Abstract

Foxp3+ regulatory T cells (Treg cells) are the central component of peripheral immune tolerance. Whereas a dysregulated Treg cytokine signature has been observed in autoimmune diseases, the regulatory mechanisms underlying pro- and anti-inflammatory cytokine production are elusive. Here, we identify an imbalance between the cytokines IFN-γ and IL-10 as a shared Treg signature present in patients with multiple sclerosis and under high-salt conditions. RNA-sequencing analysis on human Treg subpopulations revealed β-catenin as a key regulator of IFN-γ and IL-10 expression. The activated β-catenin signature was enriched in human IFN-γ+ Treg cells, as confirmed in vivo with Treg-specific β-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional Treg phenotype. Moreover, we identified prostaglandin E receptor 2 (PTGER2) as a regulator of IFN-γ and IL-10 production under a high-salt environment, with skewed activation of the β-catenin–SGK1–Foxo axis. Our findings reveal a novel PTGER2–β-catenin loop in Treg cells linking environmental high-salt conditions to autoimmunity.

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Data availability

RNA-seq data are available in the GEO repository under accession code GSE116283. The remaining data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank L. Devine, C. Wang, and H. Tomita for cell sorting; E. Cheng and M. Zhang for preparation of the RNA-seq library; K. Tanaka for microscopic imaging; and M. Shimizu, H. Taniwaki, and N. Yamanaka for technical support. FIC mice were a generous gift from S. Sakaguchi (Osaka University). This work was supported by grants from the Uehara Memorial Foundation Research Fellowship, MSD Life Science Foundation Research Fellowship, LEGEND Study Abroad Grant from BioLegend, and Tomy Digital Biology (to T.S.); the Ministry of Education, Culture, Sports, Science and Technology (MEXT), JSPS KAKENHI (grant 21229010), and the Core Research for Evolutional Science and Technology (CREST) program from the Japan Science and Technology Agency (to I.K.); and the National Institutes of Health (grants P01 AI045757, U19 AI046130, U19 AI070352, and P01 AI039671) and the Nancy Taylor Foundation for Chronic Diseases (to D.A.H.). RNA-seq was conducted at the Yale Stem Cell Center Genomics Core facility, which is supported by the Connecticut Regenerative Medicine Research Fund and the Li Ka Shing Foundation.

Author information

Author notes

    • Donald M. Rodriguez

    Present address: University of Chicago, Chicago, IL, USA

    • Margarita Dominguez-Villar

    Present address: Department of Medicine, Immunology, Imperial College London, London, UK

Affiliations

  1. Departments of Neurology and Immunobiology, Yale School of Medicine, New Haven, CT, USA

    • Tomokazu Sumida
    • , Matthew R. Lincoln
    • , Chinonso M. Ukeje
    • , Donald M. Rodriguez
    • , Margarita Dominguez-Villar
    •  & David A. Hafler
  2. Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan

    • Tomokazu Sumida
    • , Hiroshi Akazawa
    •  & Issei Komuro
  3. Department of Cell Biology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan

    • Tetsuo Noda
  4. Department of Pharmacology, Faculty of Medicine, Toho University School of Medicine, Tokyo, Japan

    • Atsuhiko T. Naito

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Contributions

T.S., D.M.R., and C.M.U. performed in vitro experiments with the help of M.R.L. and M.D.V.; T.S. and A.T.N. performed in vivo experiments with the help of H.A. and T.N.; T.S. and M.R.L. analyzed the RNA-seq data under the supervision of D.A.H.; T.S. performed data analysis and wrote the manuscript under the supervision of A.T.N., I.K., M.R.L., M.D.-V., and D.A.H.; A.T.N., I.K., M.D.-V., and D.A.H. provided supervision in the overall study.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Tomokazu Sumida.

Integrated supplementary information

  1. Supplementary Figure 1 Memory Treg cells are the main source of effector cytokines IFN-γ and IL-10.

    (a) Sorting strategy for memory and naive Treg cells from circulating human CD4+ T cells. (b) mRNA expression of IFNG and IL10 gene on memory and naive Treg cells (memory Treg cells; n = 35 subjects, naive Treg cells; n = 16 subjects). P values were calculated by two-sided Student’s t-test. Data were represented as mean + /- SD.

  2. Supplementary Figure 2 β-catenin signaling is activated in the IFN-γ-producing human Treg subset.

    (a) GSEA enrichment plots of four different Wnt signaling pathway gene sets between IFN-γSP vs. DN. (b) Flow cytometric analysis of TCF-1 expression on ex vivo Treg subpopulations relative to DN (n = 7 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (c) AXIN2 and TCF7 mRNA expression in IFN-γ+ and IFN-γ human Treg populations assessed by DNA microarray (n = 8 subjects). P values were calculated by two-sided Student’s t-test. (d) CTNNB1 gene expression on Treg cells transduced with a non-targeted shRNA or a CTNNB1 shRNA and cultured for 5 days (n = 10 subjects). P values were calculated by two-sided Student’s t-test. (e) Frequency of IFN-γ and IL-10 positive cell number. Treg cells were stimulated with anti-CD3 and anti-CD28 in the presence of SGK1 inhibitor GSK650394 (SGK1-i), IL-12 (TH1), or IL-12 and GSK650394 (TH1 + SGK1-i) (n = 6 subjects) ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Data were represented as mean + /- SD.

