Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor

Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease. Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease. The mammalian gastrointestinal tract plays host to trillions of microbes, collectively termed the microbiota, where a critical mutualism exists within the intestinal mucosa. The microbiota contributes significantly to gut homeostasis but can also contribute to establishing and maintaining mucosal disease.1Lozupone C.A. Stombaugh J.I. Gordon J.I. Jansson J.K. Knight R. Diversity, stability and resilience of the human gut microbiota.Nature. 2012; 489: 220-230Crossref PubMed Scopus (3077) Google Scholar Intestinal mucosal surfaces act as primary barriers to microbial invasion, where commensal bacteria work in a dynamic and intimate interaction with the gut epithelium and influence host cellular and immune responses.2Muniz L.R. Knosp C. Yeretssian G. Intestinal antimicrobial peptides during homeostasis, infection, and disease.Front Immunol. 2012; 3: 310Crossref PubMed Scopus (137) Google Scholar Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract that comprises Crohn disease and ulcerative colitis (UC). It is known that IBD is caused by interactions between genetic and environmental factors, and results in perturbations of the microbiota, though precisely how microbial factors affect gut homeostasis and immune response have not been extensively explored.3Xavier R.J. Podolsky D.K. Unravelling the pathogenesis of inflammatory bowel disease.Nature. 2007; 448: 427-434Crossref PubMed Scopus (3255) Google Scholar, 4Huttenhower C. Kostic A.D. Xavier R.J. Inflammatory bowel disease as a model for translating the microbiome.Immunity. 2014; 40: 843-854Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 5Frank D.N. St Amand A.L. Feldman R.A. Boedeker E.C. Harpaz N. Pace N.R. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.Proc Natl Acad Sci U S A. 2007; 104: 13780-13785Crossref PubMed Scopus (3228) Google Scholar The significance of IL-10 signaling is well established in IBD. This anti-inflammatory cytokine, which signals through the IL-10 receptor ligand-binding subunit (IL-10R1), is induced during inflammation and attenuates excessive production of proinflammatory mediators in various cell types, including intestinal epithelial cells (IEC).6Engelhardt K.R. Grimbacher B. IL-10 in humans: lessons from the gut, IL-10/IL-10 receptor deficiencies, and IL-10 polymorphisms.Curr Top Microbiol Immunol. 2014; 380: 1-18PubMed Google Scholar, 7Kominsky D.J. Campbell E.L. Ehrentraut S.F. Wilson K.E. Kelly C.J. Glover L.E. Collins C.B. Bayless A.J. Saeedi B. Dobrinskikh E. Bowers B.E. MacManus C.F. Muller W. Colgan S.P. Bruder D. IFN-gamma-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.J Immunol. 2014; 192: 1267-1276Crossref PubMed Scopus (68) Google Scholar Functional IL-10 signaling is associated with enhanced mucosal barrier function and results in maintenance and homeostasis of the epithelia.7Kominsky D.J. Campbell E.L. Ehrentraut S.F. Wilson K.E. Kelly C.J. Glover L.E. Collins C.B. Bayless A.J. Saeedi B. Dobrinskikh E. Bowers B.E. MacManus C.F. Muller W. Colgan S.P. Bruder D. IFN-gamma-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.J Immunol. 2014; 192: 1267-1276Crossref PubMed Scopus (68) Google Scholar Previous work, including our own, has shown that the epithelial IL-10R1 contributes fundamentally to resistance to intestinal inflammation and represents a component of epithelial innate immunity originally described to be induced by cytokines such as interferon-γ (IFN-γ).8Colgan S.P. Parkos C.A. Matthews J.B. D'Andrea L. Awtrey C.S. Lichtman A. Delp C. Madara J.L. Interferon-γ induces a surface phenotype switch in intestinal epithelia: downregulation of ion transport and upregulation of immune accessory ligands.Am J Physiol. 