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Cited by 403Wnt/β-catenin signaling in development and cancerInflammatory Bowel DiseaseIL-33, ST2, and ILC Pathways

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The Environmental Sensor AHR Protects from Inflammatory Damage by Maintaining Intestinal Stem Cell Homeostasis and Barrier Integrity

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Metidji, Amina, Omenetti, Sara, Crotta, Stefania, Li, Ying, Nye, Emma, Ross, Ellie, Li, Vivian, Maradana, Muralidhara R., Schiering, Chris, Stockinger, Brigitta·2018·Immunity
DOI 10.1016/j.immuni.2018.07.010PMC PMC6104739
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Abstract

The epithelium and immune compartment in the intestine are constantly exposed to a fluctuating external environment. Defective communication between these compartments at this barrier surface underlies susceptibility to infections and chronic inflammation. Environmental factors play a significant, but mechanistically poorly understood, role in intestinal homeostasis. We found that regeneration of intestinal epithelial cells (IECs) upon injury through infection or chemical insults was profoundly influenced by the environmental sensor aryl hydrocarbon receptor (AHR). IEC-specific deletion of Ahr resulted in failure to control C. rodentium infection due to unrestricted intestinal stem cell (ISC) proliferation and impaired differentiation, culminating in malignant transformation. AHR activation by dietary ligands restored barrier homeostasis, protected the stem cell niche, and prevented tumorigenesis via transcriptional regulation of of Rnf43 and Znrf3, E3 ubiquitin ligases that inhibit Wnt-β-catenin signaling and restrict ISC proliferation. Thus, activation of the AHR pathway in IECs guards the stem cell niche to maintain intestinal barrier integrity.

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Graphical abstract CC BY · PubMed Central
Figure 1 Ahr Deficiency in IEC Impairs Resistance to Citrobacter rodentium (A) Survival plot of bone marrow chimeras infected with C. rodentium (WT→ WT; Ahr−/− → WT; Ahr−/− → Ahr−/−; WT → Ahr−/−); n = 5 per group. (B) Absolute numbers of colonic RORγt+ ILC3 and IL-17A-producing TCRβ+CD4+ T cells (WT, n = 7; VillinCreAhrfl/fl, n = 7) at day 7. (C) IL-22 protein content in colon explant cultures (WT, n = 12; VillinCreAhrfl/fl, n = 12). Data represent pooled results of at least two independent experiments. (D) qPCR analysis of antimicrobial (IL-22, Reg3g, S100a9) in colon from WT and VillinCreAhrfl/fl mice at day 7. (E) C. rodentium burdens in colon, liver, and spleen. Bars show the median and each symbol represents an individual mouse. (F) Colon sections stained for E-cadherin (green), C. rodentium (red), and DAPI (blue). Scale bars, 50 μm. (G) Survival plot of mice infected with C. rodentium (WT, n = 7; VillinCreAhrfl/fl, n = 7). (H) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 (WT, n = 7; VillinCreAhrfl/fl, n = 7). (I) Representative image of AB-PAS staining in WT and VillinCreAhrfl/fl. Scale bars, 50 μm. (J) Quantification of the number of AB-PAS-positive cells per 20 crypts in WT and VillinCreAhrfl/fl. Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way ANOVA with Tukey post-test.
Figure 1 Ahr Deficiency in IEC Impairs Resistance to Citrobacter rodentium (A) Survival plot of bone marrow chimeras infected with C. rodentium (WT→ WT; Ahr−/− → WT; Ahr−/− → Ahr−/−; WT → Ahr−/−); n = 5 per group. (B) Absolute numbers of colonic RORγt+ ILC3 and IL-17A-producing TCRβ+CD4+ T cells (WT, n = 7; VillinCreAhrfl/fl, n = 7) at day 7. (C) IL-22 protein content in colon explant cultures (WT, n = 12; VillinCreAhrfl/fl, n = 12). Data represent pooled results of at least two independent experiments. (D) qPCR analysis of antimicrobial (IL-22, Reg3g, S100a9) in colon from WT and VillinCreAhrfl/fl mice at day 7. (E) C. rodentium burdens in colon, liver, and spleen. Bars show the median and each symbol represents an individual mouse. (F) Colon sections stained for E-cadherin (green), C. rodentium (red), and DAPI (blue). Scale bars, 50 μm. (G) Survival plot of mice infected with C. rodentium (WT, n = 7; VillinCreAhrfl/fl, n = 7). (H) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 (WT, n = 7; VillinCreAhrfl/fl, n = 7). (I) Representative image of AB-PAS staining in WT and VillinCreAhrfl/fl. Scale bars, 50 μm. (J) Quantification of the number of AB-PAS-positive cells per 20 crypts in WT and VillinCreAhrfl/fl. Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way ANOVA with Tukey post-test.CC BY · PubMed Central
Figure 2 AHR Pathway Is Required for Epithelial Cell Differentiation from Crypt Stem Cells (A) Images of stem cell (SC) organoids from WT, Ahr−/−, and R26Cyp1a1 mice. Scale bars, 50 μm. (B) Percentage of EdU+ cells in SC organoid cultures. (C) Percentage of EdU+ cells in SC organoid cultures treated with 5 nM FICZ or 1 μM ICZ. (D and E) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 in SC or differentiated (diff) organoids treated or not with 5 nM FICZ for 4 days. Data represent pooled results of at least two independent experiments (n = 6). Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way and two-way ANOVA with Tukey post-test.
