Bruno Lamas

Leticia Hernandez-Galan

Heather J Galipeau

Marco Constante

Alexandra Clarizio

Jennifer Jury

Natalia M Breyner

Alberto Caminero

Gaston Rueda

Christina L Hayes

Justin L Mccarville

Miriam Bermudez Brito

Julien Planchais

Nathalie Rolhion

Joseph A Murray

Philippe Langella

Linda M P Loonen

Jerry M Wells

Premysl Bercik

MD Harry Sokol
email: harry.sokol@aphp.fr

MD Elena F Verdu
email: verdue@mcmaster.ca

Aryl hydrocarbon receptor ligand production by the gut microbiota: a new therapeutic target in celiac disease

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Celiac disease (CeD) is a chronic inflammatory enteropathy that develops as a consequence of breakdown of oral tolerance to gluten in people who carry HLA-DQ2 or -DQ8 genes [START_REF] Liu | Risk of pediatric celiac disease according to HLA haplotype and country[END_REF]. Although 40% of the worldwide population expresses one or more CeD susceptibility genes, only 1% will develop the disease [START_REF] Verdu | Novel players in coeliac disease pathogenesis: role of the gut microbiota[END_REF]. It is currently accepted that additional environmental factors are needed to trigger the inflammatory response that will culminate in villous atrophy, and these may include microbial factors and non-gluten proteins [START_REF] Galipeau | Intestinal microbiota modulates gluten-induced immunopathology in humanized mice[END_REF][START_REF] Bouziat | Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease[END_REF].

The gut microbiota fosters intestinal homeostasis by modulating host immunity and physiology [START_REF] Blacher | Microbiome-Modulated Metabolites at the Interface of Host Immunity[END_REF][START_REF] Schirmer | Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity[END_REF][START_REF] Kamada | Role of the gut microbiota in immunity and inflammatory disease[END_REF]. Disruption of microbiota composition and function, which are influenced by a variety of factors including lifestyle choices and environment, has been suggested as a key contributor to the worldwide epidemics of chronic illness, including CeD [START_REF] Verdu | Novel players in coeliac disease pathogenesis: role of the gut microbiota[END_REF][START_REF] Galipeau | Intestinal microbiota modulates gluten-induced immunopathology in humanized mice[END_REF][START_REF] Kamada | Role of the gut microbiota in immunity and inflammatory disease[END_REF][START_REF] Caminero | Mechanisms by which gut microorganisms influence food sensitivities[END_REF]. Indeed, recent studies in mice have demonstrated mechanisms through which bacterial opportunistic pathogens from CeD patients and enteric viral infections directly or indirectly influence host immune responses to gluten [START_REF] Bouziat | Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease[END_REF][START_REF] Caminero | Duodenal Bacteria From Patients With Celiac Disease and Healthy Subjects Distinctly Affect Gluten Breakdown and Immunogenicity[END_REF][START_REF] Caminero | Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2[END_REF]. However, only some CeD patients have a clinical history of enteric infections, suggesting other mechanisms linking microbial cues and immunological pathways may be relevant.

By-products of microbial metabolism in the gut, such as those produced through metabolism of tryptophan (Trp) from dietary sources, are emerging as key players in hostmicrobiota cross-talk [START_REF] Nicholson | Hostgut microbiota metabolic interactions[END_REF][START_REF] Levy | Metabolites: messengers between the microbiota and the immune system[END_REF][START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF][START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF]. In the gastrointestinal tract, Trp can be metabolized by the microbiota into ligands of the aryl hydrocarbon receptor (AhR) [START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF][START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF][START_REF] Koper | Polyphenols and Tryptophan Metabolites Activate the Aryl Hydrocarbon Receptor in an in vitro Model of Colonic Fermentation[END_REF][START_REF] Roager | Microbial tryptophan catabolites in health and disease[END_REF] as well as by host cells into kynurenine via indoleamine 2,3-dioxygenase 1 (IDO 1) [START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF][START_REF] Clarke | The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner[END_REF] or into 5hydroxytryptamine via Trp hydroxylase 1 [START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF][START_REF] Yano | Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis[END_REF]. AhR is a ligand-activated nuclear transcription factor widely expressed in the body and retained in an inactive complex in the cytoplasm that after ligand binding translocates to the nucleus regulating the expression of target genes like Cyp1a1, Il22 and Il17 [START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF][START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF]. In this manner, AhR signaling orchestrates immune responses at barrier sites such as the gut mucosa, modulating intraepithelial lymphocytes (IEL), T cells and innate lymphoid cells (ILCs) group 3, that produce IL-17 and IL-22 [START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF], all of which may impact CeD pathogenesis. A recent study showed that AhR expression is down-regulated in the mucosa of active CeD patients, however the relationship with the gut microbiota and mechanisms of AhR regulation, remain unknown [START_REF] Dinallo | Protective Effects of Aryl Hydrocarbon Receptor Signaling in Celiac Disease Mucosa and in Poly I:C-Induced Small Intestinal Atrophy Mouse Model[END_REF]. Indeed, differences in composition and metabolic function of the gut microbiota between CeD patients and healthy controls have been described, including decreases in core groups known to metabolize the Trp in AhR ligands such as Lactobacillus [START_REF] Nadal | Imbalance in the composition of the duodenal microbiota of children with coeliac disease[END_REF][START_REF] Wacklin | Altered duodenal microbiota composition in celiac 28 disease patients suffering from persistent symptoms on a long-term gluten-free diet[END_REF][START_REF] D'argenio | Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients[END_REF]. This prompted us to investigate the general hypothesis that gut microbiota in active CeD has impaired capacity to stimulate the AhR pathway.

We demonstrate that an enriched Trp diet shapes the intestinal microbiota to produce AhR ligands that reduce gluten immunopathology in genetically susceptible mice. These effects were mediated by AhR activation and observed after treatment with either Lactobacillus reuteri with high capacity to produce AhR ligands [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF] or, pharmacologically, using an AhR agonist.

Finally, patients with active CeD exhibit lower levels of AhR agonist metabolites and decreased capacity of the microbiota to activate AhR compared with controls. The results open a previously unknown therapeutic target in CeD related to the modulation of the gut microbiota to enhance production of Trp metabolites that activate AhR.

