The epigenetic regulator Uhrf1 facilitates the proliferation and maturation of colonic regulatory T cells

Abstract

Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell–specific deficiency in Uhrf1 (Uhrf1fl/flCd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1fl/flCd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.

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Figure 1: Bacterial colonization induces vigorous proliferation of Treg cells in the colonic mucosa.
Figure 2: IL-2 is responsible for the vigorous proliferation of colonic Treg cells after inoculation of commensal bacteria.
Figure 3: Colonization with commensal bacteria induces Uhrf1 expression in colonic Treg cells in IL-2-dependent manner.
Figure 4: Deletion of Uhrf1 results in impaired proliferation and function of colonic Treg cells.
Figure 5: Cdkn1a is upregulated in Uhrf1-deficient Treg cells because of hypomethylation of the Cdkn1a promoter region.
Figure 6: Uhrf1-deficient mice spontaneously develop colitis characterized by activation of TH1 and TH17 responses.
Figure 7: A defect in the proliferation of colonic Treg cells is responsible for the development of spontaneous colitis in Uhrf1fl/flCd4-Cre mice.

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Acknowledgements

We thank P.D. Burrows for critical reading and editing of the manuscript; T. Mukai, M. Yoshida, P. Carnincci, Y. Shinkai and H. Kiyono for comments and suggestions; and S. Fukuda and Y. Koseki for technical support. Supported by the Japan Society for the Promotion of Science (24117723 and 25293114 to K. Ha., 24890293 to Y.F. and 252667 to Y.O.), the Japan Science and Technology Agency (PRESTO to K. Ha.), the RIKEN RCAI-IMS Young Chief Investigator program (K. Ha.), the RIKEN RCAI-IMS Open Laboratory for Allergy Research Project (T.D.), the Kato Memorial Bioscience Foundation (Y.F.), The Uehara Memorial Foundation (K. Ha.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (K. Ha.), the Toray Science Foundation (K. Ha.) and the National Center for Global Health and Medicine (21-110 and 22-205 to T.D.).

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Contributions

Y.O. and Y.F. did a large part of the experiments together with D.T., K.A., Y.F., M.T., T.I., T.O., Y.I.K. and K. Ha.; Y.O., Y.F., T.A.E. and J.S. analyzed the data; M.N., S.T. and S.H. provided materials; S.O. prepared GF mice; T.D., H.M., O.O., K. Ho., H.O. and H.K. provided experimental protocols and intellectual input into the study; T.D. and H.O. edited the manuscript; K. Ha. and H.K. conceived of the study; and K. Ha. designed the experiments, analyzed the data and wrote the manuscript (together with Y.O. and Y.F.).

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Correspondence to Koji Hase.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Time-course analysis of splenic and colonic Treg cells in exGF mice and young mice.

a, GF IQI mice were inoculated with a fecal suspension from SPF C57BL/6 mice. Splenic CD4+ T cells were analyzed for Foxp3 and Ki67 expression by flow cytometory after bacterial colonization. Data are representative of at least three independent experiments with similar results. b, CD4+ T cells in cLP of SPF C57BL/6 mice at the indicated ages were analyzed for Foxp3, IL-2 and Ki67 expression by flow cytometory. c, CD3ɛ+CD4+CD25+FR4+ Treg cells from the cLP of 2 (infant) and 11 (adult) week-old mice (n = 11 or 3 for 2 or 11 weeks old, respectively) were pooled in each group before analyzing the expression of Uhrf1 by Q-PCR. d, Splenic CD4+ T cells from SPF C57BL/6 mice at the indicated ages were analyzed for Foxp3 and Ki67 expression by flow cytometory. Data are representative of two independent experiments with similar results. e, Splenic Treg cells (CD3+CD4+CD25+FR4+) from GF or exGF mice at day 7 (n = 3 per group) were pooled in each group before analyzing Uhrf1 expression levels by Q-PCR.