Figure 2 AHR Pathway Is Required for Epithelial Cell Differentiation from Crypt Stem Cells (A) Images of stem cell (SC) organoids from WT, Ahr−/−, and R26Cyp1a1 mice. Scale bars, 50 μm. (B) Percentage of EdU+ cells in SC organoid cultures. (C) Percentage of EdU+ cells in SC organoid cultures treated with 5 nM FICZ or 1 μM ICZ. (D and E) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 in SC or differentiated (diff) organoids treated or not with 5 nM FICZ for 4 days. Data represent pooled results of at least two independent experiments (n = 6). Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way and two-way ANOVA with Tukey post-test.CC BY · PubMed Central
Figure 3 Dysregulated Ahr in IECs Leads to Enhanced Stem Cell Proliferation and Increased Inflammation (A) Flow cytometry analysis of Lgr5 and Ki-67 expression in EpCam+CD45− cells and absolute number of Lgr5+Ki-67+ cells at steady state from 5- to 8-week-old mice. (B and C) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 from sorted EpCam+ cells (WT, n = 6; VillinCreAhrfl/fl, n = 6; VillinCreR26LSL-Cyp1a1, n = 6) in young (5 to 9 weeks) and old (14 to 16 weeks) mice. (D and E) IL-6 protein content in colon explant cultures at steady state (WT, n = 6; VillinCreAhrfl/fl, n = 6; VillinCreR26LSL-Cyp1a1, n = 6) from young and old mice. (F) Number of colon tumors in WT (n = 8), VillinCreAhrfl/fl (n = 8), and VillinCreR26LSL-Cyp1a1 (n = 8) mice injected with 10 mg/kg of azoxymethane once a week for 6 weeks. Representative image of colon in mice 22 weeks after the first azoxymethane injection. Error bars, mean + SEM., ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way ANOVA with Tukey post-test. See also Figures S1A and S1B.
Figure 3 Dysregulated Ahr in IECs Leads to Enhanced Stem Cell Proliferation and Increased Inflammation (A) Flow cytometry analysis of Lgr5 and Ki-67 expression in EpCam+CD45− cells and absolute number of Lgr5+Ki-67+ cells at steady state from 5- to 8-week-old mice. (B and C) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 from sorted EpCam+ cells (WT, n = 6; VillinCreAhrfl/fl, n = 6; VillinCreR26LSL-Cyp1a1, n = 6) in young (5 to 9 weeks) and old (14 to 16 weeks) mice. (D and E) IL-6 protein content in colon explant cultures at steady state (WT, n = 6; VillinCreAhrfl/fl, n = 6; VillinCreR26LSL-Cyp1a1, n = 6) from young and old mice. (F) Number of colon tumors in WT (n = 8), VillinCreAhrfl/fl (n = 8), and VillinCreR26LSL-Cyp1a1 (n = 8) mice injected with 10 mg/kg of azoxymethane once a week for 6 weeks. Representative image of colon in mice 22 weeks after the first azoxymethane injection. Error bars, mean + SEM., ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way ANOVA with Tukey post-test. See also Figures S1A and S1B.CC BY · PubMed Central
Figure 4 Inflammation-Induced Tumorigenesis in Mice with Ahr-Deficient Epithelium (A) Number of colon tumors in WT (n = 10), VillinCreAhrfl/fl (n = 10), and VillinCreR26LSL-Cyp1a1 (n = 9) mice. (B) Representative image of colon after AOM/DSS treatment. (C) Size of colon tumors in WT (n = 10), VillinCreAhrfl/fl (n = 10), and VillinCreR26LSL-Cyp1a1 (n = 9) mice. (D) Scoring of colon tumors VillinCreAhrfl/fl (n = 10) and VillinCreR26LSL-Cyp1a1 (n = 9) mice. (E) Representative images of hematoxylin and eosin (H&E) (top) and β-catenin staining of whole intestine (middle) and focus (bottom) of the indicated mice 60 days after injection of azoxymethane. Scale bars, 50 μm. (F) qPCR analysis of Wnt-negative regulators (Znrf3 and Rnf43) and WNT target genes (Axin2, cMyc, and Ephb2) in the colon (WT, n = 5; VillinCreAhrfl/fl, n = 5; VillinCreR26LSL-Cyp1a1, n = 5) after AOM/DSS treatment. (G) Chromatin immunoprecipitation (ChIP) analysis of AHR interaction with Rnf43 promoter from lanima propria of WT, WT treated with FICZ, Ahr−/−, and Ahr−/− treated with FICZ. Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way and two-way ANOVA with Tukey post-test. See also Figures S2A–S2D.
