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Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity

Nature volume 623, pages 1044–1052 (2023)Cite this article

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Abstract

All nucleated cells express major histocompatibility complex I and interferon‐γ (IFNγ) receptor1, but an epithelial cell-specific function of IFNγ signalling or antigen presentation by means of major histocompatibility complex I has not been explored. We show here that on sensing IFNγ, colonic epithelial cells productively present pathogen and self-derived antigens to cognate intra-epithelial T cells, which are critically located at the epithelial barrier. Antigen presentation by the epithelial cells confers extracellular ATPase expression in cognate intra-epithelial T cells, which limits the accumulation of extracellular adenosine triphosphate and consequent activation of the NLRP3 inflammasome in tissue macrophages. By contrast, antigen presentation by the tissue macrophages alongside inflammasome-associated interleukin-1α and interleukin-1β production promotes a pathogenic transformation of CD4+ T cells into granulocyte–macrophage colony-stimulating-factor (GM-CSF)-producing T cells in vivo, which promotes colitis and colorectal cancer. Taken together, our study unravels critical checkpoints requiring IFNγ sensing and antigen presentation by epithelial cells that control the development of pathogenic CD4+ T cell responses in vivo.

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Fig. 1: Epithelial sensing of IFNγ protects from colitis by means of upregulation of IRF1.
Fig. 2: Epithelial sensing of IFNγ restrains pathogenic CD4 GM-CSF response.
Fig. 3: Epithelial sensing of IFNγ promotes eATPase expression in IE-T cells to restrain the colitogenic NLRP3 inflammasome.
Fig. 4: IFNγ-mediated antigen presentation by IECs promotes eATPase production by cognate IE-T cells.
Fig. 5: IFNγ-mediated antigen presentation restricts colon cancer.

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Acknowledgements

This work was supported by the US National Institutes of Health (R01DK067180) to B.J., the University of Chicago’s Center for Interdisciplinary Study of Inflammatory Intestinal Disorders (C-IID) Pilot & Feasibility Award (NIDDK P30 DK042086) to A.M. and D.S., Crohn’s and Colitis Foundation Career Development Award no. 964209 to A.M. and a G.I. Research Foundation Associates Board Award to A.M. and D.S. Whole-slide scanning was performed by S. Bond and K. Rodriguez in the Integrated Light Microscopy Core at the University of Chicago, which receives financial support from a Cancer Center Support Grant (P30CA014599), RRID:SCR_019197. We thank the University of Chicago Transgenic Mouse Facility (RRID:SCR_019171), especially L. Degenstein, for their assistance with producing the mice used in this study. We thank W. Lisicka, N. Usher, C. Ciszewski, Human Disease and Immune Discovery core (RRID:SCR_022936) and members of the B. Jabri lab at the University of Chicago. The model figure (Extended Data Fig. 11) was created with BioRender.com.

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Authors and Affiliations

  1. Department of Medicine, Committee on Immunology, Department of Pediatrics, Department of Pathology, University of Chicago, Chicago, IL, USA

    Ankit Malik, Deepika Sharma, Raúl Aguirre-Gamboa, Shaina McGrath, Sarah Zabala, Christopher Weber & Bana Jabri

  2. Department of Pathology, Department of Medicine, University of Chicago, Chicago, IL, USA

    Christopher Weber

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Contributions

A.M. and B.J. conceptualized this work. A.M., D.S., R.A.-G., C.W., S.Z. and S.M. developed the methodology. A.M., D.S., S.Z. and S.M. performed the investigation. A.M., D.S., S.M. and B.J. did the formal analysis. A.M. wrote the original draft, and D.S. and B.J. reviewed and edited it. A.M. and B.J. acquired the funding and resources. B.J. supervised the study.

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Correspondence to Ankit Malik or Bana Jabri.

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Extended data figures and tables

Extended Data Fig. 1 Progressive increase in IFN-γ signaling and antigen presentation by IECs during colitis.