1994; 267: C402-C410Crossref PubMed Google Scholar For instance, mice deficient in IL-10 or IL-10 receptor develop spontaneous severe colitis, and mice conditionally lacking intestinal epithelial IL-10R1 show increased susceptibility to colitis.7Kominsky D.J. Campbell E.L. Ehrentraut S.F. Wilson K.E. Kelly C.J. Glover L.E. Collins C.B. Bayless A.J. Saeedi B. Dobrinskikh E. Bowers B.E. MacManus C.F. Muller W. Colgan S.P. Bruder D. IFN-gamma-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.J Immunol. 2014; 192: 1267-1276Crossref PubMed Scopus (68) Google Scholar, 9Kuhn R. Lohler J. Rennick D. Rajewsky K. Muller W. Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3659) Google Scholar, 10Chaudhry A. Samstein R.M. Treuting P. Liang Y. Pils M.C. Heinrich J.M. Jack R.S. Wunderlich F.T. Bruning J.C. Muller W. Rudensky A.Y. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation.Immunity. 2011; 34: 566-578Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar Metabolomic analysis has revealed that gut bacteria impact host immunity through a variety of metabolites, including indole metabolites,11Wikoff W.R. Anfora A.T. Liu J. Schultz P.G. Lesley S.A. Peters E.C. Siuzdak G. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites.Proc Natl Acad Sci U S A. 2009; 106: 3698-3703Crossref PubMed Scopus (1802) Google Scholar which originate from the microbial metabolism of tryptophan. Indole-3-propionic acid (IPA) and indole-3-aldehyde (IAld), which are tryptophan metabolites produced by intestinal bacteria, are known for their intercellular signaling activity. Further, IAld has recently been identified as an aryl hydrocarbon receptor (AHR) ligand.12Zelante T. Iannitti R.G. Cunha C. De Luca A. Giovannini G. Pieraccini G. Zecchi R. D'Angelo C. Massi-Benedetti C. Fallarino F. Carvalho A. Puccetti P. Romani L. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22.Immunity. 2013; 39: 372-385Abstract Full Text Full Text PDF PubMed Scopus (1291) Google Scholar AHR is a ligand-dependent transcription factor activated by a variety of synthetic and biological molecules that plays an important role in immunological response and inhibition of inflammation.13Hubbard T.D. Murray I.A. Bisson W.H. Lahoti T.S. Gowda K. Amin S.G. Patterson A.D. Perdew G.H. Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles.Sci Rep. 2015; 5: 12689Crossref PubMed Scopus (207) Google Scholar AHR contributes to immune homeostasis through various methods, including T-cell differentiation and Th17 development,14Mascanfroni I.D. Takenaka M.C. Yeste A. Patel B. Wu Y. Kenison J.E. Siddiqui S. Basso A.S. Otterbein L.E. Pardoll D.M. Pan F. Priel A. Clish C.B. Robson S.C. Quintana F.J. Metabolic control of type 1 regulatory T cell differentiation by AHR and HIF1-alpha.Nat Med. 2015; 21: 638-646Crossref PubMed Scopus (305) Google Scholar, 15Kimura A. Naka T. Nohara K. Fujii-Kuriyama Y. Kishimoto T. Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells.Proc Natl Acad Sci U S A. 2008; 105: 9721-9726Crossref PubMed Scopus (408) Google Scholar as well as the up-regulation of IL-22 production.16Monteleone I. Rizzo A. Sarra M. Sica G. Sileri P. Biancone L. MacDonald T.T. Pallone F. Monteleone G. Aryl hydrocarbon receptor-induced signals up-regulate IL-22 production and inhibit inflammation in the gastrointestinal tract.Gastroenterology. 2011; 141 (248.e1): 237-248Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar Recently, several studies have shown that the host microbiota provides a consistent source of endogenous AHR ligands with distinct effects on immune homeostasis.16Monteleone I. Rizzo A. Sarra M. Sica G. Sileri P. Biancone L. MacDonald T.T. Pallone F. Monteleone G. Aryl hydrocarbon receptor-induced signals up-regulate IL-22 production and inhibit inflammation in the gastrointestinal tract.Gastroenterology. 