Pathobiont-triggered induction of epithelial IDO1 drives regional susceptibility to Inflammatory Bowel Disease

Abstract

The structure and function of the mammalian gut vary by region, yet why inflammatory diseases manifest in specific regions and not others remains unclear. We use a TNF-overexpressing Crohn's disease (CD) model (Tnf^ΔARE/+), which typically presents in the terminal ileum (TI), to investigate how environmental factors interact with the host's immune susceptibility to drive region-specific disease. We identified Chlamydia muridarum, an intracellular bacterium and murine counterpart to the human sexually transmitted C. trachomatis, as necessary and sufficient to trigger disease manifestation in the ascending colon (AC), another common site of human CD. Disease manifestation in the AC depends on indoleamine 2,3-dioxygenase (IDO1) activity induced by hypersensitive surface secretory cells in genetically susceptible hosts. Single-cell and microbial analyses of human specimens also implicates this pathobiont-epithelial IDO1 pathway in patients with a history of CD in the AC. Our findings demonstrate that genetic and microbial factors can independently drive region-specific disease and provide a unique model to study CD specific to the AC.

Figure 1.. Crohn’s-like disease presents in the ascending colon as a function of murine housing facilities.

(A) Representative H&E-stained intestinal sections (terminal ileum, ascending colon, distal colon) from wildtype (N = 3) and Tnf^ΔARE/+ mice from SPF-B (N = 4) and CONV facilities (N = 5). Wildtype samples are from the CONV facility and all mice are age-matched (34–42 w of age). (B) Colitis scores from histopathological analysis of colons from CONV wildtype, SPF-B Tnf^ΔARE/+, and CONV Tnf^ΔARE/+ mice from the cohort in (A). Mean plus standard error of the mean (SEM) are shown and statistical significance was determined using an ordinary one-way ANOVA with CONV wildtype as the control group. (C - D) Colitis scores from histopathological scoring of colons from age-matched CONV Tnf^ΔARE/+ mice (N = 4 for 12w of age in (C), N = 5 for 34–42w of age in (D)), separated by ascending and distal colon regions. Mean plus SEM are shown and statistical significance was determined using paired t-tests. (E) Representative H&E-stained ascending colon sections from SPF-B wildtype Tnf^ΔARE/+ mice transferred and co-housed/fostered as pups in the CONV facility with a wildtype or Tnf^ΔARE/+ foster dam along with the dam’s age-matched biological pups. Pups were transferred and fostered within the first 3 days of birth and co-housed until experimental collection (37w of age, N = 3 wildtype, N = 4 Tnf^ΔARE/+). (F) Representative H&E-stained ascending colon sections from SPF-B adult mice (N = 2 wildtype, N = 3 Tnf^ΔARE/+) transferred and co-housed in the CONV facility in mixed-sex conditions until experimental collection (32–54w of age). CONV donors are of either wildtype or Tnf^ΔARE/+ genotype. All scale bars = 200 μm. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001. See also Figure S1.

Figure 2.. Chlamydia muridarum is associated with colonic inflammation in the context of TNF overexpression.