  3. Supplementary Figure 3 Treg-specific activation of β-catenin induces an IFN-γ-secreting dysfunctional phenotype with Scurfy-like autoimmunity.

    (a) Schematic of the wild-type and targeted CTNNB1 allele. (b) Flow cytometric analysis of β-catenin and Foxp3 in peripheral lymph nodes, spleen, and thymus CD4+ T cells from Foxp3Cre and Ctnnb1ΔEx3; Foxp3Cre mice. (c) Expression for classical helper cytokines and transcription factors in both Treg cells and T effector cells (CD4+ CD25neg; non-Treg) assessed by qPCR (n = 4 mice). *P < 0.05, **P < 0.01 (two-sided Student’s t-test). (d) Flow cytometric analysis of Foxp3 and Helios expression on CD4+ T cells in peripheral lymph nodes and spleen from Foxp3Cre and Ctnnb1ΔEx3; Foxp3Cre mice at the age of 3 weeks. Percentages of Foxp3+ and/or Helios+ CD4+ T cells isolated from lymph nodes are shown at the bottom. (n = 5-6 mice) ***P < 0.001 (two-way ANOVA with Sidak’s multiple comparisons test). (e) GSEA enrichment plot between Foxp3Cre and Ctnnb1ΔEx3; Foxp3Cre Treg cells using the gene set that is positively regulated by Foxo1 (left) and negatively regulated by Foxo1 (right) identified from the comparison between wild-type vs. Foxo1 KO Treg cells (GSE40655). Normalized enrichment score (NES) and false discovery rate (FDR) are indicated. Data were represented as mean + /- SD.

  4. Supplementary Figure 4 High salt activates the β-catenin–SGK1–Foxo axis in IFN-γ-producing human Treg cells.

    (a) Flow cytometric analysis of p-SGK1 (Thr256) (left) and p-Foxo1 (Ser256) (right) level in human Treg subsets. Treg cells were stimulated with anti-CD3 and anti-CD28 for 96 h followed by 4 h PMA plus iomomycin stimulation, and the expression of p-SGK1 and p-Foxo1 were determined by intracellular staining in each subset. (p-SGK1; n = 12, p-Foxo1; n = 10 subjects) (b) Representative immunofluorescence images of human Treg cells with PLA signal for β-catenin-Foxo1 interaction (red) and IFN-γ staining (green). Nuclei were stained with DAPI (blue). PLA signal (arrowheads) was observed in an IFN-γ positive cell (arrow). Scale bar 5 μm. Data were representative of three experiments. (c) Flow cytometric analysis of active β-catenin, p-SGK1, and p-Foxo1 expression in human IL-10 producing Treg cells. Treg cells were stimulated with anti-CD3 and anti-CD28 in the presence (NaCl) or absence (Control) of additional 40 mM NaCl for 96 h followed by PMA plus iomomycin stimulation for 4 h (active β-catenin; n = 15, p-SGK1; n = 13, p-Foxo1; n = 10 subjects). (d) Flow cytometric analysis of TCF-1 expression in human IFN-γ producing Treg cells. Treg cells were cultured as in (c) (n = 3 subjects). P values were calculated by two-sided Student’s t-test for (c, d). (e) Relative frequency of IFN-γ and IL-10 positive cell number (fold of control condition) in human Treg cells cultured as in Fig. 6c (n = 11 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (f) Relative frequency of IFN-γ and IL-10 positive cell number (fold of control condition) in human Treg cells cultured as in Fig. 6d (n = 9 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (g) Flow cytometric analysis of p-SGK1 expression in human IFN-γ producing Treg cells. Treg cells were cultured as in Fig. 6d (n = 6 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (h) Relative frequency of IFN-γ and IL-10 positive cell number in human Treg cells stimulated in the presence (NaCl) or absence (Control) of additional 40 mM NaCl with and without Wnt/inhibitor GSK650394 (SGK1-i) for 96 h (n = 11 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (i) Flow cytometric analysis of p-Foxo1 expression in human IFN-γ producing Treg cells. Treg cells were cultured as in (h) (n = 8 subjects). *P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). Data were represented as mean + /- SD.

  5. Supplementary Figure 5 The β-catenin–SGK1–Foxo axis is also activated in Teff and human Jurkat T cells under high-salt conditions.