2011; 141 (248.e1): 237-248Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 17Gaitanis G. Magiatis P. Stathopoulou K. Bassukas I.D. Alexopoulos E.C. Velegraki A. Skaltsounis A.L. AhR ligands, malassezin, and indolo[3,2-b]carbazole are selectively produced by Malassezia furfur strains isolated from seborrheic dermatitis.J Invest Dermatol. 2008; 128: 1620-1625Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 18Vlachos C. Schulte B.M. Magiatis P. Adema G.J. Gaitanis G. Malassezia-derived indoles activate the aryl hydrocarbon receptor and inhibit Toll-like receptor-induced maturation in monocyte-derived dendritic cells.Br J Dermatol. 2012; 167: 496-505Crossref PubMed Scopus (64) Google Scholar However, the precise role of indole metabolites in the gastrointestinal tract remains elusive. Together, these data lead us to hypothesize that microbial-derived indole metabolites promote intestinal homeostasis through AHR-mediated regulation of the IEC IL-10R1. For metabolomics analysis, C57BL/6-129 mice were administered 3% (wt/vol) dextran sodium sulfate (DSS) (molecular weight 36,000 to 50,000; MP Biochemicals, Burlingame, CA) in drinking water for 5 days, followed by a 2-day recovery period. Normal tap water was returned during these 2 days before tissue collection. Control mice were maintained on tap water for 7 days. In subsequent animal experiments, 8– to 12-week–old C57BL/6 mice were administered water or 2.5% DSS ad libitum for 9 days. This administration approach varied slightly to account for a change in death susceptibility to DSS. DSS was then removed and mice were allowed to recover for 2 days before euthanasia. For IPA treatment experiments, the addition of IPA at 0.1 mg/mL was administered to water- and DSS-treated animals. Mice were housed in accordance with guidelines from the American Association for Laboratory Animal Care and Research Protocols, and all animal work was approved by the Institutional Animal Care and Use Committee of the University of Colorado. Human T84 IEC were cultured in 1:1 Dulbecco's modified Eagle's medium–Ham's F12 with 2.5 mmol/L l-glutamine and 10% fetal bovine serum. Cells were maintained at 37°C with 5% CO2. Transepithelial electrical resistances were monitored using an EVOM2 Voltohmmeter (World Precision Instruments, Sarasota, FL). Cytokines were purchased from R&D Systems (Minneapolis, MN). IAld, IPA, and AHR inhibitor, CH-223191, were obtained from Sigma-Aldrich (St. Louis, MO). 6-Formylindole[3,2-b] carbazole was obtained from Tocris Bioscience (Bristol, UK). All compounds were used at indicated concentrations. Human intestinal organoids (HIOs) were derived following previously described methods19McCracken K.W. Howell J.C. Wells J.M. Spence J.R. Generating human intestinal tissue from pluripotent stem cells in vitro.Nat Protoc. 2011; 6: 1920-1928Crossref PubMed Scopus (303) Google Scholar and kindly provided by Dr. Jason Spence (University of Michigan, Ann Arbor, MI). HIOs were maintained by embedding in Matrigel (BD Biosciences, San Jose, CA) and applying Advanced DMEM-F12 medium (Invitrogen, Carlsbad, CA) containing 1X B27 supplement (Invitrogen), 1X GlutaMAX (Life Technologies, Carlsbad, CA), 10 μmol/L HEPES, 10% penicillin/streptomycin, 100 ng/mL rhNoggin (R&D Systems), 100 ng/mL epidermal growth factor (R&D Systems), and approximately 500 ng/mL R-Spondin1 (RSPO1). RSPO1 was obtained from conditioned media collected from a HEK293 cell line that was stably transfected and zeocin-selected for the RSPO1 expression vector. The medium was changed every 2 to 4 days, and HIOs were transferred to fresh Matrigel once a