(A-B) Shotgun metagenomic data, filtered for only the eubacteria kingdom, of proximal (ascending) colon luminal contents. Data is represented as relative abundance of mapped phyla for individual wildtype and Tnf^ΔARE/+ mice in SPF-B and CONV facilities. N = 4 per condition from 8 cages. Mean plus standard error of the mean (SEM) are shown and statistical significance was determined using multiple unpaired t tests with false discovery rate (FDR) of 1%. (C) Shotgun metagenomic data with reads mapped to C. muridarum species. Mean plus standard error of the mean (SEM) are shown and statistical significance was determined using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (D) Fecal DNA-based PCR testing for C. muridarum in Tnf^ΔARE/+ mice from SPF-B (N = 4) and CONV (N = 3) facilities. Original gels shown in Data S1. (E) Fecal DNA-based PCR testing for C. muridarum in SPF-B Tnf^ΔARE/+ mice before and after co-housing with Tnf^ΔARE/+ mice from the CONV facility. N = 3 SPF-B Tnf^ΔARE/+ recipients shown before and after co-housing with N = 3 donors. (F) Representative immunofluorescence (IF) images of Chlamydia major outer membrane protein (MOMP - green) and nuclei (Hoechst - blue) co-staining on colonic sections from wildtype (N = 3 at 8w of age, N = 4 at 35–37w of age) and Tnf^ΔARE/+ (N = 3 at 8w of age, N = 3 at 35–37w of age) mice from the CONV facility. Scale bars = 200 μm. (G) Quantification of Chlamydia from IF images, separated by ascending colon (AC) and distal colon regions. Mean plus standard error of the mean (SEM) are shown and statistical significance was determined using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (H) Representative confocal high-magnification IF stained images of Chlamydia major outer membrane protein (MOMP - green) and nuclei (Hoechst - blue) co-staining. Lumen side indicated in each image. N = 3 per condition. Scale bars = 10 μm. (I) Representative IF images of Chlamydia major outer membrane protein (MOMP - green) and nuclei (Hoechst - blue) co-staining with epithelial cell type-specific markers (CHGA – enteroendocrine cells, CLCA1 – goblet cells, DCLK1 – tuft cells) on colonic sections from age-matched CONV facility Tnf^ΔARE/+ mice (N = 5, 35–37w of age). White arrows point to infected enteroendocrine, goblet, or tuft cells. Scale bars = 50 μm. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001. See also Figure S2, Data S1, and Table S1.

Figure 3.. Chlamydia muridarum is necessary and sufficient to drive ascending colonic inflammation in the Tnf^ΔARE/+ model of Crohn’s-like disease.