Figure 4 Inflammation-Induced Tumorigenesis in Mice with Ahr-Deficient Epithelium (A) Number of colon tumors in WT (n = 10), VillinCreAhrfl/fl (n = 10), and VillinCreR26LSL-Cyp1a1 (n = 9) mice. (B) Representative image of colon after AOM/DSS treatment. (C) Size of colon tumors in WT (n = 10), VillinCreAhrfl/fl (n = 10), and VillinCreR26LSL-Cyp1a1 (n = 9) mice. (D) Scoring of colon tumors VillinCreAhrfl/fl (n = 10) and VillinCreR26LSL-Cyp1a1 (n = 9) mice. (E) Representative images of hematoxylin and eosin (H&E) (top) and β-catenin staining of whole intestine (middle) and focus (bottom) of the indicated mice 60 days after injection of azoxymethane. Scale bars, 50 μm. (F) qPCR analysis of Wnt-negative regulators (Znrf3 and Rnf43) and WNT target genes (Axin2, cMyc, and Ephb2) in the colon (WT, n = 5; VillinCreAhrfl/fl, n = 5; VillinCreR26LSL-Cyp1a1, n = 5) after AOM/DSS treatment. (G) Chromatin immunoprecipitation (ChIP) analysis of AHR interaction with Rnf43 promoter from lanima propria of WT, WT treated with FICZ, Ahr−/−, and Ahr−/− treated with FICZ. Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as calculated by one-way and two-way ANOVA with Tukey post-test. See also Figures S2A–S2D.CC BY · PubMed Central
Figure 5 Dietary AHR Ligands Prevent Tumorigenesis and Restore Regulation of the WNT Pathway (A) qPCR analysis of WNT negative regulators (Znrf3 and Rnf43) and WNT target genes (Axin2, cMyc, and Ephb2) in the colon of VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet (n = 5). (B) Number of colon tumors in WT (n = 10), VillinCreAhrfl/fl, and VillinCreR26LSL-Cyp1a1 (n = 10), fed purified or I3C diet after AOM/DSS treatment. (C) Representative images of hematoxylin and eosin (H&E) and β-catenin of colon tumors in VillinCreR26LSL-Cyp1a1 fed purified or I3C diet. Scale bars, 50 μm. (D) qPCR analysis of Muc2 and Car4 in colon from WT (n = 5) and VillinCreR26LSL-Cyp1a1 (n = 5), fed purified or I3C diet. (E) Representative images of Ki-67 of colon tumors in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. Scale bars, 50 μm. (F) Quantification of the number of Ki-67+ cells in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, as calculated by unpaired t test and two-way ANOVA with Tukey post-test. See also Figures S3A and S3B.
