Epithelial Indoleamine 2,3-Dioxygenase 1 Modulates Aryl Hydrocarbon Receptor and Notch Signaling to Increase Differentiation of Secretory Cells and Alter Mucus-Associated Microbiota

Abstract

Background & aims: Inflammation, injury, and infection up-regulate expression of the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in the intestinal epithelium. We studied the effects of cell-specific IDO1 expression in the epithelium at baseline and during intestinal inflammation in mice.

Methods: We generated transgenic mice that overexpress fluorescence-tagged IDO1 in the intestinal epithelium under control of the villin promoter (IDO1-TG). We generated intestinal epithelial spheroids from mice with full-length Ido1 (controls), disruption of Ido1 (knockout mice), and IDO1-TG and analyzed them for stem cell and differentiation markers by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. Some mice were gavaged with enteropathogenic Escherichia coli (E2348/69) to induce infectious ileitis, and ileum contents were quantified by polymerase chain reaction. Separate sets of mice were given dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid to induce colitis; intestinal tissues were analyzed by histology. We utilized published data sets GSE75214 and GDS2642 of RNA expression data from ilea of healthy individuals undergoing screening colonoscopies (controls) and patients with Crohn's disease.

Results: Histologic analysis of small intestine tissues from IDO1-TG mice revealed increases in secretory cells. Enteroids derived from IDO1-TG intestine had increased markers of stem, goblet, Paneth, enteroendocrine, and tuft cells, compared with control enteroids, with a concomitant decrease in markers of absorptive cells. IDO1 interacted non-enzymatically with the aryl hydrocarbon receptor to inhibit activation of NOTCH1. Intestinal mucus layers from IDO1-TG mice were 2-fold thicker than mucus layers from control mice, with increased proportions of Akkermansia muciniphila and Mucispirillum schaedleri. Compared to controls, IDO1-TG mice demonstrated an 85% reduction in ileal bacteria (P = .03) when challenged with enteropathogenic E coli, and were protected from immune infiltration, crypt dropout, and ulcers following administration of dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid. In ilea of Crohn's disease patients, increased expression of IDO1 correlated with increased levels of MUC2, LYZ1, and aryl hydrocarbon receptor, but reduced levels of SLC2A5.

Conclusions: In mice, expression of IDO1 in the intestinal epithelial promotes secretory cell differentiation and mucus production; levels of IDO1 are positively correlated with secretory cell markers in ilea of healthy individuals and Crohn's disease patients. We propose that IDO1 contributes to intestinal homeostasis.

Keywords: Kynurenine; Metabolism; Microbiome; Organoids; Ulcerative Colitis.

Figure 1:. IDO1-TG mice overexpress functional IDO1 in the intestinal epithelium.

A) Transgenic mouse line pVil-EGFP/hIDO1 (IDO1-TG) schematic. B) Representative indirect immunofluorescence images showing the EGFP fluorescence as a surrogate marker for IDO1 expression (Top) and with epithelial cells marker E-cadherin (E-cad) staining (Bottom). Bar = 50μm. C) HuIDO1 RNA is detectable in whole tissue from transgenic mouse ileum and colon, and is comparable to endogenous IDO1 expression levels produced in response to models of inflammation: immunostimulatory DNA (CpG), TNBS, and DSS. RNA relative abundances were calculated relative to Gapdh levels from N=3-5 mice. D) Enzymatic activity of IDO1-TG was confirmed by measuring the Tryptophan and Kynurenine in spheroid culture medium. Representative data from two independent experiments. **, P<0.01; ***, P<0.001 by Student’s t-test.

Figure 2:. IDO1 expression promotes intestinal secretory cell differentiation in vivo.

Representative images of formalin-fixed, paraffin-embedded tissue sections stained with PAS stain identifying secretory cells of the ileum (A) and colon (B) of WT, IDO1-TG, and KO mice. H&E or PAS stained sections were used to quantify crypt metrics, goblet and Paneth cells. CHGA and DCLK1 immunohistochemistry was used to quantify enteroendocrine and tuft cells respectively. Data is presented as mean ± standard deviation from N=3–6 mice/genotype from at least two experimental replicates. Bars in micrographs = 50μm. *, P<0.05; **, P<0.01 by Mann-Whitney test. n.s., not significant.

Figure 3:. IDO1 expression promotes secretory lineage differentiation in vitro.