(A) Experimental paradigm for doxycycline administration to Tnf^ΔARE/+ mice in the CONV facility. (B) Fecal DNA-based PCR testing for C. muridarum in Tnf^ΔARE/+ mice from doxycycline administration, where fecal DNA was tested pre-treatment, within 1 week of cessation of doxycycline, and prior to harvesting tissue. N = 4 mice per condition, age-matched at 11–12w of age at harvest. Original gels shown in Data S1. (C) Representative IF images of Chlamydia major outer membrane protein (MOMP - green) and nuclei (Hoechst - blue) co-staining on ascending colon sections from Tnf^ΔARE/+ mice treated with doxycycline or vehicle. N = 4 mice per condition, age-matched at 11–12w of age at harvest. (D) Representative H&E-stained ascending colon sections from Tnf^ΔARE/+ mice treated with doxycycline or vehicle. N = 4 mice per condition, age-matched at 11–12w of age at harvest. (E) Colitis scores from histopathological scoring of colons from Tnf^ΔARE/+ mice in D. Mean plus SEM are shown, and statistical significance was determined using an unpaired t test. (F) Colitis subscores that contribute to overall colitis score in E. Mean plus SEM are shown, and statistical significance was determined using multiple unpaired t tests with FDR of 1%. LP = lamina propria. PMNs = polymorphonuclear leukocytes. (G) Shotgun metagenomic data, filtered for only the eubacteria kingdom, of proximal (ascending) colon luminal contents from Tnf^ΔARE/+ mice treated with doxycycline or vehicle. Data is represented as relative abundance of mapped phyla for individual mice and grouped by doxycycline (N = 3) or vehicle treated (N = 4) conditions from 7 cages. Samples are from Tnf^ΔARE/+ mice of mixed ages (11–23w of age). (H) Shotgun metagenomic data from G with reads specifically mapped to C. muridarum species. Mean plus standard error of the mean (SEM) are shown and statistical significance was determined using an unpaired t test. (I) Shotgun metagenomic data from G represented as relative abundance of mapped phyla for individual samples. Mean plus standard error of the mean (SEM) are shown, and statistical significance was determined using multiple unpaired t tests with false discovery rate (FDR) of 1%. (J) Experimental paradigm for CM001-GFP inoculation (3x10⁶ IFUs) of Tnf^ΔARE/+ mice from the SPF-B facility. (K) Fecal DNA-based PCR testing for C. muridarum in Tnf^ΔARE/+ mice from CM001-GFP inoculation, where fecal DNA was tested prior to harvesting tissue. N = 4 mice per condition, age-matched at 16–20w of age at harvest. (L) Body weights of mice from the CM001-GFP or sham-inoculated Tnf^ΔARE/+ mice, where weight change was calculated at harvest as a percent of the individual mouse’s weight prior to treatment. N = 10 mice per condition, age-matched at 16–20w of age at harvest. (M) Colitis scores from histopathological scoring of colons from Tnf^ΔARE/+ mice inoculated with CM001-GFP or sham. Mean plus SEM are shown, and statistical significance was determined using an unpaired t test. (N) Representative H&E-stained ascending colon sections from Tnf^ΔARE/+ mice inoculated with CM001-GFP or sham. N = 10 mice per condition, age-matched at 16–20w of age at harvest. (O) Representative IF images of Chlamydia major outer membrane protein (MOMP - green), endogenous GFP from CM001-GFP (GFP – red), and nuclei (Hoechst - blue) co-staining on ascending colon sections from Tnf^ΔARE/+ mice that are sham or CM001-GFP-inoculated. N = 5 mice per condition, age-matched at 16–20w of age at harvest. Inset to show colocalization of GFP and MOMP signal in CM001-GFP-inoculated conditions. (P) Shotgun metagenomic data, filtered for only the eubacteria kingdom, of proximal (ascending) colon luminal contents from Tnf^ΔARE/+ mice in sham or CM001-GFP-inoculated conditions. Data is represented as relative abundance of mapped phyla for individual Tnf^ΔARE/+ mice and grouped by CM001-GFP-inoculated (N = 5) or sham (N = 4) conditions from 4 cages. Samples are from Tnf^ΔARE/+ mice aged-matched at 16–20w of age at collection. (Q) Shotgun metagenomic data from O with reads specifically mapped to C. muridarum species. Mean plus standard error of the mean (SEM) are shown and statistical significance was determined using an unpaired t test. (R) Shotgun metagenomic data from O represented as relative abundance of mapped phyla for individual samples. Mean plus standard error of the mean (SEM) are shown, and statistical significance was determined using multiple unpaired t tests with false discovery rate (FDR) of 1%. (S) Venn diagram depicting enriched species from shotgun metagenomic samples that are shared across all experimental paradigms. All scale bars = 200 μm. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001. See also Figure S3, Figure S4, Table S2, Table S3, and Data S1.

Figure 4.. Chlamydia muridarum colonization upregulates ascending colonic goblet cell IDO1 expression.