Supplementary Figure 2 Composition of B lymphocytes, T lymphocytes and Treg cells in lymphoid tissues and cLP of Uhrf1+/+Cd4-Cre and Cd4creUhrf1fl/flCd4-Cre mice.

a, Schematic representation of the genomic structure of wild-type murine Uhrf1 gene, the resultant mutant allele generated by homologous recombination and carrying the neomycin resistance gene (Neo), and the locus after removal of the Neo gene. Exons are indicated by boxes. b-d, Lymphocytes in cLP, mesenteric lymph node (MLN), spleen and thymus of Cd4CreUhrf1+/+ (+/+) and Cd4CreUhrf1fl/fl (fl/fl) mice at 5 weeks of age were characterized by flow cytometry. The frequency of Treg cells were analyzed by flow cytometory (d). Data are representative of at least three independent experiments with similar results

Supplementary Figure 3 Bone marrow–chimera experiment for the analysis of Treg cells and Teff cells.

a, In preliminary experiments, after adoptive transfer of an equivalent number of CD45.1+ WT and Uhrf1-deficient BM cells into irradiated Rag1-deficient recipients, the WT CD4+ T cells became dominant (data not shown), indicating that Uhrf1 confers competitive fitness on T cells during T cell development in the thymus. We therefore increased the ratio of Uhrf1-deficient BM cells to gain comparable numbers of peripheral CD45.2+ Uhrf1-deficient and CD45.1+ CD4+ WT T cells as described. b, The ratio of CD45.1+ and CD45.2+ cells in the cLP of the mixed bone marrow chimeric mice were shown. c-d, CD4+ T cells in cLP from mixed BM chimera mice were analyzed for Foxp3 (c) or cytokine expression (d) at 6 weeks after the BM transfer. Representative FACS plots are shown. Data are representative of two independent experiments with similar results. Error bars: s.e.m. (n = 8). **P < 0.01, as calculated by Mann-Whitney U test. NS: not significant.

Supplementary Figure 4 Deficiency in Uhrf1 does not affect the differentiation of colonic Treg cells.

a-b, In vivo generation of Foxp3+ T cells after adoptive transfer of naive CD4+ T cells from Cd4creUhrf1+/+ and Cd4creUhrf1fl/fl mice. A schematic diagram of in vivo differentiation assay of Foxp3+ T cells (a). CD4+CD44loCD62hi naive T cells from Cd4creUhrf1+/+ and Cd4creUhrf1fl/fl mice were intravenously injected into congenic Foxp3hCD2 reporter mice. The cLP cells in the recipient mice were analyzed for Foxp3 expression at day 14 after transfer (b). Representative FACS plots are shown. Data are representative of two independent experiments with similar results.

Supplementary Figure 5 Ablation of Uhrf1 impairs the expression of functional molecules by Treg cells.

a, Expression levels of functional molecules by proliferative and non-proliferative Treg cells in cLP. Ki67+ and Ki67- Treg cells from cLP of 16-week-old C57BL/6 Foxp3hCD2 reporter mice were analyzed for CTLA-4, ICOS and GITR expression by flow cytometry. Mean fluorescent intensity (MFI) in each population is shown in the histograms. b, Foxp3+ Treg cells in cLP of Cd4CreUhrf1+/+ (+/+) and Cd4CreUhrf1fl/fl (fl/fl) mice were analyzed for PD-1, ICOS and GITR expression by flow cytometry. Histograms gated on CD3ɛ+CD4+hCD2+ cells are shown. Data are representative of two independent experiments with similar results.