Results

Enriched tryptophan diet shapes the microbiota to produce AhR ligands

We first investigated the capacity of enriched Trp diet to shape the microbiota after 3 weeks of dietary intervention in NOD/DQ8 mice before gluten treatment. Mice were fed a diet containing either low or enriched Trp concentration (Fig. 1A) for 3 weeks and fecal microbiota composition was analyzed. The principal coordinate analysis revealed a difference in microbiota profiles between mice fed low and enriched Trp diet (Fig. 1B) but no difference in alpha diversity was observed (fig. S1A). Mice fed the enriched Trp diet had lower relative abundance of bacteria belonging to Proteobacteria phylum such as Bilophila, Desulfovibrio and Enterobacteriaceae, and higher abundance in bacteria belonging to Firmicutes phylum such as Lactobacillus, Aerococcus, Facklamia, Jeotgalicoccus and Staphylococcus (Fig. 1, C andD & fig. S1B). These data suggest that high Trp diet in a gluten-naive mouse, favors the growth of bacteria considered beneficial, such as Lactobacillus, able to metabolize Trp into AhR ligands [START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF][START_REF] Roager | Microbial tryptophan catabolites in health and disease[END_REF][START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF], at the expense of potentially pro-inflammatory bacteria belonging to Proteobacteria [START_REF] Rizzatti | Proteobacteria: A Common Factor in Human Diseases[END_REF][START_REF] Shin | Proteobacteria: microbial signature of dysbiosis in gut microbiota[END_REF]. Trp concentration in feces was higher in mice fed the enriched Trp diet (Fig. 1E). This was associated with increased concentrations of AhR agonists including, tryptamine, indole-3aldehyde and indole-3-lactic acid (Fig. 1E and fig. S1, C to E). A higher concentration of Trp and AhR agonists was also observed in the serum of mice fed the enriched Trp diet (fig. S1, F to I). In contrast, the concentration of kynurenine (Kyn), a Trp metabolite produced mainly by IDO 1 enzyme in host cells and implicated in chronic inflammation [START_REF] Prendergast | Indoleamine 2,3dioxygenase as a modifier of pathogenic inflammation in cancer and other inflammationassociated diseases[END_REF], was higher in feces of mice fed the low Trp diet (Fig. 1F). Finally, the IDO activity determined by Kyn/Trp ratio, was higher in mice fed the low Trp diet (Fig. 1F). Thus, the metabolomic profile indicates that an enriched Trp diet shapes the gut microbiota to produce AhR ligands and potentially decreases intestinal IDO 1 activity.

To investigate the functional relevance of these findings, we determined the capacity of the microbiota to activate AhR. Fecal and small intestinal contents from mice fed the enriched Trp diet induced greater activation of AhR than contents from mice fed the low Trp diet (Fig. 1G). Expression of Cyp1a1, an AhR target gene [START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF], was higher in the duodenum of mice fed the enriched Trp diet (Fig. 1H). Fecal lipocalin-2, duodenal Il6 expression, IEL counts, ion transport and paracellular permeability were lower in mice fed the enriched Trp diet, suggesting an anti-inflammatory effect of the diet (Fig. 1, I to L). Thus, the production of AhR agonists by the gut microbiota of high Trp diet-fed naïve mice activates AhR, which may promote intestinal homeostasis. S4A), but principal coordinate analysis revealed different clustering between the two groups (Fig. 2B). Differential analysis confirmed this effect with a relative increase in bacteria belonging to the Bacteroidetes and Firmicutes phyla at the expense of bacteria from the Proteobacteria and Verrucomicrobia phyla (Fig. 2, C to D). At species level, abundance of Bilophila, Desulfovibrio, Suturella and Erwinia, from the Proteobacteria phylum, was lower in the mice fed the enriched Trp diet (Fig. 2C). Among Firmicutes, Ruminococcus gnavus (Fig. 2C), known to produce AhR ligands [START_REF] Roager | Microbial tryptophan catabolites in health and disease[END_REF][START_REF] Williams | Discovery and characterization of gut microbiota decarboxylases that can produce the neurotransmitter tryptamine[END_REF], was increased in mice treated with the enriched Trp diet. In accordance, Trp and AhR agonist concentrations in feces and serum were higher in enriched Trp diet-fed mice (Fig. 2E and fig. S4, B andC). We also observed a decrease in kynurenine concentration and IDO activity in feces of enriched Trp diet-fed mice compared with low Trp diet-fed mice (Fig. 2F). Taken together these results suggest that gluten exposure was a major driver of microbiota shift, independently of Trp content in diet; however, the enriched Trp diet maintained the functional capacity to produce AhR ligands and promoted a microbiota structural profile associated with beneficial bacteria capable of AhR ligand production. The capacity of small intestine and colon content to activate AhR was higher in enriched Trp diet-fed mice compared with low Trp dietfed mice, (Fig. 2G) and this was paralleled by higher duodenal expression of Ahr, and AhR target genes such as Cyp1a1 and Il22 (Fig. 2H). The enriched Trp diet was also associated with higher villus-to-crypt (V/C) ratios, lower IEL counts, intestinal paracellular permeability, serum fecal lipocalin-2 and duodenal Il6 expression, but no differences in ion secretion (Fig. 2, I to L and fig. S4D). Collectively, these results support that an enriched Trp diet improves gluten immunopathology in NOD/DQ8 mice by inducing and maintaining an intestinal microbiota able to produce AhR agonists.

Enriched tryptophan diet sustains microbial AhR activation in gluten treated mice

Lactobacillus reuteri or pharmacological-activation of AhR reduces gluten-immunopathology in mice

To investigate microbial modulation of AhR activity in the model, two previously isolated Lactobacillus reuteri strains that naturally exhibit high AhR-ligand production [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF] (fig. S4E) were administered to gluten treated NOD/DQ8 mice fed either a low or enriched Trp diet (Fig. 3A). L. reuteri supplementation increased the capacity of the small intestinal microbiota to activate AhR in mice fed both diets, but the increase was only significant in mice fed the enriched Trp diet (Fig. 3B). L. reuteri supplementation decreased IEL counts in mice fed a low Trp diet compared with mice treated with media (Fig. 3C). L. reuteri also improved V/C ratios in mice on a low Trp diet (Fig. 3D). Taken together, these results suggest that even in the context of a low Trp diet, supplementation with L. reuteri may be sufficient to produce AhR ligands that modulate gluten immunopathology.