C57BL/6 J mice were infected with Citrobacter rodentium. (A) Citrobacter colonization in the colon (n = 5 per group) and (B) quantification of Ifng transcript in colon tissue by RT-PCR (n = 4 for d0 and d21, n = 6 for d6 and n = 5 for d12). STAT1 pTyr701 analyses in IECs by (C) immunoblotting, representative of two independent experiments and (D) flow cytometry (n = 3 for d0, n = 6 for d6 and n = 5 for d12 and n = 4 for d21). (E) Quantification of Irf1 transcript in purified IECs (EpCAM+) by RT-PCR (n = 4 for d0, d6 and d21, n = 5 for d12). Flow cytometric analyses for (F and G) MHCI (n = 4 for d0, n = 5 for d6 and d12) and (H and I) MHCII (n = 4 for d0, n = 5 for d6 and d12) in the IECs from colons of mice at indicated day post-infection. (J) Colon histology score in Ifngr1fl/fl and Ifngr1fl/fl VilCre mice. The horizontal bar represents the median, and each symbol and each lane in the immunoblot represents an individual mouse. Data were analyzed by (A, B, D, E, G and I) Brown-Forsthye and Welsch ANOVA tests followed by the Dunnet’s post-hoc test.

Source data

Extended Data Fig. 2 Epithelial sensing of IFN-γ orchestrates T cell responses during colitis.

(A-I) Ifngr1fl/fl and Ifngr1fl/fl VilCre mice were infected with Citrobacter rodentium (n = 5 per group). (A-B) Enumeration of I.E. CD4+ T cells and (C-G) evaluation of production of indicated factors by them at indicated days post-infection. Enumeration of (H-L) indicated cells in the epithelium. (M) Comparison of GM-CSF production from ILCs and CD4+ T cells (n = 7 per group) and (N) GM-CSF MFI in ILCs and CD4+ T cells in epithelium of Ifngr1fl/fl VilCre mice at day 12 p.i. (n = 7 per group). (O) The indicated factors were gated globally on the live cell gate and then analyzed for the contribution of CD8αβ+ or CD4+ colonic IE-T cells in their production. Each symbol represents an individual mouse, and the bar indicates the median. Data were analyzed by (A-L) ANOVA followed by the Holm–Sidak post-hoc test or (M-N) Mann-Whitney U test (two-sided). MFI, median fluorescence intensity.

Source data

Extended Data Fig. 3 Generation of the ovalbumin-expressing strain of C. rodentium and characterization of ova-specific IE-T cells.

Ovalbumin coding sequence was cloned into the NocI site of pOBX18 plasmid containing the artificial and constitutive Enterobacteriaceae-specific promoter and terminator sequence into the temperature-sensitive transposase 7 attachment site (attTn7) targeting vector (vector described in59) (A) plasmid map and (B) gel picture of the transformed vector. Then, this vector was electroporated into C. rodentium. Growth at permissive temperature along with constitutive expression of the recombination machinery led to the integration of the promoter-ova-terminator sequence into the attTn7 site. Next, the plasmid was cured by subsequent growth at non-permissive temperatures. Site-specific integration was confirmed by (C) PCR and sequencing from the neighboring glmS region of the attTntn7 locus and the expression of ova was confirmed by (D) RT-PCR, representative of two independent clones. (E) Ifngr1fl/fl and Ifngr1fl/fl VilCre mice were infected with ova+ Citrobacter and (F) Ifngrfl/fl VilcreERT2 OvaTg and Ifngrfll+ VilcreERT2 OvaTg mice were infected with wild-type Citrobacter and administered tamoxifen on days 3-6 p.i and evaluated for IFN-γ production from the ova-specific CD4+ IE-T cells (n = 4 per group). (G) Flow cytometric phenotyping of ova-specific CD4+ IE-T cells at day 12 post-infection with ova+ Citrobacter. (H-K) Ifngr1fl/fl, Ifngr1fl/fl VilCreERT2, and Ifngr1fl/fl VilCreERT2 R26LSL Irf1 mice were infected with ova+ Citrobacter rodentium on day 0, injected with tamoxifen at days 3-6 post-infection, and analyzed on day 12 for CD4+ IE-T cells and (I) Citrobacter colonization in the colon (n = 5 per group). (L) Ifngr1fl/fl and Ifngr1fl/flVilCre mice were infected with Citrobacter and treated with isotype or anti-CD4 antibody every 2 days starting at day 7 analyzed for CD4+ T cells on day 12 p.i. (n = 8 per group). The horizontal bar represents the median, and each symbol represents an individual mouse. Data were analyzed by (E and F) Mann-Whitney U test (two-sided) or (H-L) Kruskal-Wallis test followed by Dunn’s post-test.