Supplementary Figure 6 The immunosuppressive function of Uhrf1-deficient Treg cells is attenuated both in vitro and in vivo.

a, CFSE-labeled responder cells were co-cultured with Treg cells from MLN of Cd4CreUhrf1+/+ (+/+) or Cd4CreUhrf1fl/fl (fl/fl) mice at 1:1 ratios in the presence of APCs and anti-CD3 antibody. The numbers in the histograms indicate the percentage of cells that had undergone one or more divisions. Data are representative of three independent experiments with similar results. Error bars: s.d. (n = 3). **P < 0.01, as calculated by the one-way ANOVA followed by Tukey's test. b-c, Experimental colitis was induced in Rag1-/- mice by adoptive transfer of CD4+CD25-CD45RBhi T cells from CD45.1+ C57BL/6 mice. CD4+CD25+ T cells from CD45.2+ Cd4creUhrf1+/+ (+/+) or Cd4creUhrf1fl/fl (fl/fl) mice were co-transferred in the group shown in the middle or right panels, respectively. Histological examination by hematoxylin-eosin (upper) and Alcian blue staining (lower) are shown (b). Scale bars: 200 μm. CD4+ T cells in cLP were analyzed for CD45.2 and Foxp3 expression at 6 weeks after the transfer (c). Representative FACS plots are shown. Data are representative of two independent experiments with similar results.

Supplementary Figure 7 Identification of the molecule responsible for cell-cycle arrest in Uhrf1-deficient Treg cells.

a-c, Global gene expression profiles of colonic Tconv and Treg from Cd4CreUhrf1+/+ (+/+) or Cd4CreUhrf1fl/fl (fl/fl) mice. Heat map shows the upregulated genes in colonic Treg cells from Cd4CreUhrf1fl/fl (fl/fl) mice (a). Functional category analysis of the profiled 251 genes classified a group of genes termed ‘cellular growth and proliferation’ at the top of the list (b). Subsequent computational gene network analysis identified a molecular network composed of 16 genes (c).

Supplementary Figure 8 Uhrf1fl/flCd4-Cre mice under GF conditions do not develop colitis.

a, Cd4creUhrf1fl/fl (fl/fl) and control littermate (+/+) mice were maintained under SPF conditions until 12 weeks of age. Macroscopic observation of colon are shown. b-c, fl/fl and +/+ mice were maintained under GF conditions until they were 10 weeks old. Macroscopic observation (b), histochemical examination by hematoxylin-eosin (upper) and Alcian blue staining (lower) (c), and immunofluorescent images with nuclei counter staining (blue) (d) are shown. Scale bars: 200 μm. Data are representative of three independent experiments (total 3 mice per group) with similar results.

Supplementary Figure 9 Histological examination of various peripheral tissues in Uhrf1fl/flCd4-Cre mice and control littermates.

The tissue sections from the indicated tissues of 13-16 week-old Cd4CreUhrf1+/+ (+/+) or Cd4CreUhrf1fl/fl (fl/fl) mice were stained with hematoxylin-eosin for histological examination. Data are representative of three individual mice for each group. Scale bars: 200 μm.

Supplementary Figure 10 Uhrf1 deficiency does not influence the development of TH1 or TH17 cells.

a-b, Splenic naive CD4+ T cells were cultured under stimulation with anti-CD3 and anti-CD28-mAbs and IL-12 (a) or IL-1β, IL-6 and TGF-β (b) to induce differentiation into TH1 or TH17 cells, respectively. Data are representative of two independent experiments. Error bars: s.d. (n = 3). NS: not significant (Mann-Whitney U test). c-f, DNA methylation status on the indicated genes in Tconv (CD3ɛ+CD4+CD25-hCD2-) and Treg (CD3ɛ+CD4+CD25+hCD2+) cells from mesenteric lymph nodes of Cd4creUhrf1fl/fl (fl/fl) and control littermate (+/+) mice was analyzed by MeDP-Seq.

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Obata, Y., Furusawa, Y., Endo, T. et al. The epigenetic regulator Uhrf1 facilitates the proliferation and maturation of colonic regulatory T cells. Nat Immunol 15, 571–579 (2014). https://doi.org/10.1038/ni.2886

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