Because L. reuteri may have anti-inflammatory effects independent of AhR ligands, we further investigated the therapeutic potential of intestinal AhR activation, using either an AhR agonist, the 6-formylindolo (3,2-b) carbazole (Ficz) or its vehicle (Fig. 4A). After gluten treatment, mice developed higher IEL counts, lower V/C ratios, increased Il15 expression, and barrier dysfunction (Fig. 4, B to G). Compared with basal conditions (day 0), gluten-treated mice had higher intestinal expression of Ahr and AhR target genes such as Cyp1a1, Il22 and Il17, suggesting an overall state of immune activation (Fig. 4D). However, the degree of increase in Ahr and AhR target genes in gluten-treated NOD-DQ8 mice remained below those that were observed in control C67Bl/6 mice, particularly for Ahr and Il22 (fig. S5). Moreover, higher expression of these AhR target genes was observed in NOD/DQ8 mice treated with Ficz compared with those receiving vehicle, confirming activation of AhR by the agonist (Fig. 4D).

Ficz did not affect intestinal IL-15 expression (Fig. 4D), but it improved IEL counts, V/C ratios, paracellular permeability, ion transport, and other markers of overall gut inflammation (Fig. 4,B to G). Taken together, these results demonstrate that pharmacological modulation of the AhR pathway ameliorates gluten immunopathology in NOD/DQ8 mice.

Impaired AhR signaling and microbiota metabolites in patients with CeD

To study the relevance of gut microbiota-derived AhR ligands in CeD pathogenesis, we analyzed the concentrations of specific microbiota metabolites known to activate AhR in feces from patients with active CeD, patients with CeD on a gluten-free diet (GFD) for 2 years and non-celiac controls. Patients with active CeD had lower concentrations of AhR agonists including tryptamine, indole-3-aldehyde and indole-3-lactic acid (Fig. 5A and fig. S6, A to C). A higher concentration of Trp was also observed in non-celiac controls compared with patients with CeD (Fig. 5B). In contrast, xanthurenic acid and kynurenic acid, products of Trp metabolism through the kynurenine pathway, were higher in CeD patients compared with controls (Fig. 5C and fig. S6, D andE). In CeD patients treated with GFD, no difference in fecal levels of the AhR agonists tryptamine, indole-3-aldehyde and indole-3-lactic acid was observed compared with patients with active CeD (Fig. 5A and fig. S6,A to C). However, CeD patients treated with GFD had higher concentrations of indole-3-acetic acid (IAA), known to be an AhR agonist (Fig. 5D) [START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF][START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF][START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF][START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF]. The reason for decreased fecal Trp in treated CeD patients (Fig. 5B) is unclear, but could result on the one hand, from a partially restored capacity of the gut microbiota to metabolize Trp into indoles and on the other, from a residual inflammation associated with higher Trp metabolism through the kynurenine pathway (Fig 5C andfig. S6, D andE).

To investigate the functional relevance of these findings, we studied the capacity of the microbiota to activate AhR. Consistent with the impaired production of AhR ligands, fecal samples from patients with active CeD had a lower capacity to active AhR (Fig. 5E). The capacity of the microbiota to activate AhR was rescued in CeD patients treated with the GFD (Fig. 5E). Duodenal expression of Ahr and AhR pathway genes such as Cyp1a1, Il22 and Il17 was lower, while that of Il15, a key cytokine in CeD, was higher in active CeD patients compared with controls (Fig. 5F). Consistent with the increased production of the AhR ligand, IAA, by the microbiota of treated CeD patients, their expression of AhR pathway genes, such as Cyp1a1 and Il22, was increased. Taken together, the results suggest that patients with active CeD have, on one hand impaired Trp metabolism by the gut microbiota and defective AhR activation, and on the other, higher Trp metabolism by host immune cells leading to proinflammatory kynurenine metabolites [START_REF] Prendergast | Indoleamine 2,3dioxygenase as a modifier of pathogenic inflammation in cancer and other inflammationassociated diseases[END_REF][START_REF] Ciorba | Indoleamine 2,3 dioxygenase in intestinal disease[END_REF] (fig. S7). In patients with CeD, the gluten-free diet partly corrects the impaired Trp metabolism by reducing the inflammation-driven kynurenine production and increasing the microbiota-dependent AhR agonist production leading to AhR activation and notably Il22 expression, which could aid in intestinal recovery (fig. S7). These clinical data should be confirmed in a larger cohort of patients but demonstrate altered production of microbiota-derived AhR ligands in active CeD patients, suggesting a role in its pathogenesis.

Discussion

The capacity of the microbiota to produce tryptophan-based AhR ligands influences the balance between intestinal homeostasis [START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF][START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF][START_REF] Roager | Microbial tryptophan catabolites in health and disease[END_REF][START_REF] Rothhammer | The aryl hydrocarbon receptor: an environmental sensor integrating immune responses in health and disease[END_REF] and chronic inflammation, notably through impairment of IL-22-mediated anti-inflammatory mechanisms [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF]. With respect to CeD, shifts in intestinal microbial composition have been described in patients with active disease [START_REF] Nadal | Imbalance in the composition of the duodenal microbiota of children with coeliac disease[END_REF][START_REF] Wacklin | Altered duodenal microbiota composition in celiac 28 disease patients suffering from persistent symptoms on a long-term gluten-free diet[END_REF][START_REF] D'argenio | Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients[END_REF] but its link with AhR pathways, is unknown. Using a mouse model that develops moderate inflammation upon gluten sensitization and challenge, we show that a diet enriched in Trp, shifts the gut microbiota towards a higher abundance of Lactobacillus and Ruminococcus gnavus, which are known AhR ligand producers [START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF][START_REF] Roager | Microbial tryptophan catabolites in health and disease[END_REF][START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Williams | Discovery and characterization of gut microbiota decarboxylases that can produce the neurotransmitter tryptamine[END_REF]. Metabolomic and transcriptomic analysis indicates that this shift is accompanied by an increased production of AhR ligands, as well as an activation of the AhR pathway, which promotes homeostasis and ameliorates gluten immunopathology in mice expressing CeD risk genes. Experimental modulation of the pathway with either a combination of AhR ligand producers L. reuteri CNCM-I5022 and CNCM-I5429 [START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF][START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF], or pharmacological stimulation using Ficz, improves gluten immunopathology. Finally, relevance to human disease is demonstrated by the reduced amounts of specific microbiota metabolites, known to activate AhR, in fecal samples from patients with active CeD versus non-celiac controls. Some of these parameters are rescued by the gluten-free diet (GFD).