week until they reached approximately 0.5 mm to 1 mm in size for experiments and RNA isolation. Lentiviral particles encoding shRNA directed against aryl hydrocarbon receptor nuclear translocator (ARNT; TRC MISSION shRNA, University of Colorado Functional Genomics Facility) were used to transduce T84 cells using standard protocols. Stable integration was achieved by puromycin selection at 6 μg/mL. Knockdown was confirmed by real-time quantitative PCR analysis, indicating 80% to 85% depletion of ARNT levels. Total RNA was extracted from cells using TRIzol (Invitrogen) and from tissue using RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was prepared using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time PCR to measure transcripts was performed in 1 × Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) using an ABI 7300 thermocycler (Applied Biosystems). Fold-change in expression of target mRNA relative to β-actin mRNA was calculated as previously described.20Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25528) Google Scholar Total RNA from HIOs was isolated using Direct-zol RNA Miniprep Kit (Zymo Research Corp, Irvine, CA). Fold-change in expression of mRNA transcript from HIO experiments was calculated relative to HSPCB (heat shock protein 90 α family class B member 1). Human and mouse primer sequences are listed in Table 1.Table 1Primer Sequences for Real-Time Quantitative PCRGeneForward primerReverse primerHuman ACTB5′-CCTGGCACCCAGCACAAT-3′5′-GCCGATCCACACGGAGTACT-3′ HSPCB5′-TCTGGGTATCGGAAAGCAAGCC-3′5′-GTGCACTTCCTCAGGCATCTTG-3′ IL10R15′-CACATCCTCCACTGGACACC-3′5′-CAAGGTCACTGCGGTAAGGT-3′ SOCS35′-GGCCACTCTTCAGCATCTC-3′5′-ATCGTACTGGTCCAGGAACTC-3′ CYP1A15′-ACCCGCCACCCTTCGACAGTTC-3′5′-TGCCCAGGCGTTGCGTGAGAAG-3′ CYP1B15′-CTGGCACTGACGACGCCAAGA-3′5′-TGGTCTGCTGGATGGACAGC-3′ MDR15′-AACGGAAGCCAGAACATTCC-3′5′-AGGCTTCCTGTGGCAAAGAG-3′Mouse Actb5′-TACGGATGTCAACGTCACAC-3′5′-AAGAGCTATGAGCTGCCTGA-3′ Il10r15′-CCCATTCCTCGTCACGATCTC-3′5′-TCAGACTGGTTTGGGATAGGTTT-3′ Open table in a new tab Whole-cell and tissue lysates were extracted in Tris-Lysis buffer on ice and disrupted by sonication. Protein content was quantified using BCA protein assay reagent (Thermo Fisher Scientific, Waltham, MA) and 30 μg of whole-cell or tissue extract was boiled in Laemmli buffer in reducing conditions subjected to SDS-PAGE. The SDS-PAGE was transferred onto a polyvinylidene difluoride membrane and probed for IL-10R1 using a rabbit polyclonal IL-10RA antibody (dilution 1:1000; Thermo Fisher Scientific) and β-actin (dilution 1:10,000; Abcam, Cambridge, UK). Distal colon tissue (1 cm) was collected from control and DSS-treated mice. Tissues were flash-frozen and placed at −80°C. Global metabolomics was performed by Metabolon, Inc. (Durham, NC). Briefly, tissue samples were thawed and weighed in tared cryovials, and 80% ice-cold methanol was added at a ratio of 75 μL of solvent per mg of sample, then incubated overnight at 4°C to extract biochemicals. Internal standards were included to control for extraction efficiency. Following methanol extraction, colon samples were processed and analyzed as previously described.21Albrecht E. Waldenberger M. Krumsiek J. Evans A.M. Jeratsch U. Breier M. Adamski J. Koenig W. Zeilinger S. Fuchs C. Klopp N. Theis F.J. Wichmann H.E. Suhre K. Illig T. Strauch K. Peters A. Gieger C. Kastenmuller G. Doering A. Meisinger C. Metabolite profiling reveals new insights into the regulation of serum urate in humans.Metabolomics. 