Figure 5 Dietary AHR Ligands Prevent Tumorigenesis and Restore Regulation of the WNT Pathway (A) qPCR analysis of WNT negative regulators (Znrf3 and Rnf43) and WNT target genes (Axin2, cMyc, and Ephb2) in the colon of VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet (n = 5). (B) Number of colon tumors in WT (n = 10), VillinCreAhrfl/fl, and VillinCreR26LSL-Cyp1a1 (n = 10), fed purified or I3C diet after AOM/DSS treatment. (C) Representative images of hematoxylin and eosin (H&E) and β-catenin of colon tumors in VillinCreR26LSL-Cyp1a1 fed purified or I3C diet. Scale bars, 50 μm. (D) qPCR analysis of Muc2 and Car4 in colon from WT (n = 5) and VillinCreR26LSL-Cyp1a1 (n = 5), fed purified or I3C diet. (E) Representative images of Ki-67 of colon tumors in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. Scale bars, 50 μm. (F) Quantification of the number of Ki-67+ cells in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. Error bars, mean + SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, as calculated by unpaired t test and two-way ANOVA with Tukey post-test. See also Figures S3A and S3B.CC BY · PubMed Central
Figure 6 Dietary AHR Ligands Can Halt Progression of Tumorigenesis (A) VillinCreR26LSL-Cyp1a1 were injected with 10 mg/kg of AOM followed with one cycle of 1% DSS on standard chow diet. For the second cycle of DSS, mice were put either on a purified diet or I3C diet until the end of the experiment. (B) Number of colon tumors in WT (n = 10), VillinCreAhrfl/fl, and VillinCreR26LSL-Cyp1a1 (n = 10) mice, fed purified or I3C diet after AOM/DSS treatment. (C) Representative image of colon after AOM/DSS treatment. (D and E) Size (D) and score (E) of tumors in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. (F) Representative images of hematoxylin and eosin (H&E) colon tumors in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. Scale bars, 50 μm. Error bars, mean + SEM. ∗∗∗p < 0.001, as calculated by paired t test.
Figure 6 Dietary AHR Ligands Can Halt Progression of Tumorigenesis (A) VillinCreR26LSL-Cyp1a1 were injected with 10 mg/kg of AOM followed with one cycle of 1% DSS on standard chow diet. For the second cycle of DSS, mice were put either on a purified diet or I3C diet until the end of the experiment. (B) Number of colon tumors in WT (n = 10), VillinCreAhrfl/fl, and VillinCreR26LSL-Cyp1a1 (n = 10) mice, fed purified or I3C diet after AOM/DSS treatment. (C) Representative image of colon after AOM/DSS treatment. (D and E) Size (D) and score (E) of tumors in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. (F) Representative images of hematoxylin and eosin (H&E) colon tumors in VillinCreR26LSL-Cyp1a1 mice fed purified or I3C diet. Scale bars, 50 μm. Error bars, mean + SEM. ∗∗∗p < 0.001, as calculated by paired t test.CC BY · PubMed Central

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Authors
Metidji, A., Omenetti, S., Crotta, S., Li, Y., Nye, E., Ross, E., Li, V., Maradana, M. R., Schiering, C., & Stockinger, B.
Affiliations
  • The Francis Crick Institute (GB)
  • University of London (GB)
Corresponding author
  • Schiering, Chris
  • Stockinger, Brigitta
ORCID
Venue
Immunity
Publisher
Elsevier BV
Published
2018-08
Volume
49
Issue
2
Pages
353-362.e5
ISSN
1074-7613 (ISSN-L)
DOI
10.1016/j.immuni.2018.07.010
PMC
PMC6104739
OpenAlex
W2887221348
Cited by
402
References
45
Citations by year
2026: 37 · 2025: 50 · 2024: 56 · 2023: 63 · 2022: 55 · 2021: 60 · 2020: 50 · 2019: 30
FWCI
12.45
Type
journal article
Language
en
Update notices
erratum (2019-06-18)
Open access
Licence
Locations
  • Immunity (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
  • Cancer Research UK (FC001105, FC001159, 103156/B/13/Z, 19691)
  • Fondation Acteria
  • Medical Research Council
  • Wellcome Trust (100910/Z/13/Z)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
  • Health Sciences › Medicine › Gastroenterology › Inflammatory Bowel Disease
  • Life Sciences › Immunology and Microbiology › Immunology › IL-33, ST2, and ILC Pathways
Keywords
aryl hydrocarbon receptor, intestinal stem cells, intestinal barrier integrity, intestinal homeostasis, intestinal inflammation, RNF43, ZNRF3, Wnt/β-catenin signaling, E3 ubiquitin ligase, stem cell niche, intestinal tumorigenesis
MeSH terms
Animals, Cell Differentiation, Cell Line, Enterobacteriaceae Infections, Epithelial Cells, Humans, Intestinal Mucosa, Mice, Inbred C57BL, Stem Cells, Receptors, Aryl Hydrocarbon, Mice, Knockout, Tight Junctions, Citrobacter rodentium, Ubiquitin-Protein Ligases, Cell Proliferation, Mice, Basic Helix-Loop-Helix Proteins, Basic Helix-Loop-Helix Transcription Factors, HEK293 Cells, Wnt Signaling Pathway, Carcinogenesis

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