An AhR pathway role in CeD is indirectly supported by altered IL-17 and IL-22 production by gluten-specific T cells in a macaque model of CeD [START_REF] Xu | Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease[END_REF] and in biopsies of patients with CeD [START_REF] Bodd | HLA-DQ2-restricted glutenreactive T cells produce IL-21 but not IL-17 or IL-22[END_REF]. In accordance with this, we found lower expression of key genes mediating the physiological activation of AhR pathway (Cyp1a1, Il22 and Il17) in the duodenum of patients with CeD when compared with controls. Additional support comes from Ahr -/-mice which exhibit changes in TCRαβ + CD4 + and CD8 + IEL phenotypes in the colon, a subset that increases in active CeD and that responds to the gluten-free diet [START_REF] Li | Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation[END_REF]. In NOD/DQ8 mice, previously described to have a genetic impairment in natural killer cells [START_REF] Suwanai | A defective Il15 allele underlies the deficiency in natural killer cell activity in nonobese diabetic mice[END_REF], we observed an increase in CD3 + IELs during the low Trp diet, suggesting that a lower AhR activating capacity of the microbiota in the absence of dietary substrate may affect this key CeD cell subset. Finally, a recent study has shown lower AhR expression in CD4 + , CD8 + (T cells) and CD56 + (natural killer cells) cells isolated from both the intestinal lamina propria and the intraepithelial compartment of patients with active CeD [START_REF] Dinallo | Protective Effects of Aryl Hydrocarbon Receptor Signaling in Celiac Disease Mucosa and in Poly I:C-Induced Small Intestinal Atrophy Mouse Model[END_REF]. However, the drivers for these changes, remain unknown. We hypothesized that previously described shifts of the gut microbiota in active CeD [START_REF] Nadal | Imbalance in the composition of the duodenal microbiota of children with coeliac disease[END_REF][START_REF] Wacklin | Altered duodenal microbiota composition in celiac 28 disease patients suffering from persistent symptoms on a long-term gluten-free diet[END_REF][START_REF] D'argenio | Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients[END_REF] influence AhR pathways through impaired production of AhR ligands.

Lower Lactobacillus abundance, which include strains with known capacity to produce AhR ligands [START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF][START_REF] Roager | Microbial tryptophan catabolites in health and disease[END_REF][START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF], has been described in the small intestine and feces of patients with active CeD [START_REF] Nadal | Imbalance in the composition of the duodenal microbiota of children with coeliac disease[END_REF][START_REF] Wacklin | Altered duodenal microbiota composition in celiac 28 disease patients suffering from persistent symptoms on a long-term gluten-free diet[END_REF][START_REF] D'argenio | Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients[END_REF]. On the other hand, Proteobacteria, have been reported to be overrepresented in several intestinal and extra-intestinal diseases with an inflammatory phenotype [START_REF] Rizzatti | Proteobacteria: A Common Factor in Human Diseases[END_REF][START_REF] Shin | Proteobacteria: microbial signature of dysbiosis in gut microbiota[END_REF]. Our previous work showed that Proteobacteria members in duodenal content of patients with CeD, such as Pseudomonas aeruginosa, degrade gluten differently than bacteria from healthy subjects, producing peptides that stimulate gluten-specific T cells [START_REF] Caminero | Duodenal Bacteria From Patients With Celiac Disease and Healthy Subjects Distinctly Affect Gluten Breakdown and Immunogenicity[END_REF]. In this study, enriched Trp diet shifted the microbiota of naïve NOD/DQ8 mice, towards a higher abundance of Lactobacillus, and decreased Proteobacteria, leading to higher production of AhR ligands that promoted intestinal homeostasis. Gluten sensitization and challenge was a major independent driver of the microbiota shift, but the capacity of the microbiota to produce AhR ligands was preserved in mice that had received the enriched Trp diet. On the other hand, the concentration of kynurenine, a Trp metabolite produced by host cells through IDO 1 and implicated in chronic inflammatory diseases [START_REF] Prendergast | Indoleamine 2,3dioxygenase as a modifier of pathogenic inflammation in cancer and other inflammationassociated diseases[END_REF][START_REF] Ciorba | Indoleamine 2,3 dioxygenase in intestinal disease[END_REF], was increased in the fecal samples of mice fed the low Trp diet, as well as in the CeD patients [START_REF] Torres | Tryptophan metabolism and indoleamine 2,3-dioxygenase expression in coeliac disease[END_REF]. Although kynurenine is an AhR agonist when used at high concentration in vitro, its low concentration in different animal tissues and fluids makes it unlikely to be a major AhR agonist in vivo, especially compared to the indoles derivatives [START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF][START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF][START_REF] Stockinger | The aryl hydrocarbon receptor: multitasking in the immune system[END_REF]. The ability of the microbiota or host cells to metabolize Trp into AhR ligands or kynurenine respectively, depends on the availability of Trp in the gut (13-15, 24, 25). Taken together, this suggests that both impaired Trp metabolism by the gut microbiota and higher Trp metabolism by host cells can lead to defective AhR signaling and increased production of kynurenine metabolites that are associated with an inflammatory state. Based on previous studies, it is also possible that the microbiota changes induced by the enriched Trp diet impact other beneficial effects related to the microbial metabolism of immunogenic wheat proteins [START_REF] Caminero | Duodenal Bacteria From Patients With Celiac Disease and Healthy Subjects Distinctly Affect Gluten Breakdown and Immunogenicity[END_REF][START_REF] Caminero | Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2[END_REF][START_REF] Caminero | Lactobacilli Degrade Wheat Amylase Trypsin Inhibitors to Reduce Intestinal Dysfunction Induced by Immunogenic Wheat Proteins[END_REF].