2014; 10: 141-151Crossref PubMed Scopus (38) Google Scholar Indole derivatives were quantified in mouse and human samples using reversed-phase high-performance liquid chromatography with electrochemical coulometric array detection (EC-HPLC) (CoulArray; Thermo Fisher Scientific). Archived human serum samples from patients diagnosed with IBD or from healthy individuals were obtained under research protocols approved by the Colorado Multi-Institutional Review Board. Study participant demographics are listed in Table 2. Tissue and serum samples were extracted in 80% methanol, and protein precipitate was removed by centrifugation at 15,000 × g. Separation was achieved using an Acclaim Polar Advantage II C18 column (Thermo Fisher Scientific) at a flow rate of 1 mL/minute on a gradient of 10% to 55% acetonitrile in 50 mmol/L sodium phosphate buffer (pH 3), containing 0.42 mmol/L octanesulphonic acid as an ion-pairing agent. Calibration curves were composed by performing linear regression analysis of the peak area versus the analyte concentration. The data were quantified using the peak area in comparison to standards.Table 2Study Participant DemographicsCharacteristicControlUC remissionUlcerative colitisNumber202015Sex Male111213 Female982Age, years58.6 ± 10.946.5 ± 18.640.4 ± 13.4Disease location Left-sidedNA61 ExtensiveNA1412 ProctitisNA02NA, not applicable. Open table in a new tab NA, not applicable. Escherichia coli (E. coli) strains were obtained from GE Dharmacon (Lafayette, CO). An E. coli K12 parent strain [E. coli BW25113; wild type (WT)], along with strains containing a deletion of tnaA (E. coli JW3686; ΔtnaA) and tnaB (E. coli JW5619; ΔtnaB) were used for experiments. All strains were grown in Lysogeny broth (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract) or on Lysogeny broth–agar plates supplemented with appropriate antibiotics (kanamycin, 50 μg/mL), if needed. Cultures were grown overnight at 37°C to stationary phase and tested for the presence of indole by the addition of 200 μL of Kovac's reagent (Sigma-Aldrich) to 2 mL bacterial culture. For T84 cell experiments, cultures were grown overnight at 37°C to stationary phase. Briefly, cultures were spun, supernatants collected, and afterward serially diluted and placed onto cells for 24 hours. Bacterial supernatants used for EC-HPLC analysis were placed through a 0.22-μm filter before the run. For gnotobiotic colonization experiments, 5- to 8-week–old germ-free C57BL/6 mice were gavaged with a 100-μL bacterial suspension for mono-association (109 colony forming units of each bacterial strain collected from a stationary-phase culture and re-suspended in phosphate-buffered saline). Mice were colonized for 2 weeks before euthanasia. Fresh fecal pellets were collected periodically, weighed, homogenized, and then serially diluted in phosphate-buffered saline phosphate-buffered saline for plating to determine bacterial colony forming units per g of feces. Colon samples were fixed in 10% neutral buffered formalin and paraffin embedded before staining with hematoxylin and eosin. All histologic quantitation was performed blinded by the same individual (S.P.C.) using a scoring system previously described.22Dieleman L.A. Palmen M.J. Akol H. Bloemena E. Pena A.S. Meuwissen S.G. Van Rees E.P. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.Clin Exp Immunol. 1998; 114: 385-391Crossref PubMed Scopus (858) Google Scholar Briefly, the three independent parameters measured were severity of inflammation (0 to 3: none, slight, moderate, severe), extent of injury (0 to 3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0 to 4: none, basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact, entire crypt and epithelium lost). