Feedback control of AHR signalling regulates intestinal immunity

Abstract

The aryl hydrocarbon receptor (AHR) recognizes xenobiotics as well as natural compounds such as tryptophan metabolites, dietary components and microbiota-derived factors1,2,3,4, and it is important for maintenance of homeostasis at mucosal surfaces. AHR activation induces cytochrome P4501 (CYP1) enzymes, which oxygenate AHR ligands, leading to their metabolic clearance and detoxification5. Thus, CYP1 enzymes have an important feedback role that curtails the duration of AHR signalling6, but it remains unclear whether they also regulate AHR ligand availability in vivo. Here we show that dysregulated expression of Cyp1a1 in mice depletes the reservoir of natural AHR ligands, generating a quasi AHR-deficient state. Constitutive expression of Cyp1a1 throughout the body or restricted specifically to intestinal epithelial cells resulted in loss of AHR-dependent type 3 innate lymphoid cells and T helper 17 cells and increased susceptibility to enteric infection. The deleterious effects of excessive AHR ligand degradation on intestinal immune functions could be counter-balanced by increasing the intake of AHR ligands in the diet. Thus, our data indicate that intestinal epithelial cells serve as gatekeepers for the supply of AHR ligands to the host and emphasize the importance of feedback control in modulating AHR pathway activation.

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Figure 1: CYP1A1 controls AHR ligand availability.
Figure 2: Depletion of natural AHR ligands leads to impaired immunity to C. rodentium.
Figure 3: IECs serve as gatekeeper for the supply of AHR ligands to the host.
Figure 4: Dietary AHR ligands restore immunity to C. rodentium.

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Acknowledgements

This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, The UK Medical Research Council and the Wellcome Trust. We would like to acknowledge the Biological Research Facility at the Francis Crick Institute for expert breeding and maintenance of our mouse strains, the Histopathology Facility for help with sections and staining and the Flow Cytometry Facility. We thank G. Frankel (Imperial College) for the gift of antiserum to C. rodentium and Genentech, South San Francisco, California, for providing anti-IL-22 and IL-22–Fc. The study was supported by a Wellcome Advanced Investigator Grant (B.S.) and a Sir Henry Wellcome Fellowship and the Fondation Acteria (C.S.). C.H. and C.R.W. are funded by Cancer Research UK Programme Grant C4639/A10822. E.W. is funded by the Swedish Research Council FORMAS and D.N. by National Institute of Environmental Health Sciences, NIH grant R01 ES014403.

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Authors

Contributions

C.S. designed, performed and analysed most of the experiments with input from A.M. A.I., Y.L., S.O. and E.W. performed the metabolic studies. A.P. assisted in designing the construct for R26Cyp1a1 mice, C.J.H. and C.R.W. provided Cyp1a1 reporter mice, and D.W.N. provided Cyp1-knockout mice. B.S. conceived the project and wrote the manuscript together with C.S.

Corresponding author

Correspondence to Brigitta Stockinger.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks H. Sokol and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Generation of R26Cyp1a1 allele.

a, The endogenous Rosa26 locus, the gene-targeting vector, the targeted Rosa26 allele including the Neo resistance gene cassette (R26Cyp1a1-neoR), the targeted allele (R26LSL-Cyp1a1) after FLPe-mediated recombination and the ubiquitously expressed R26Cyp1a1 are schematically depicted to scale. A minigene composed of the coding sequences of mouse Cyp1a1 and rat Thy1 connected by a 2A sequence followed by the Woodchuck hepatitis virus derived regulatory element (WPRE) and a bovine growth hormone polyadenylation site (bGH pA). b, Expression of rat THY1 in indicated cell types in the colon of R26Cyp1a1, IECCyp1a1 (Vil1-cre-R26LSL-Cyp1a1), Rag1Cyp1a1 (Rag1-cre-R26LSL-Cyp1a1) strains.

Extended Data Figure 2 Altered AHR ligand availability affects IL-22 production.

Flow cytometry analysis of IL-17A and IL-22 expression in in vitro differentiated TH17 cells from indicated genotypes (day 4) exposed to DMSO, 0.01 nM FICZ or 1 nM FICZ from the start of culture.