To directly investigate bacterial modulation of AhR pathway in the model, we used, a combination of two L. reuteri strains with known AhR ligand producing capacity [START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF][START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF] in the context of low and enriched Trp diet. We found that L. reuteri supplementation increased the capacity of the small intestinal microbiota to activate AhR, particularly in mice fed the enriched Trp diet, indicating a synergism between the substrate and its metabolizer. The slight increase in AhR activity in mice fed a low Trp diet and L. reuteri was sufficient to improve V/C ratios and decrease IEL counts. On the other hand, L. reuteri supplementation in mice fed the enriched Trp diet did not improve further the beneficial effects of the enriched Trp diet, suggesting that the increased microbiota-derived endogenous production of AhR agonists induced by Trp supplementation is enough to reduce gluten immune-pathology and that further activating the pathway with L. reuteri does not improve the disease severity. All together, these results demonstrate that gluten immunopathology is ameliorated by dietary Trp as well as L. reuteri and suggest both independent, and likely shared pathways of these interventions. Our results agree with previous studies in colitis and metabolic syndrome showing that Lactobacillus strains capable of metabolizing Trp into AhR ligands, improve disease outcomes [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF]. Interestingly, it has recently been shown that L. reuteri is able to reprogram gut CD4 + IEL cells into immunoregulatory T cells in an AhR-dependent manner [START_REF] Cervantes-Barragan | Lactobacillus reuteri induces gut intraepithelial CD4+CD8αα+ T cells[END_REF], suggesting a possible mechanism for improvement of intestinal barrier integrity in the context of CeD. However, we cannot rule out additional beneficial effects by L. reuteri, such as barrier enhancement or wheat immunogenic peptide detoxification, as previously demonstrated for other Lactobacillus strains including L. reuteri [START_REF] Caminero | Duodenal Bacteria From Patients With Celiac Disease and Healthy Subjects Distinctly Affect Gluten Breakdown and Immunogenicity[END_REF][START_REF] Caminero | Lactobacilli Degrade Wheat Amylase Trypsin Inhibitors to Reduce Intestinal Dysfunction Induced by Immunogenic Wheat Proteins[END_REF][START_REF] Herrán | Gluten-degrading bacteria are present in the human small intestine of healthy volunteers and celiac patients[END_REF].

To further investigate the role of AhR activation in the improvement of gluten immunopathology, we pharmacologically modulated the AhR pathway using Ficz. Our results show that Ficz treatment improved intestinal permeability and inflammation in NOD/DQ8 treated with gluten. Studies have shown that intestinal AhR activation in mice by Ficz and/or a diet-derived AhR ligands, decrease development of colon cancer [START_REF] Gronke | Interleukin-22 protects intestinal stem cells against genotoxic stress[END_REF], experimental colitis [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Monteleone | Aryl hydrocarbon receptor-induced signals up-regulate IL-22 production and inhibit inflammation in the gastrointestinal tract[END_REF] and Citrobacter rodentium colitis severity [START_REF] Schiering | Feedback control of AHR signalling regulates intestinal immunity[END_REF][START_REF] Qiu | Group 3 innate lymphoid cells inhibit T-cell-mediated intestinal inflammation through aryl hydrocarbon receptor signaling and regulation of microflora[END_REF] through IL-22 production [START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF].

Production of AhR ligands by the microbiota is required for IL-22 production [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF], which is involved in mucosal wound healing [START_REF] Pickert | STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing[END_REF] and production of antimicrobial proteins by intestinal epithelial cells [START_REF] Sonnenberg | Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22[END_REF][START_REF] Stelter | Salmonella-induced mucosal lectin RegIIIβ kills competing gut microbiota[END_REF]. Several sources of IL-22 have been identified in the gut, including Th22, TCR γδ IELs and ILCs [START_REF] Sonnenberg | Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22[END_REF][START_REF] Spits | Innate lymphoid cells--a proposal for uniform nomenclature[END_REF]. In this study, we observed an increased duodenal Il22 expression after AhR signaling by Ficz or the enriched Trp diet, suggesting that the microbiota/AhR/IL-22 axis could play a role in CeD pathogenesis.

Our results are relevant to humans as impaired microbial production of AhR ligands was detected in a cohort of patients with active CeD. This was associated with reduced microbiota AhR agonist activity and reduced duodenal expression of Ahr and AhR pathway genes like Il22 and Il17. Our results agree with a previous study showing lower AhR expression in inflamed mucosa of CeD patients [START_REF] Dinallo | Protective Effects of Aryl Hydrocarbon Receptor Signaling in Celiac Disease Mucosa and in Poly I:C-Induced Small Intestinal Atrophy Mouse Model[END_REF] and identify a previously unreported defect in microbiota-specific AhR activation in CeD. These results raise the hypothesis that inflammation in active CeD promotes a gut microbiota shift with impaired AhR agonist production that affects IL-22 levels.

Likewise, a diet low in factors that serve as substrates for microbial derived AhR ligands could contribute to CeD. This hypothesis is supported by the fact that a GFD increases the production of AhR ligands by the microbiota, improves activation of the AhR pathway in the intestine and increases the expression of the Il22, known to participate in intestinal recovery [START_REF] Lamas | Aryl hydrocarbon receptor and intestinal immunity[END_REF][START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF][START_REF] Pickert | STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing[END_REF][START_REF] Sonnenberg | Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22[END_REF]. Our study has some limitations, related to the small cohort of patients investigated as well as the fact that all the patients included in the GFD group were clinically in remission. Future studies should confirm the results in a larger cohort of patients, and also include non-responders to the GFD to investigate persistent AhR pathway abnormalities, as this would be a population of interest to target therapeutically.

In summary, our study identifies a mechanism related to the impaired production of AhR ligands by the intestinal microbiota in CeD. In mice, we show that gluten-induced pathology can be reversed through a combination of an enriched tryptophan diet and next-generation probiotics that produce AhR ligands. Therefore, in addition to providing key evidence of the importance of the microbiota composition and function in CeD pathogenesis, this study suggests that Trp catabolites derived from the metabolic activity of the intestinal microbiota could be used as biomarkers for dysbiosis and may be targeted for the development of new therapeutic approaches in CeD. These include tryptophan supplementation in combination with next generation probiotic organisms, such as L reuteri strains, that produce AhR ligands from the dietary substrate.

Currently, the only treatment for CeD is a strict, life-long adherence to a gluten-free diet, and many suffer from persistent symptoms, despite following a GFD. The results should prompt interventional studies in humans to evaluate this therapeutic strategy in CeD patients nonresponding to a GFD, but also as a preventative strategy in at-risk populations. Further interventional studies in humans are necessary to evaluate the proposed therapeutic strategy.