    (a) Flow cytometric analysis of active β-catenin expression in human T effector cells (Teffs) and human Jurkat T cells. Human Teff were stimulated as well as Treg cells for 96 h (n = 8 subjects). Human Jurkat T cells were cultured without TCR stimulation for 120 h (n = 12). Both were cultured in the presence (NaCl) or absence (Control) of additional 40 mM NaCl. P values were calculated by two-sided Student’s t-test. (b) Relative frequency of IFN-γ and IL-10 positive cell number (fold of control condition) in human Teffs cultured as in (a) (n = 8 subjects). P values were calculated by two-sided Student’s t-test. (c, d) Flow cytometric analysis of p-SGK1 (Thr256) (c) and p-Foxo1 (Ser256) (d) in human Jurkat T cells. Human Jurkat T cells were transduced with scramble gRNA (CRISPR/Scramble) or CTNNB1 targeted gRNA (CRISPR/CTNNB1) with Cas9. Both cell lines were cultured in the presence (NaCl) or absence (Control) of additional 40 mM NaCl without TCR stimulation for 120 h (n = 4). **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). Data were represented as mean + /- SD.

  6. Supplementary Figure 6 High-salt-induced β-catenin activation via AKT is independent of Wnt ligands.

    (a) Relative frequency of IFN-γ and IL-10 positive cell number (fold of control condition) in human Treg cells. Treg cells were stimulated with anti-CD3 and anti-CD28 in the presence of Fzd8-FC (Fzd), additional 40 mM NaCl (NaCl), or Fzd8-FC and NaCl (NaCl + Fzd) (n = 7 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (b) Relative expression level of active β-catenin in human Treg cells cultured as in (a). (n = 7 subjects) *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (c) GSEA enrichment plots of PI3K/AKT pathway gene sets between IFN-γSP vs. IL10SP. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated at the bottom of each plot. (d) Relative expression level of p-AKT on Treg subsets. Treg cells were stimulated with anti-CD3 and anti-CD28 for 4 days, followed by 4 h PMA/iomomycin stimulation and intracellular cytokine staining for IFN-γ and IL-10 (n = 5 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (e) Flow cytometric analysis of β-catenin phosphorylation at s522 in human Jurkat T cells. Human Jurkat T cells were stimulated in the presence of AKT inhibitor MK2206 (AKT-i), additional 40 mM NaCl (NaCl), or MK2206 and NaCl (NaCl + AKT-i) (n = 4). (f) Flow cytometric analysis of GSK3β phosphorylation at s9 and AKT phosphorylation at s473 in human Jurkat T cells. Human Jurkat T cells were prepared as in Supplementary Fig. 6c. (n = 4). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). Data were represented as mean + /- SD.

  7. Supplementary Figure 7 The PTGER2–β-catenin loop is activated by high-salt stimulation.

    (a) PTGER2 expression assessed by RNA-seq on ex vivo Treg subpopulations (n = 8 subjects). (b) Flow cytometric analysis of PTGER2 in human Jurkat T cells. Human Jurkat T cells were prepared as in Supplementary Fig. 6c. (n = 4). **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (c) Flow cytometric analysis of active β-catenin in human Jurkat T cells. Human Jurkat T cells were transduced with a scramble shRNA or a PTGER2 shRNA and cultured in normal media (Control) or media supplemented with additional 40 mM NaCl (NaCl) for 120 h. (n = 4) *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (d) Representative flow cytometric analysis of IFN-γ and IL-10 production in human Treg cells cultured in the normal media (Control) or media supplemented with additional 40 mM NaCl (NaCl) with anti CD3 (2 μg/ml) and different concentration of anti CD28 (1, 2, 5 μg/ml) for 96 h. Relative frequency of IFN-γ and IL-10 producing Treg cells are shown at the bottom (n = 4 subjects). *P < 0.05, **P < 0.01 (two-way ANOVA with Sidak’s multiple comparisons test). Data were represented as mean + /- SD.

  8. Supplementary Figure 8 Schematic model of the role of PTGER2 and the AKT–β-catenin–SGK1–Foxo axis in the production of IFN-γ and IL-10 in Treg cells.

    AKT-β-catenin signaling balances between IFN-γ and IL-10 production in Treg cells. Under high salt conditions, PTGER2 was increased and established the positive feed forward loop with β-catenin, resulted in amplified activation of the β-catenin-SGK1-Foxo axis in IFN-γ-producing Treg cells.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–8

  2. Reporting Summary

  3. Supplementary Table 1: Upstream regulator analysis in IPA between each Treg subpopulation

    Lists of the top-ranking genes identified by IPA analysis as upstream regulators between each Treg subpopulations. Tables show statistically significant (overlap P value <0.05) upstream regulators in each comparison (genes that could not be calculated for fold change were blank). CTNNB1 gene, which encodes β-catenin protein, was highlighted in red.

  4. Supplementary Table 2: Clinical characteristics of evaluated patients with MS.

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https://doi.org/10.1038/s41590-018-0236-6