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1: 0% to 25%, ×2: 26% to 50%, ×3: 51% to 75%, ×4: 76% to 100%), and all numbers were summed. Maximum possible score was 40. For cytokine analysis, colon tissue was extracted in Tris-Lysis buffer by sonication and protein homogenates were quantified using BCA protein assay reagent (Thermo Fisher Scientific). Tissue concentrations of cytokines were measured using a proinflammatory cytokine screen (Meso Scale Discovery, Rockville, MD). Assays were performed according to the manufacturer's instructions. Cytokine concentrations were normalized to total protein concentration. Data are expressed as means ± SEM. Statistical analyses were performed with GraphPad Prism software version 7.0 (GraphPad Software, La Jolla, CA) using a two-tailed unpaired t-test for direct comparisons, and one-way or two-way analysis of variance with Tukey's test for multiple comparisons. Statistical differences are reported as significant when P < 0.05. To better understand microbe-derived factors that contribute to intestinal homeostasis, a comprehensive, unbiased screen of serum and colonic tissue metabolites was performed from healthy and DSS colitic mice. Mice were administered DSS via drinking water, and serum and colonic tissue were collected during peak disease (day 7). Of the microbe-derived metabolites, most notable were decreases in tryptophan-indole metabolites in both serum and colons of colitic mice compared with mice administered water alone (Figure 1A). Tryptophan is metabolized through various pathways, including the indole pathway, resulting in derivatives that are produced from metabolism by gut microbiota (Figure 1B). Colitis profoundly altered tryptophan metabolism, specifically revealing a selective decrease in indole metabolites. To validate this mass spectrometry–based metabolite screen, an HPLC-based protocol was developed using electrochemical detection methods (Figure 1C). Eight- to 10-week–old C57BL/6 mice were administered water or 2.5% DSS ad libitum for 9 days. DSS was then removed and mice were allowed to recover for 2 days before euthanasia. Serum indole metabolites were profiled by EC-HPLC (Figure 1D). Serum indole and IPA levels were significantly decreased in actively colitic animals (P < 0.05). A marked decrease in serum IAld was also observed (P = 0.09). It is notable that overall, tryptophan levels in DSS colitis actually increase (Figure 1A), suggesting that the findings of indole depletion are not a result of diminished tryptophan absorption. Guided by results of indole depletion in active murine colitis, we attempted to translate our results to human patients. Here, the EC-HPLC method was used to quantify various indole metabolites in serum samples from patients with UC. For these purposes, serum samples from healthy controls (n = 20), subjects with active UC (n = 15), and subjects with UC in remission (n = 20) were profiled (Table 2). This analysis revealed that serum IPA was deceased by nearly 60% in subjects with active UC compared with healthy controls (P < 0.05) (Figure 1E). Notably, this IPA deficiency normalized in UC patients in remission, implicating IPA as both a biomarker for active UC as well as an indicator of disease remission in human UC. Based on these findings, the role of indole and metabolites IPA and IAld on intestinal epithelial function was further studied. We have previously shown that tryptophan metabolites are important regulators of IL-10R1 in IEC.23Lanis J.M. Alexeev E.E. Curtis V.F. Kitzenberg D.A. Kao D.J. Battista K.D. Gerich M.E. Glover L.E. Kominsky D.J. Colgan S.P. Tryptophan metabolite activation of the aryl hydrocarbon receptor regulates IL-10 receptor expression on intestinal epithelia.Mucosal Immunol. 2017; 10: 1133-1144Crossref PubMed Scopus (114) Google Scholar Guided by our unbiased metabolomic profile of serum and colon tissue from healthy and DSS-colitic mice, and our recent work identifying the epithelial interleukin-10 receptor as the dominant signature for resolution of inflammation in DSS colitis,6Engelhardt K.R. Grimbacher B. IL-10 in humans: lessons from the gut, IL-10/IL-10 receptor deficiencies, and IL-10 polymorphisms.Curr Top Microbiol Immunol. 