Extended Data Figure 3 Altered AHR ligand availability affects intestinal AHR-dependent ILC populations.

a, CYP1A1 enzyme activity, measured by EROD assay, in intestinal tissue homogenates of steady-state mice. b, Flow cytometry analysis of NKp46 and RORγt expression in CD45+ lineage negative (TCRβCD3TCRγδCD19CD11bGr1) Thy1+ live cells in the small intestine (upper panel) and phenotypic analysis of RORγt+NKp46 innate lymphoid cells (lower panel). Results are representative of three independent experiments (n = 3). Error bars, mean + s.e.m. ***P < 0.001, as calculated by one-way ANOVA with Tukey post-test. Source data

Extended Data Figure 4 Effects of Cyp1a1 overexpression on liver development.

a, Liver weight represented as percentage of body weight. b, Liver (left lobe) weight represented as percentage of body weight. Results are representative of two independent experiments. Bars are the mean, each symbol represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, as calculated by one-way ANOVA with Tukey post-test. Source data

Extended Data Figure 5 IL-22–Fc improves immunity to C. rodentium infection in R26Cyp1a1 mice.

Mice of indicated genotypes were infected orally with ~2 × 109 C. rodentium CFU and killed 7 days after infection or monitored for survival. a, C. rodentium burdens in the colon and caecum. Bars are the median, each symbol represents an individual mouse. b, Colon sections stained for E-cadherin (green), C. rodentium (red) and DAPI (blue). Scale bars, 100 μm. c, Survival plot (WT, n = 4; R26Cyp1a1 + control Ig, n = 6; R26Cyp1a1 + IL-22–Fc, n = 5). Results are representative of three independent experiments. NS, not significant, **P < 0.01, ***P < 0.001, as calculated by one-way ANOVA with Tukey post-test. Source data

Extended Data Figure 6 Cyp1-deficiency enhances AHR pathway activation.

a, CD4+ T cells from indicated genotypes were cultured under TH17-cell-inducing conditions and exposed to FICZ from the start of culture. Intracellular levels of FICZ were determined by HPLC and normalized to total protein content at the indicated time points (n = 3 per time point). b, Frequencies of IL-22-producing cells after 4 days of culture under TH17-cell-inducing conditions in presence of indicated concentrations of FICZ. Results are representative of three independent experiments. Error bars, mean + s.e.m. **P < 0.01, ***P < 0.001, as calculated by two-way ANOVA with Dunnett’s post-test (a) or one-way ANOVA with Tukey post-test (b). c, Flow cytometry analysis of Cyp1a1 (eYFP) expression by TH17 cells differentiated from indicated genotypes. Plots are gated on IL-17A+ cells and numbers indicate frequencies. d, Representative flow cytometry plots of IL-17A and IL-22 expression in in vitro differentiated TH17 cells from indicated genotypes (day 4) exposed to DMSO, 0.01 nM FICZ or 1 nM FICZ from the start of culture. e, Mice of indicated genotypes were infected orally with ~2 × 109 C. rodentium CFU and bacterial burdens measured in the faeces at various time points. f, Pathology scores of distal colon. Bars are the mean, symbol represents an individual mouse. Results are representative of at least two independent experiments. NS, not significant. **P < 0.01, ***P < 0.001, as calculated by Student’s t-test. Source data

Extended Data Figure 7 Effects of Rag1-cre mediated Cyp1a1 expression on immunity to C. rodentium.

Mice of indicated genotypes were infected orally with ~2 × 109 C. rodentium CFU and killed 14 days after infection or monitored for survival. a, Survival plot. b, C. rodentium burdens in the colon, caecum, liver and spleen. c, Pathology scores of distal colon and caecum. Bars are the mean, each symbol represents an individual mouse (b, c). d, Absolute numbers of cytokine-producing TCRβ+CD4+ T cells in the colon of mice infected with C. rodentium. Error bars, mean + s.e.m. Results are representative of two independent experiments (n = 5 per group). NS, not significant. **P < 0.01, as calculated by Student’s t-test. Source data

Extended Data Figure 8 CYP1A1-mediated metabolism of DIM, ICZ and FICZ.

ac, CYP1A1-mediated metabolism of DIM (a), ICZ (b) and FICZ (c) was studied over time in the presence of human recombinant CYP1A1 (3.5 nM) and the co-factor NADPH (1.0 mM). At indicated time points, samples from respective incubations were extracted and analysed by means of HPLC. All chemicals were quantified according to separate standard curves. Left panels show relative amount of compound remaining at each given time point compared to parallel incubations without co-factor present. Right panels show HPLC chromatograms at 40 min enzyme-incubation, with and without co-factor present. All three compounds were detected on the basis of their fluorescence properties (fluorescence units, FLU). Results are representative of two independent experiments with two biological replicates at each experiment. Error bars, mean + s.d. Source data

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Schiering, C., Wincent, E., Metidji, A. et al. Feedback control of AHR signalling regulates intestinal immunity. Nature 542, 242–245 (2017). https://doi.org/10.1038/nature21080

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