(A) UMAP co-embedding of scRNA-seq samples of AC epithelial cells from CONV wildtype (N = 3), CONV young Tnf^ΔARE/+, (N = 4), CONV aged Tnf^ΔARE/+ (N = 4), SPF-B wildtype (N = 4), and SPF-B young Tnf^ΔARE/+ (N = 5) mice. Sample type overlay is indicated by color. (B) UMAP co-embedding of scRNA-seq data with cell type overlay indicated by color. (C) Bar graph of cell type proportions from scRNA-seq data, separated by sample type. (D) Statistical comparison of cell type proportions from scRNA-seq data separated by sample type. Mean plus standard error of the mean (SEM) are shown, and statistical significance was determined using multiple unpaired t tests with false discovery rate (FDR) of 1% between the following samples: CONV wildtype vs CONV young Tnf^ΔARE/+, CONV young Tnf^ΔARE/+ vs SPF-B young Tnf^ΔARE/+, and CONV young Tnf^ΔARE/+ vs CONV aged Tnf^ΔARE/+. Only statistically significant results are shown. (E) Gene Ontology of Biological Process terms derived from over-representation analysis (ORA) of upregulated genes in cells from the SPF-B facility (top) or CONV facility (bottom). Input to ORA included all upregulated genes identified by differential expression performed for each cell type between CONV and SPF-B facilities, pooling all genotypes (wildtype and Tnf^ΔARE/+) and age (young and aged) conditions in each facility. Enrichment ratio is shown and statistical significance was determined using false discovery rate (FDR) < 0.05. (F) Dot plot of gene expression from scRNA-seq data separated by sample type. Selected marker genes for surface goblet cells, progenitors, and goblet cells. Color indicates expression level while circle size represents percent of cells expressing the gene. (G) Violin plot of Ido1 expression in each cell type from scRNA-seq data separated by sample type. (H) Representative IF images of IDO1 (magenta), Chlamydia major outer membrane protein (green), and nuclei (Hoechst - blue) co-staining on ascending colon sections from wildtype and Tnf^ΔARE/+ mice from the SPF-B and CONV facilities. N = 3 mice, age-matched at 34–42w of age at harvest. Scale bars = 100 μm. (I) Representative IF images of IDO1 (magenta), UEA1 lectin (yellow), and nuclei (Hoechst - blue) co-staining on ascending colon sections from wildtype and Tnf^ΔARE/+ mice from the CONV facility. Inset image to show colocalization of UEA1 lectin, a goblet and secretory granule marker, with IDO1. N = 3 mice, age-matched at 16–17w of age at harvest. Scale bars = 100 μm. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001. See also Figure S5, Figure S6, Table S4, Table S5, Table S6, and Table S7.

Figure 5.. Perturbation of the IDO1 pathway reduces Chlamydia-driven ascending colon inflammation in the Tnf^ΔARE/+ model.

(A) Representative IF images of IDO1 (magenta), Chlamydia major outer membrane protein (MOMP - green), and nuclei (Hoechst - blue) co-staining on ascending colon sections from CONV Tnf^ΔARE/+ mice treated with doxycycline or vehicle. N = 4 mice per condition, age-matched at 11–12w of age at harvest. Scale bars = 100 μm. (B) Representative IF images of IDO1 (magenta), Chlamydia major outer membrane protein (MOMP - green), and nuclei (Hoechst - blue) co-staining on ascending colon sections from SPF-B Tnf^ΔARE/+ mice that are sham or CM001-GFP-inoculated. N = 5 mice per condition, age-matched at 16–20w of age at harvest. Scale bars = 200 μm. (C) Experimental paradigm for secretory cell ablation in Tnf^ΔARE/+ mice from the SPF-B facility, subsequently transferred to the CONV facility to be co-housed for 3 weeks with Chlamydia-positive cagemates. (D) Representative H&E-stained ascending colon sections from Tnf^ΔARE/+ mice with or without secretory cell ablation. N = 5 control Tnf^ΔARE/+ mice, N = 3 Lrig1-Atoh1-KO Tnf^ΔARE/+ mice, age-matched at 20–27w of age at harvest. Scale bars = 200 μm. Insets to show lack of goblet cell granules in the Lrig1-Atoh1-KO condition. (E) Representative IF images of IDO1 (magenta), Chlamydia major outer membrane protein (MOMP - green), and nuclei (Hoechst - blue) co-staining on ascending colon sections from Tnf^ΔARE/+ mice with or without secretory cell ablation. N = 5 control Tnf^ΔARE/+ mice, N = 3 Lrig1-Atoh1-KO Tnf^ΔARE/+ mice, age-matched at 20–27w of age at harvest. Scale bars = 100 μm. (F) Colitis scores from histopathological scoring of colons from Tnf^ΔARE/+ mice from the secretory cell ablation experiment. Mean plus SEM are shown, and statistical significance was determined using an unpaired t test. (G) Colitis subscores that contribute to overall colitis score. Mean plus SEM are shown, and statistical significance was determined using multiple unpaired t tests with FDR of 1%. (H) Experimental paradigm for administration of tryptophan-deficient diet to Tnf^ΔARE/+ mice from the CONV facility. (I) Representative H&E-stained ascending colon sections from CONV Tnf^ΔARE/+ mice fed a control diet or a tryptophan-deficient diet. N = 7 Tnf^ΔARE/+ mice on control diet, N = 5 Tnf^ΔARE/+ mice on tryptophan-deficient diet, age-matched at 11–12w of age at harvest. Scale bars = 200 μm. (J) Colitis scores from histopathological scoring of colons from CONV Tnf^ΔARE/+ fed a control diet or a tryptophan-deficient diet. Mean plus SEM are shown, and statistical significance was determined using an unpaired t test. (K) Colitis subscores that contribute to overall colitis score. Mean plus SEM are shown, and statistical significance was determined using multiple unpaired t tests with FDR of 1%. (L) Representative IF images of IDO1 (magenta), Chlamydia major outer membrane protein (MOMP - green), and nuclei (Hoechst - blue) co-staining on ascending colon sections from CONV Tnf^ΔARE/+ mice fed a control diet or a tryptophan-deficient diet. N = 7 Tnf^ΔARE/+ mice on control diet, N = 5 Tnf^ΔARE/+ mice on tryptophan-deficient diet, age-matched at 11–12w of age at harvest. Scale bars = 100 μm. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001. See also Figure S7 and Figure S8.