2014; 380: 1-18PubMed Google Scholar, 7Kominsky D.J. Campbell E.L. Ehrentraut S.F. Wilson K.E. Kelly C.J. Glover L.E. Collins C.B. Bayless A.J. Saeedi B. Dobrinskikh E. Bowers B.E. MacManus C.F. Muller W. Colgan S.P. Bruder D. IFN-gamma-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.J Immunol. 2014; 192: 1267-1276Crossref PubMed Scopus (68) Google Scholar it was hypothesized that indole-containing tryptophan metabolites regulate intestinal homeostasis via regulation of epithelial IL-10R1 expression. To examine the induction of IL-10R1 in response to indole metabolites, T84 IEC were exposed to IPA or IAld for 6 hours; real-time quantitative PCR of IL-10R1 transcript levels showed a concentration-dependent induction of IL-10R1 (Figure 2A). These findings were not limited to colonic cancer cell lines. HIOs are complex, three-dimensional spheroid tissues derived from human pluripotent stems cells19McCracken K.W. Howell J.C. Wells J.M. Spence J.R. Generating human intestinal tissue from pluripotent stem cells in vitro.Nat Protoc. 2011; 6: 1920-1928Crossref PubMed Scopus (303) Google Scholar and contain the majority of functional epithelial cell types (ie, enterocytes, goblet, Paneth, enteroendocrine, intestinal stem cells) and structures (ie, brush borders of microvilli, crypt-like structures, and a mesoderm layer) comprising the human small intestine.24Spence J.R. Mayhew C.N. Rankin S.A. Kuhar M.F. Vallance J.E. Tolle K. Hoskins E.E. Kalinichenko V.V. Wells S.I. Zorn A.M. Shroyer N.F. Wells J.M. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.Nature. 2011; 470: 105-109Crossref PubMed Scopus (1295) Google Scholar HIOs treated with IPA exhibited increasing levels of IL-10R1 transcript, with significant induction at 24 hours (P < 0.001) (Figure 2B). Further analysis revealed a prominent induction of IL-10R1 protein expression in T84 IEC exposed to IPA and IAld (Figure 2C). The influence of the metabolite IAld on barrier formation in T84 cell monolayers was further examined, as our previous work has demonstrated that IL-10 signaling is important in IEC barrier development and maintenance.7Kominsky D.J. Campbell E.L. Ehrentraut S.F. Wilson K.E. Kelly C.J. Glover L.E. Collins C.B. Bayless A.J. Saeedi B. Dobrinskikh E. Bowers B.E. MacManus C.F. Muller W. Colgan S.P. Bruder D. IFN-gamma-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.J Immunol. 2014; 192: 1267-1276Crossref PubMed Scopus (68) Google Scholar, 23Lanis J.M. Alexeev E.E. Curtis V.F. Kitzenberg D.A. Kao D.J. Battista K.D. Gerich M.E. Glover L.E. Kominsky D.J. Colgan S.P. Tryptophan metabolite activation of the aryl hydrocarbon receptor regulates IL-10 receptor expression on intestinal epithelia.Mucosal Immunol. 2017; 10: 1133-1144Crossref PubMed Scopus (114) Google Scholar Cells exposed to both IAld and IL-10 exhibited significantly increased barrier formation at 72 hours compared with untreated cells as measured by transepithelial electrical resistances (P < 0.01) (Figure 2D). Further, IAld in combination with IL-10 (10 ng/mL) significantly induced suppressor of cytokine signaling 3 (SOCS3), an IL-10–responsive gene that has been found to be protective in intestinal inflammation25Suzuki A. Hanada T. Mitsuyama K. Yoshida T. Kamizono S. Hoshino T. Kubo M. Yamashita A. Okabe M. Takeda K. Akira S. Matsumoto S. Toyonaga A. Sata M. Yoshimura A. CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation.J Exp Med. 2001; 193: 471-481Crossref PubMed Scopus (422) Google Scholar (P < 0.05) (Figure 2E). From this perspective, it is notable that SOCS3 was not induced by IL-10 alone as a result of nearly undetectable levels