Materials and Methods

Study design

The objectives of the study were to investigate whether AhR ligand production by the gut microbiota influences gluten immunopathology. Several approaches were used to address this question. Transgenic SPF mice expressing the CeD DQ8 susceptibility gene were used and subjected to interventions aimed at enhancing the AhR pathways: 1) treatment with a low or high Trp diet, before and after gluten-treatment, 2) oral supplementation with AhR-ligand producing Lactobacillus during gluten treatment, or 3) treatment with either an AhR agonist or vehicle during gluten treatment. Gluten treatment consisted of a previously described method of sensitization and challenge, described below. Microbiota composition, AhR activation by intestinal contents, and fecal AhR ligand production were investigated in mice on low or high Trp diet, and following gluten-treatment. Signs of intestinal inflammation, AhR activation, and gluten immunopathology were examined following gluten treatment and exposure to high or low Trp diet, treatment with Lactobacillus, or treatment with an AhR agonist. Studies were planned with the minimum number of animals per group (n=4) to observe significant differences reproducibly. Mice were randomly assigned to groups. Statistical outliers or samples where technical issues were encountered were removed from analysis. Specific number of mice used in each experimental group are included in figure legends. In order to provide more clinical relevance, we also analyzed the concentrations of specific microbiota metabolites known to activate AhR in feces from patients with active CeD, patients with CeD on a GFD, and nonceliac controls. Subjects with inflammatory bowel, peptic ulcer, or reflux disease, as well as those taking immunosuppressants, glucocorticosteroids, antibiotics or probiotics were excluded.

Written informed consent was obtained, and the study was approved by the Hamilton Integrated Research Ethics Board. Analysis of data was performed in a blinded fashion when possible. Louis, MO) by oral gavage once a week for 3 weeks, to break oral tolerance to gliadin [START_REF] Galipeau | Intestinal microbiota modulates gluten-induced immunopathology in humanized mice[END_REF][START_REF] Galipeau | Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice[END_REF].

Gluten sensitization and challenge

Mice were then challenged by oral gavage with 10 mg of gluten (Sigma-Aldrich) dissolved in acetic acid three times a week for 3 weeks ("gluten treatment") [START_REF] Galipeau | Intestinal microbiota modulates gluten-induced immunopathology in humanized mice[END_REF][START_REF] Galipeau | Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice[END_REF]. Mice were sacrificed 18 to 24 hours following the final gluten challenge. In additional experiments, male and female 8-12 week-old SPF C57Bl/6 mice maintained on a normal gluten-containing chow (Envigo 2918) were included.

Probiotic and pharmacological treatments

NOD/DQ8 mice were injected i.p. with 6-formylindolo(3,2-b) carbazole (Ficz, Enzo Life Sciences, 1 μg/mouse) or vehicle (dimethyl sulfoxide, Sigma-Aldrich) at day 1, 10, 20 and 30 during gluten sensitization and challenge. For the treatments with bacteria known to produce AhR ligands [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF] mice were gavaged 6 times a week for 3 weeks with 10 9 colony-forming units of a combination of Lactobacillus reuteri CNCM-I5022 and L. reuteri CNCM-I5429. Bacteria were grown in MRS broth supplemented with cysteine (0.5mg/mL) and Tween 80 (1mg/mL) for 18-20 h. Oral gavage with MRS was performed in control mice.

Custom tryptophan (Trp) rodent diet

NOD/DQ8 mice were fed a customized version of Envigo TD.01084 in which tryptophan concentration was adjusted at 0.1% (low Trp diet) or 1% (high Trp diet) (table S1). Three weeks after the beginning of the diets, mice were gluten sensitized and challenged.

Measurement of AhR activity

The AhR activity of human and animal stool samples was measured using a luciferase reporter assay method, as described previously [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF]. Briefly, H1L1.1c2 cells, which contained the dioxin response element-driven firefly luciferase reporter plasmid pGudLuc1.1, were seeded into a 96-well plate and stimulated with human or animal stool suspensions or small intestinal contents for 24 h. Luciferase activity was measured using a luminometer, and the results are reported as fold changes based on the negative luciferase activity of the control. All values were normalized based on the cytotoxicity of the samples using the Lactate Dehydrogenase Activity Assay (Promega, Madison, USA).

Metabolite measurements

The metabolite concentrations in stool and serum samples were determined by a specific method using HPLC-coupled to high resolution mass spectrometry as previously described [START_REF] Lefèvre | Validation of a global quantitative analysis methodology of tryptophan metabolites in mice using LC-MS[END_REF][START_REF] Hendrikx | Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice[END_REF][START_REF] Miani | Gut Microbiota-Stimulated Innate Lymphoid Cells Support β-Defensin 14 Expression in Pancreatic Endocrine Cells, Preventing Autoimmune Diabetes[END_REF]. IDO activity was assessed by measurement of Kyn/Trp ratio [START_REF] Natividad | Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome[END_REF][START_REF] Laurans | Genetic deficiency of indoleamine 2,3-dioxygenase promotes gut microbiota-mediated metabolic health[END_REF].

16S rRNA gene sequencing

Total DNA was extracted from mouse fecal samples and data were sequenced as previously described [START_REF] Caminero | Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2[END_REF] and analyzed as previously described [START_REF] Constante | Dietary Heme Induces Gut Dysbiosis, Aggravates Colitis, and Potentiates the Development of Adenomas in Mice[END_REF]. Briefly, the V3 region of the 16S ribosomal RNA gene was amplified and sequenced on the Illumina MiSeq sequencing system. Sequences obtained were aligned to each other using the Paired-End Read (PEAR) merger and Clustering of reads into operational taxonomic units was performed using the Quantitative Insights into Microbial Ecology (QIIME) version 1.8.0 (55) and data was loaded into R using the phyloseq package for downstream processing. A total of 4,012,804 reads were obtained with an average of 74,311.19 per sample and ranged from 11512 to 169,626 per sample.

Measurement of gut barrier function

Barrier function was assessed by Ussing chamber technique as previously described (49).

Briefly, sections of jejunum from each mouse were mounted in an Ussing Chamber with an opening of 0.6 cm 2 . Net active transport across the epithelium was measured via a short circuit current response (Isc, µA) injected through the tissue under voltage-clamp conditions. Baseline Isc (µA/cm 2 ) was recorded at equilibrium 20 min after mounting jejunum sections. Mucosal-toserosal transport of macromolecules was assessed by adding 51 Cr-EDTA in the luminal side of the chamber. 51 Cr-EDTA fluxes were calculated by measuring the proportion of radioactive 51 Cr-EDTA detected in the serosal side of the chamber after 2 hours compared to the radioactive 51 Cr-EDTA placed in the luminal side at the beginning of experiment.