Source data

Extended Data Fig. 4 Epithelial sensing of IFN-γ restrains NLRP3 inflammasome activation in macrophages.

(A-K) Ifngr1fl/fl and Ifngr1fl/fl VilCre mice were infected with Citrobacter rodentium. (A and B) Evaluation of indicated cytokines in colon explants (n = 4 for d0, d6 and n = 8 for d12, per group) and (C) immunoblotting for caspase-1 p10 in colon lysates, representative of two independent experiments. (D) Evaluation of IL-1α in colon explants, (E-F) Colon histology analyses and (G) Citrobacter colonization after isotype (n = 8), αβ-ATP (n = 8), apyrase (n = 6), MCC950 (n = 6) or anti-IL1R (n = 6) treatment. (H-K) Ifngr1fl/fl and Ifngr1fl/fl VilCre mice were infected and given 500ug of anti-CSF1R or isotype control antibody on days 7, 9 and 11 p.i and sacrificed on day 12 p.i. (n = 8 per group). (H) Flow cytometric plot for colon macrophages from Ifngr1fl/fl VilCre mice treated with anti-CSF1R or isotype control antibody. (I) Evaluation of indicated cytokines in colon explants. (J) Flow-cytometric analyses of CD4+ IE-T cells and GM-CSF production from them and (K) colon length at day 12 p.i. Data were analyzed by Brown-Forsythe and Welch’s ANOVA followed by Dunnett’s post-test. Scale bar = 100 um.

Source data

Extended Data Fig. 5 Antigen presentation by CX3CR1 + APCs promote TH1 immunity.

H2abfl/fl and H2abΔiCX3CR1 mice were infected with Citrobacter, injected with tamoxifen on days 3–6 and sacrificed on day 12 p.i. (A) Flow cytometric analyses of MHCII expression in CD172a+ cells from the MLNs of H2abfl/fl and H2abΔiCX3CR1 mice (n = 9 per group). (B) enumeration of GM-CSF- IL-17A+ CD4+ IE-T cells from the MLNs of H2abfl/fl»IfngrΔiIEC and H2abΔiCX3CR1»IfngrΔiIEC chimeric mice after treatments described in Fig. 3g at day 12 p.i. (n = 7 per group). (C) H2abfl/fl and H2abΔiCX3RC1 mice were treated with tamoxifen for 4 consecutive days, followed by infection with Citroova 3 days after the last injection. Mice were injected with CTV-labelled 1 × 106 Rag-OTII cells on day 5 p.i., treated with 20ug of FTY720 i.p. on days 6-8 and sacrificed on day 9 (n = 6 per group). (C) Experimental scheme (D) OTII cells from MLNs were analyzed for T-bet induction. Data were analyzed by (A and D) by Mann-Whitney U test (two-sided) or (B) Kruskal-Wallis test followed by Dunn’s post-test.

Source data

Extended Data Fig. 6 Epithelial sensing of IFN-γ promotes eATPase production from IE-T cells.