Figure 6.. Inflammation and secretory function of the small intestine are independent of ascending colon inflammation.

(A) Representative H&E-stained ascending colon sections from CONV Tnf^Δreg/+ (N = 5), CONV Tnf^ΔARE/+ (N = 6), rederived Chlamydia-negative CONV Tnf^Δreg/+ (N = 5), and doxycycline-treated CONV Tnf^ΔARE/+ (N = 4) mice. Mice are age-matched at 16–17w of age at harvest. Scale bars = 200 μm. (B) Colitis scores of ascending colons of TNF mutant mice and wildtype mice (N = 4). Mean plus SEM are shown, and statistical significance was determined using an ordinary one-way ANOVA with multiple comparisons. (C) Representative H&E-stained terminal ileum sections from CONV Tnf^Δreg/+ (N = 5), CONV Tnf^ΔARE/+ (N = 6), rederived Chlamydia-negative CONV Tnf^Δreg/+ (N = 5), and doxycycline-treated CONV Tnf^ΔARE/+ (N = 4) mice. Mice are age-matched at 16–17w of age at harvest. Scale bars = 50 μm. (D) Ileitis scores of terminal ilea of TNF mutant mice and wildtype mice (N = 4). Mean plus SEM are shown, and statistical significance was determined using an ordinary one-way ANOVA with multiple comparisons. (E) Representative IF images of IDO1 (magenta), Chlamydia major outer membrane protein (MOMP - green), and nuclei (Hoechst - blue) co-staining of ascending colon sections from CONV Tnf^Δreg/+, CONV Tnf^ΔARE/+, rederived Chlamydia-negative CONV Tnf^Δreg/+, and doxycycline-treated CONV Tnf^ΔARE/+ mice. N = 3 mice per condition, age-matched at 16–17w of age at harvest. Scale bars = 100 μm. (F) Representative IF images of IDO1 (magenta), UEA1 lectin (yellow), and nuclei (Hoechst - blue) co-staining of ascending colon sections from CONV Tnf^Δreg/+ mice. Inset image to show colocalization of UEA1 lectin, a goblet and secretory granule marker, with IDO1. N = 3 mice, age-matched at 16–17w of age at harvest. Scale bars = 100 μm. (G) Representative H&E-stained ascending colon sections from control Tnf^ΔARE/+ (N = 4) and PC-DTA Tnf^ΔARE/+ (N = 4) mice. Mice are from the CONV facility and are age-matched at 8w of age at harvest. Scale bars = 200 μm. (H) Colitis scores of ascending colons from control Tnf^ΔARE/+ and PC-DTA Tnf^ΔARE/+ mice. Mean plus SEM are shown, and statistical significance was determined using an unpaired t test. (I) Representative H&E-stained ascending colon sections from control Tnf^ΔARE/+ (N = 3) and PC-Atoh1-KO Tnf^ΔARE/+ (N = 4) mice. Mice are from the CONV facility and are age-matched at 6–10w of age at harvest. Scale bars = 200 μm. (J) Colitis scores of ascending colons from control Tnf^ΔARE/+ and PC-Atoh1-KO Tnf^ΔARE/+ mice. Mean plus SEM are shown, and statistical significance was determined using an unpaired t test. (K) Representative H&E-stained ascending colon sections from aged control Tnf^ΔARE/+ (N = 4) and aged PC-Atoh1-KO Tnf^ΔARE/+ (N = 4) mice. Mice are from the CONV facility and are age-matched at 23–64w of age at harvest. Scale bars = 200 μm. (L) Colitis scores of ascending colons from aged control Tnf^ΔARE/+ and aged PC-Atoh1-KO Tnf^ΔARE/+ mice. Mean plus SEM are shown, and statistical significance was determined using an unpaired t test. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001. See also Figure S9, Figure S10, and Figure S11.