Gene expression analysis using quantitative reverse-transcription PCR

Total RNA was isolated from human or animal duodenum samples using RNeasy Mini Kit (Qiagen, Hilden, Germany) with the DNase treatment, according to the manufacturer's instructions. RNA concentration was determined using a NanoDrop spectrophotometer (Qiagen).

Quantitative RT-PCR was performed using iScript Reverse Transcriptase (Bio-Rad) and then a SsoFast EvaGreen Supermix (Bio-Rad) in a Mastercycler ep realplex apparatus (Eppendorf) with specific human or mouse oligonucleotides. Additional information on the primers used is available in table S2. qPCR data were analyzed using the 2 -ΔΔCt quantification method with human or mouse Gapdh as the endogenous control.

Histology and immunohistochemistry

Cross-sections of the proximal small intestine were fixed in 10% formalin and embedded in paraffin. Enteropathy was determined by measuring villus-to-crypt (V/C) ratios in a blinded fashion, as previously described [START_REF] Galipeau | Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice[END_REF]. The presence of IELs in the sections was performed by immunostaining for CD3 + cells as described previously [START_REF] Galipeau | Intestinal microbiota modulates gluten-induced immunopathology in humanized mice[END_REF][START_REF] Galipeau | Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice[END_REF]. Briefly, sections were stained with polyclonal rabbit anti-mouse CD3 (Dako) and in each section, and intraepithelial lymphocytosis was determined by counting CD3 + IELs per 20 enterocytes in five randomly chosen villus tips.

Subjects and clinical cohort

Eleven patients (7 females, mean age of 36. S3. Not all patients were used for all determinations due to sample availability. Specific numbers of patients included in each parameter are included in figure legends.

Statistical analysis

GraphPad Prism version 6.0 (San Diego, CA, USA) was used for all analyses and preparation of graphs. The data are presented as median (interquartile range) or mean ± SEM. Multiple comparisons were evaluated statistically by one-way ANOVA and post hoc Tukey test or nonparametric Kruskal-Wallis test followed by a post hoc Dunn's test. For comparisons between two groups, significance was determined using the two-tailed Student's two-tailed t-test or nonparametric Mann-Whitney test. In all determinations, statistical outliers (using the ROUT method), or samples where technical issues were encountered, such as poor RNA quality, poor tissue quality for Ussing chambers, or poor histological orientation were removed from analysis.

Exact numbers and tests used are provided in each figure legend. Differences corresponding to P<0.05 were considered significant.

For microbiota analysis, R Statistics, with the stats and vegan packages, was used to perform the statistical analysis. Data transformation was used when required and possible to achieve a normal distribution (logarithmic, square root, inversion, and inverted logarithm).

Differences between whole bacterial communities were tested by permutational multivariate analysis of variance (PERMANOVA) calculated using an unweighted UniFrac distance.

Multiple comparisons were evaluated statistically by one-way ANOVA or Kruskall-Wallis. Statistically significant differences were then evaluated by two-tailed Student's two-tailed t-test or Wilcoxon rank-sum test and multiple testing was corrected via false discovery rate (FDR) estimation. Each dot represents an individual mouse and data presented as median with interquartile range and whiskers extending from minimum to maximum, or as mean + SEM. Differences in counts (IEL/100 enterocytes) in gluten treated NOD/DQ8 mice fed a low (n=6) or enriched tryptophan diet (n=6). K. Small intestinal paracellular permeability to 51 Cr-EDTA (% hot sample/h/cm 2 ) in gluten treated NOD/DQ8 mice fed with low (n=11) or enriched tryptophan diet (n=12). L. Fecal lipocalin-2 quantification in gluten treated NOD/DQ8 mice fed a low (n=14) or enriched tryptophan diet (n=14). Each dot represents an individual mouse and data presented as median with interquartile range and whiskers extending from minimum to maximum or as mean + SEM. Differences in microbial composition calculated by PERMANOVA using unweighted UniFrac distance, and significant differences between taxa evaluated by two-tailed Student's ttest or Wilcoxon test and multiple testing was corrected via false discovery rate (FDR) estimation. Statistical significance in other parameters determined by Student's two-tailed t-test, or Mann-Whitney. Unc CeD, celiac disease; GFD, gluten-free diet; TG2, tissue transglutaminase *Result of >4 using TG2 ELISA from Immco Diagnostics was considered positive.

• • • • • • • • • • • • • • • • • • • P= 0.

  Gluten sensitization and challenge (D59; "gluten-treatment"; Fig 2A), regardless of dietary intervention, shifted fecal microbiota composition (fig. S2A) and alpha diversity (fig. S2, B and C) leading to increased abundance of bacteria from the Firmicutes phylum and lower abundance of bacteria belonging to the Bacteroidetes phylum (fig. S2, D to G). This was accompanied by higher AhR agonists in mice fed the enriched Trp diet (fig S3). Compared with the low Trp diet, the enriched Trp diet did not impact alpha diversity (fig.

Female and male 8 -

 8 12 weeks old SPF NOD AB° DQ8 (NOD/DQ8) mice were bred in a conventional SPF facility at McMaster University and maintained on a gluten-free diet (Envigo, TD.05620) unless otherwise stated. All mice had unlimited access to food and water. All experiments were carried out in accordance with the McMaster University animal utilization protocols and with approval from the McMaster University Animal Care Committee and McMaster Animal Research Ethics Board (AREB). NOD/DQ8 mice were sensitized with 500 µg of pepsin-trypsin digest of gliadin (PT-gliadin) and 25 µg of cholera toxin (Sigma-Aldrich, St

  8 years) with positive anti-tissue transglutaminase (TG2) IgA test at diagnosis attending the Adult GI Diseases Clinic at McMaster University were recruited after confirming active CeD by the presence of duodenal atrophy (>Marsh IIIa). Patients with concurrent or past history of inflammatory bowel disease (Crohn's disease, ulcerative or undetermined colitis), as determined by laboratory, endoscopic or bowel imaging were excluded. Eighteen other patients (10 females, mean age of 51.7 years) undergoing endoscopy, in which organic disease including peptic ulcer, reflux disease, inflammatory bowel disease and celiac disease were ruled out, were recruited as non-celiac controls. Six patients (3 with active CeD and 3 non-celiac controls) were taking a proton pump inhibitor in the month before enrolment. An additional six CeD patients on a GFD for >2 years (4 females, mean age of 35.3) in clinical remission (with a negative anti-TG2 IgA) attending the Adult GI Disease Clinic at McMaster University were included as treated CeD patients. Patients taking immunosuppressants, glucocorticosteroids, antibiotics or probiotics were excluded. When possible, genetic analysis was performed for celiac risk genes. All the participants signed the written informed consent; fecal samples were collected and kept frozen and stored until analysis. The study was approved by the Hamilton Integrated Research Ethics Board (REB# 12-599-T). For subject demographics see table

  Normal distribution was determined by D'Agustino-Pearson omnibus normality test, Shapiro-Wilk test and Kolmogorov-Smirnov test with Dallal-Wilkinson-Lillie correction. For data sets that failed normality tests, nonparametric tests were used to analyze significant differences.

