity and expressed as median ± quartiles of fold change towards unstimulated cells. Data represented at least 3 independent experiments. P value: *P<0.05, **P<0.005, ***P<0.001.
<caption>774 **Figure 6:**</caption>

775 Butyrate mediated impact on IDO-1 is independent of its receptors GPR41, GPR43 and
776 GPR109a. (A) HT-29-IDO-1 reporter cells were incubated for 24h with selective GPR
777 agonists: GPR41: AR420626 (1µM) and 1-MCPC (1mM); GPR43: 4-CMTB (1µM) and
778 Tiglic acid (1mM); GPR109a: Niacin (1mM) and MK1903 (1µM) or with DMSO (vehicle),
779 butyrate (But 2mM) or Control (RPMI). (B) HT-29-IDO-1 reporter cells were incubated for
780 24h with 2mM butyrate +/- GPRs sub-unit inhibitors: Pertussis toxin (Ptx, 0.2µg/ml), U73122
781 (10µM) or glycerol (vehicle). IDO-1 expression was measured by luciferase activity and
782 expressed as median ± quartiles of fold change towards un-stimulated cells. Data represented
783 at least 3 independent experiments. P value: *P<0.05, **P<0.005, ***P<0.001.

<caption>784 **Figure 7:**</caption>

785 HDAC inhibitor mimicked the butyrate-dependent down-regulation of IDO-1 expression in a
786 SP1, PPARγ and AP-1 independent manner. (A). HT-29-IDO-1 reporter cells were incubated
787 for 24h with butyrate (But 2mM), SAHA (5µM), Trichostatin A (TSA 1µM) or Valproic acid
788 (VAP 5mM) ± SP1 inhibitor (Mitramycin A; MitA 0.1µM). (B), HT-29-IDO-1 reporter cells
789 were stimulated for 24h with two PPARγ activators: Pioglitazone (Pio 5µM); Rosiglitazone
790 (Rosi, 10µM) or the specific PPARγ inhibitor GW9662 (10µM) ± butyrate (But 2mM). (C)
791 HT-29-IDO-1 reporter cells were incubated for 24h with butyrate (But 2mM) and/or the AP1
792 inhibitor, SR11302 (10µM). IDO-1 expression was measured by luciferase activity and
793 expressed as median ± quartiles of fold change towards un-stimulated cells. Data represented
794 at least 3 independent experiments. P value: *P<0.05, **P<0.005, ***P<0.001.
A

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