Figure 7.. Analysis of human Crohn’s disease in the AC reveals epithelial IDO1 upregulation coupled to alternative microbial colonization.

(A) UMAP co-embedding of human scRNA-seq data of AC epithelial cells from normal (N = 15), inactive CD (N = 34), and active CD (N = 19) specimens. Sample type overlay is indicated by color. Normal AC is composed of healthy control specimens, inactive CD AC is composed of CD specimens histopathologically scored as normal or quiescent, and active CD AC is composed of CD specimens histopathologically scored as mild, moderate, or severe. (B) Cell type breakdown of AC scRNA-seq data: UMAP with cell type overlay indicated by color (left), barplot with cell type proportion indicated by color, separated by sample type (middle), UMAP with LND cell overlay indicated by color (right). (C) Statistical comparison of LND cell type proportion amongst all epithelial cells from AC scRNA-seq data separated by sample type. Mean plus standard deviation are shown, and statistical significance was determined using independent t tests. (D) Statistical comparison of IDO1-expressing cell proportions amongst all epithelial cells from AC scRNA-seq data separated by sample type. Mean plus standard deviation are shown, and statistical significance was determined using independent t tests. (E) UMAP of AC scRNA-seq data with overlay of IDO1 gene expression indicated by the color gradient. Inset to show IDO1 expression in the LND cluster. (F) Statistical comparison of the proportion of IDO1-expressing LND cells amongst all epithelial cells from AC scRNA-seq data separated by sample type. Mean plus standard deviation are shown, and statistical significance was determined using independent t tests. (G) Representative IF images of IDO1 (magenta) and nuclei (Hoechst - blue) co-staining on AC biopsy sections from CD specimens with normal (N = 3) or active (N = 3) histopathological scoring. Inset and individual channels to show IDO1 expression in epithelial cells. Scale bars = 200 μm. (H) Shotgun metagenomic data, filtered for only the eubacteria kingdom, of AC biopsies from CD specimens with normal (N = 5) or active (N = 3) histopathological scoring. Data is represented as relative abundance of mapped species for individual specimens. “CD of SI” indicates AC specimens from CD patients with history of small intestinal inflammation and no history of AC inflammation, while “CD of SI and colon” indicates AC specimens from CD patients with a history of ileocolonic inflammation. p-value * < 0.05, ** < 0.01, *** < 0.001. **** < 0.0001. See also Figure S12 and Table S8.