Figure legends : Fig. 1 .

 :1 Figure legends:

Fig. 3 .

 3 Fig. 3. Lactobacillus reuteri rescues AhR activation and attenuates gluten immunopathology

Fig. 4 .

 4 Fig. 4. Treatment with a pharmacological AhR agonist reduces gluten immunopathology in

Fig. 5 .

 5 Fig. 5. Celiac patients (CeD) show decreased AhR activation and lower concentration of

Figure S2 .

 S2 Figure S2. Gluten treatment shifts gut microbiota of NOD/DQ8 mice regardless of diet

Figure S3 Figure S3 .

 S3S3 Figure S3

Figure S4 Figure S4 .

 S4S4 Figure S4

Figure S5 Figure S5 .

 S5S5 Figure S5

Figure S6 Figure S6 .

 S6S6 Figure S6

  03

		Protocol	Principal coordinate analysis			Significant taxonomy (species)
				R=0.46	P=0.001
									Firmicutes	Cyanobacteria
	3 weeks NOD/DQ8 mice	Axis.2 [22.8%]					Row Z-score -4 0 4	Proteobacteria Staphylococcus sp. Actinobacteria Lactobacillus sp.
		D21							Unc. Enterobacteriaceae
									Enterococcus sp.
				Axis.1 [52.3%]			Jeotgalicoccus psychrophilus
									Aerococcus sp.
		Taxa summary (phylum)						Facklamia sp.
		100							Jeotgalicoccus sp.
	Relative abundance (%)	50 25 75		Bacteroidetes Firmicutes Proteobacteria	Verrucomicrobia Other		Bilophila sp. Unc. YS2 Dorea sp. Gemella sp. Corynebacterium stationis Desulfovibrio C21-c20
		0
	) pmol/mg dry (x10 4						pmol/mg dry	P= 0.02	Kyn/Trp ratio	P= 0.05
		Low trp	High trp	Low trp	High trp		Low trp	High trp	Low trp	High trp
									Gene expression
		P= 0.0003		P= 0.05	Relative expression		P= 0.006	Low trp diet P= .05 High trp diet
		Low trp	High trp	Low trp High trp
								Ion secretion	Paracellular permeability
	pg/mg of feces	P= 0.05	CD3+/100 enterocytes		P= 0.01			% hot sample/h/cm 2
		Low trp	High trp	Low trp High trp		Low trp	High trp	Low trp	High trp

Table S3 . Characteristics of the subjects and clinical cohort.

 S3

	Diagnosis	HLA	Age Sex Anti-TG2 IgA*	Marsh scale	Bristol Scale
	CeD-active Not determined	33	M	Positive	3c	6
	CeD-active Not determined	26	F	Positive	3a	5
	CeD-active DQ2 homozygous	46	M	Positive	3a	5
	CeD-active Not determined	34	F	Positive	3a	6
	CeD-active DR3-DQ2 heterozygous	55	M	Positive	3c	4
	CeD-active	DR3-DQ2 heterozygous/ DR4-DQ8 heterozygous	37	F	Positive	3a	7
	CeD-active DR3-DQ2	30	F	Positive	3a	4
	CeD-active DQ2 homozygous	31	F	Positive	3b	5
	CeD-active DR3-DQ2	52	M	Positive	3a	5
	CeD-active DQ2 homozygous	29	F	Positive	3a	6,7
	CeD-active DQ2 homozygous	32	F	Positive	3a	2,3,4
	CeD-GFD	DR3/DR7-DQ2 homozygous	23	F	Negative	Normal	2
	CeD-GFD	DR3-DQ2 heterozygous	27	F	Negative	Normal	6,7
	CeD-GFD	DR3-DQ2 heterozygous	52	M	Negative	Normal	6
	CeD-GFD	DR3-DQ2 homozygous	54	M	Negative	Normal	5
	CeD-GFD	DR3-DQ2 heterozygous	29	F	Negative	Normal	3,6
	CeD-GFD	DR7-DQ2/DQ7 heterozygous	27	F	Negative	1	5
	Control	Not determined	45	F	Negative	Normal	6
	Control	Not determined	50	M	Negative	Normal	4
	Control	Not determined	53	M	Negative	Normal	2
	Control	Not determined	39	F	Negative	Normal	6
	Control	Not determined	42	M	Negative	Normal	6
	Control	DR3-DQ2 heterozygous	72	F	Negative	Normal	4
	Control	DR4-DQ8 heterozygous	72	F	Negative	Normal	6
	Control	Not determined	63	F	Negative	Normal	4
	Control	Negative	60	M	Negative	Normal	2
	Control	Not determined	51	F	Negative	Normal	6
	Control	Not determined	63	F	Negative	Normal	2
	Control	Not determined	63	M	Negative	Normal	2
	Control	Not determined	45	M	Negative	Normal	1
	Control	Not determined	18	F	Negative	Normal	4
	Control	Negative	45	F	Negative	Normal	6
	Control	DR7-DQ2 heterozygous	36	M	Negative	Normal	6
	Control	Not determined	63	M	Negative	Normal	3
	Control	DR11-DQ7 heterozygous	51	F	Negative	Normal	6

Acknowledgments: We thank the members of the animal facilities of McMaster University for their assistance in mouse care; and S.M. Collins, G. De Palma, M.I. Pinto-Sanchez, A. Nardelli, N. Causada Calo, J. Lu, R. Borojevic, C. Shimbori, P. Vivek, S. Rahmani, J. Dong and J.J Park for fruitful discussions and technical help. The H1L1.1c2 cell line was provided by M.S. Denison (University of California, Davis). EFV holds a Canada Research Chair in Nutrition, Inflammation and Microbiota. Funding: The work was supported by a Michael G. DeGroote

Supplementary Materials