Enteroids were derived from the ileum of WT, IDO1-TG and KO mice and examined for expression of (A) stem cell, (B) enterocyte, and (C) secretory lineage markers. qRT-PCR data presented as fold change relative to WT differentiated state. Data represents mean ± standard deviation from at least three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001 by Student’s t-test. n.s., not significant.

Figure 4:. IDO1-TG spheroids exhibit increased secretory cells in number and fluorescence intensity.

Enteroids from WT and IDO1-TG mice were analyzed by immunofluorescence after 48h in differentiation medium. Representative immunofluorescence images, quantification of positive cells (number of positive cells per total nucleated cells) and fluorescence intensity using markers of (A) goblet cells (TFF3), (B) Paneth cells (LYZ1), and (C) enteroendocrine cells (CHGA). Data represents mean ± standard deviation from two independent experiments. Bars in micrographs = 50μm.*, P<0.05 by Student’s t-test; #, P<0.05; ###, P<0.001 by Student’s t-test with Welch’s correction; ◊◊◊, P<0.001 by Mann-Whitney test.

Figure 5:. IDO1 interacts with AHR to enhance secretory lineage differentiation by inhibiting Notch signaling.

A) Muc2 expression in enteroids differentiated in the presence of two metabolic inhibitors of IDO1, 1-methyl-L-tryptophan (1-mT, 500μM) and Epacadostat (Epa, 5μM). B) Muc2 expression in enteroids differentiated in the presence of IDO1 metabolite-L-Kynurenine (Kyn, 100μM). C) Expression of Ahr and two transcriptional targets, Cyp1a1 and Cyp1b1. Data presented as fold change relative to WT. D) Reduction in Muc2 and Lyz1 expression in enteroids differentiated in the presence of CH-223191, a specific inhibitor of AHR (20μM), relative to untreated enteroids. qPCR data represents mean ± standard deviation from at least 3 independent experiments. E) Immunoblot of protein from undifferentiated WT (−) and IDO1-TG (+) spheroids cultured in the presence of DAPT (10uM), Epa (5μM), or CH-223191 (20μM). Densitometry of immunoblot is displayed below. Data is representative of 3 independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001 by Student’s t-test; ###, P<0.001 by one-way ANOVA.

Figure 6:. IDO1 expression enhances mucus barrier and modulates microbiota.

A) Representative immunofluorescence micrographs of methacarn-fixed WT and IDO1-TG ileum tissue depicting the preserved mucus layers. Bar=50μm. B) Quantification of the mucus layers measured from the top of the villi to the periphery of the MUC2-positive region. C) Lysozyme activity was measured from stool extracts from WT and IDO1-TG mice. D) qPCR using species-specific primers was used to quantify muciniphilic bacteria in the ileum mucus-associated microbiota from WT and IDO1-TG mice. Abundance was calculated relative to bacterial gene rpolB. E) Presence (left) and quantification (right) of enteropathogenic E. coli E2348/69 (EPEC) in the ileum of WT, IDO1-TG, and KO mice on day 3 normalized to luminal content and inoculation quantity. F,G) Representative micrographs of colon tissue from WT and IDO1-TG mice treated with 3% DSS in drinking water for 5 days followed by 3 days of water (F) or 2.5% TNBS in 50% ethanol and harvested after 3 days (G) stained with H&E. Representative micrograph of Ki67-positive cells in the peri-ulcer region of WT and IDO1-TG mice (center); Colitis severity as scored from histological sections. N=3-10 mice per genotype in at least two independent experiments. Bar = 500μm (G, left) or 200μm. Black triangle: epithelial ulceration; yellow triangle: edema. *, P<0.05; **, P<0.01 by Student’s t test; #, P<0.05 by one-way ANOVA with Bonferroni’s multiple comparison test.

Figure 7:. IDO1 expression correlates with reduced absorptive and enhanced secretory cell markers in human ileum from control and Crohn’s disease.

A) Ileal RNA expression data from healthy control patients (Control), Crohn’s disease patients with active inflammation (Active), and Crohn’s disease patients without active inflammation (inactive). Log2 expression values for each gene were grouped according to disease state. Data represents mean ± standard deviation. B) Log2 expression values of select enterocyte (SLC2A5), goblet (MUC5B), and Paneth cell (LYZ1) markers, and AHR was assessed for correlation with IDO1 expression. n.s., not significant; *, P<0.05; **, P<0.01; ***, P<0.001 by nonparametric one-way ANOVA with Dunn’s multiple comparison test.