(A-I) Ifngr1fl/fl and Ifngrfl/fl VilCre mice were infected with Citrobacter rodentium. (A and B) RT-PCR analyses for indicated factors in colon epithelial tissue fraction (n = 4 for d0, d6 per group and n = 8 for Ifngr1fl/fl and n = 6 for Ifngr1fl/fl VilCre for d12). (C, E-I) flow cytometric (n = 3 for d0, n = 5 for d6 and d12 per group) and (D) immunofluorescence evaluation of CD39 expression, representative of two independent experiments and (G) RT-PCR analyses of Entpd5 expression from the purified IE-T cells and IECs (n = 6 per group). (I) Ifngr1fl/fl and Ifngr1fl/fl VilCre mice were infected with ova+ Citrobacter and the ova-specific CD8αβ+ and CD4+ IE-T cells were evaluated for CD39 expression and (J) comparison of GM-CSF and CD39 producing CD4+ colonic IE-T cells in Ifngr1fl/fl mice at day 12 p.i. (K-M) Ifngr1fl/fl and Ifngr1fl/fl VilCre mice were infected with Citrobacter rodentium and treated with 250ug isotype or CD8β-depleting antibodies every 2 days after the infection and sacrificed on day 12 p.i. (n = 8 per group). (K) FACS plot for CD8+ IE-T cells from mice given isotype or CD8β-depleting antibodies. (L) Colon explants were analyzed for indicated analytes and (M) colon length measurement. (N) Ifngr1fl/fl, Ifngr1fl/fl VilCreERT2 and Ifngr1fl/fl VilCreERT2 R26LSLIrf1 mice were infected with Citrobacter rodentium on day 0, injected with tamoxifen at days 3–6 post-infection and analyzed on day 12 for CD39 expression from CD8αβ+ and CD4+ IE-T cells (n = 5 per group). The horizontal bar represents the median, and each symbol represents an individual mouse. Data were analyzed by (A, B) by Brown-Forsythe and Welch’s ANOVA followed by Dunnett’s post-test or (E-I) Mann-Whitney U test (two-sided) or (L-N) Kruskal-Wallis test followed by Dunn’s post-test. Scale bar = 100 um.

Source data

Extended Data Fig. 7 Epithelial IFN-γ signaling controls antigen presentation by IECs.

Flow cytometric enumeration of MHCII (A and C) or MHCI (B) or SIINFEKL-MHCI complex (D-E) on the surface of the colonic IECs after infection with CitroWT (A and E) or CitroOva (B-D) at indicated day p.i. (A) or on day 12 p.i. (n = 5 per group). (B-E). Tamoxifen treatment was applied to induce the expression of the transgenes (B-E) at days 3-6 p.i. (F) Ifngr1fl/fl and Ifngr1fl/flVilCre mice were infected with Citrobacter and treated with anti-CD4 (n = 6) or isotype antibodies (n = 4) every 2 days starting at day 6 and sacrificed on day 12 p.i. The horizontal bar represents the median, and each symbol represents an individual mouse. Data were analyzed by (A) by ANOVA followed by Holm-Sidak post-test or (B-D and F) Kruskal-Wallis test followed by Dunn’s post-test or (E) Mann-Whitney U test (two-sided).

Source data

Extended Data Fig. 8 Antigen presentation via MHCI by IECs promotes eATPase production from CD8ab+ IE-T cells.

(A) Design and genotyping of B2mfl/fl mouse, representative of two independent lines. (B-K) B2mfl/fl and B2mfl/flVilCreERT2 mice were infected with ova-expressing Citrobacter on day 0, treated with tamoxifen on days 3–6 and sacrificed on day 12. Colonic IECs on day 12 were analyzed for (B) MHCI expression (n = 5 for B2mfl/fl and n = 7 B2mfl/flVilCreERT2) and (C) IE-T cells for CD39 expression (n = 5 per group) (D-E) (n = 3 for d0 and n = 5 for d6, d12 per group) by flow cytometry. (F-I) quantification of indicated analytes from colon explants (n = 8 per group) and (J) GM-CSF production from the ova-specific CD4+ IE-T cells and (K) Citrobacter colonization in the colon at day 12 p.i. (n = 5 per group). (L-N) B2mfl/fl and B2mfl/flVilCreERT2 mice were infected with ova-expressing Citrobacter on day 0, treated with tamoxifen on days 3–6, injected with anti-NK1.1 or isotype antibodies on days 2, 4, 6, 8 and 10 and sacrificed on day 12 (n = 5 per group). (L) IFN-γ production from NK1.1+ TCRαβ− TCRγδ− cells (M) CD45+CD49b+ cells and (N) colon length at day 12. The horizontal bar represents the median, and each symbol represents an individual mouse. Data were analyzed by (B, D, E and N) ANOVA test followed by Sidak’s post-test or (C, F-L) Kolmogorov-Smirnov test.

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Extended Data Fig. 9 Antigen presentation by IECs promotes eATPase production from CD4+ IE-T cells.

H2abfl/fl and H2abfl/flVilCreERT2 mice were infected with ova-expressing Citrobacter on day 0, treated with tamoxifen on days 3-6, and sacrificed on day 12 (n = 5-8 per group). (A) Colonic IECs and myeloid cells were analyzed for MHCII expression on day 12 and (C-F) IE-T cells for CD39 expression by flow cytometry (n = 5 per group). (G) Entpd5 expression by RT-PCR after purification (n = 6 per group). (H-K) quantification of indicated analytes from colon explants (n = 8 pe group). (L-M) GM-CSF production from the CD4+ IE-T cells (n = 5 per group). (N) colon length and (O) Citrobacter colonization in the colon at day 12 p.i. (n = 8 per group). The horizontal bar represents the median, and each symbol represents an individual mouse. Data were analyzed by (A-O) Mann-Whitney U test (two-sided).

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Extended Data Fig. 10 Antigen presentation by IECs restrains pathogenic CD4 T cells that promote chemical colitis.

(A-I) Ifngr1fl/fl and Ifngr1fl/flVilCre were treated with 1% DSS for 9 days followed by regular drinking water and sacrificed on day 12. (A) Expression of MHCI and (B) MHCII on the epithelial cell surface (n = 6 per group). Expression of CD39 on (C) CD8αβ+ and (D) CD4+ IE-T cells (n = 5 per group). (E) Quantification of ATP in the colon explants (n = 8 per group) and (F) production of GM-CSF from the CD4+ IE-T cells at day 12 (n = 5 per group). (G) Enumeration of neutrophils in the epithelium and (H) Bodyweight change and (I) colon length (n = 6 per group) and representative picture at day 12. (J-M) B2mfl/fl and B2mfl/flVilCreERT2 mice (n = 5 per group) were treated with 1% DSS for 9 days followed by regular drinking water and sacrificed on day 12. Mice were also treated with tamoxifen on days 3-6. (N) Colon lengths from Ifngr1fl/fl and Ifngr1fl/flVilCre mice treated isotype (n = 10), α-CD4 (n = 8), α-GM-CSF (n = 8), αβ-ATP (n = 10), apyrase (n = 10), MCC950 (n = 10) or α-IL1R (n = 10) during DSS administration and sacrificed on day 12. Each symbol represents an individual mouse, and the bar represents the median except for (H) where each dot indicated the mean and bar indicates the SEM. Data were analyzed by (A-G, I-M) Mann-Whitney U test (two-sided) or (H) ANOVA followed by Sidak post-test or (N) Kruskal-Wallis test followed by Dunn’s post-test.

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Extended Data Fig. 11 Model.

Upon sensing IFN-γ, colonic epithelial cells productively present pathogen-derived and self-antigens to the cognate intraepithelial T cells, critically situated at the epithelial barrier. Antigen presentation by the epithelial cells induces extracellular adenosine triphosphatase (ATPase) expression in the cognate intraepithelial T cells, which limits accumulation of extracellular ATP and resultant activation of the NLRP3 inflammasome in macrophages resident in the tissue. In contrast, macrophage antigen presentation alongside inflammasome-associated interleukin 1 alpha (IL-1α) and IL-1β production elicits pathogenic transformation of CD4-positive T cells into granulocyte-macrophage colony stimulating factor (GM-CSF)-producing T cells in vivo, promoting colitis and colorectal carcinogenesis. The figure was created using BioRender (https://biorender.com).

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Malik, A., Sharma, D., Aguirre-Gamboa, R. et al. Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity. Nature 623, 1044–1052 (2023). https://doi.org/10.1038/s41586-023-06721-1

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