Journal of Molecular Medicine

, 87:1191

Induction of Foxp3 demethylation increases regulatory CD4+CD25+ T cells and prevents the occurrence of diabetes in mice

Original Article

DOI: 10.1007/s00109-009-0530-8

Cite this article as:
Zheng, Q., Xu, Y., Liu, Y. et al. J Mol Med (2009) 87: 1191. doi:10.1007/s00109-009-0530-8

Abstract

CD4+CD25+ regulatory T cells (Treg), a subpopulation of CD4+ T cells, regulate immune responses. Foxp3 is a key transcription factor for the development and function of Treg cells. During T-cell activation in vitro, a DNA demethylation agent 5-Aza-2′-deoxycytydine (DAC) can induce Foxp3 expression in CD4+CD25 Foxp3 cells via altering methylation status of a conserved element in the 5′-untranslated region of the Foxp3 gene. However, the effects of this agent on the development of Foxp3+ Treg cells in the thymus and in vivo are poorly understood. In the present study, a short-term treatment with a low dose of DAC significantly increased the ratios of thymic CD4+CD8 CD25+ cells or CD4+CD8 Foxp3+ cells to CD4+CD8 cells, and the total numbers of thymic CD4+CD8Foxp3+ Treg cells or CD4+CD8CD25+Foxp3+ Treg cells in the thymus in mice. DAC-treatment induced the Foxp3 expression and the significant demethylation of a CpG island in the first intron of the Foxp3 gene in CD4+CD8CD25+ cells predominantly. Furthermore, CD4+CD8CD25+ thymocytes in DAC-treated mice exhibited enhanced immunosuppressive function than those in control mice. In addition, DAC treatment in vivo was effective in improving the clinical course of diabetes in cyclophosphamide (CY)-potentiated non-obese diabetic mice (CY-NOD). Thus, the in vivo treatment with DAC can significantly promote the development of natural thymic CD4+CD25+Foxp3+ Treg cells through Foxp3 demethylation, implicating a therapeutic application of DAC in patients suffering from autoimmune diseases.

Keywords

ThymocytesRegulatory T cellsEpigeneticDevelopmentAutoimmune disease

Abbreviations

Treg

Regulatory T cell

DP

Double positive

DN

Double negative

SP

Single positive

Foxp3

Forkhead box protein 3

CTLA-4

Cytotoxic T-lymphocyte-associated protein 4

GITR

Glucocorticoid-induced tumor necrosis factor receptor

FCM

Flow cytometry

FITC

Fluorescein isothiocyanate

PE

Phycoerythrin

PI

Propidium iodide

LNs

Lymph nodes

PBMC

Peripheral blood mononuclear cells

MFI

Median fluorescence intensity

bp

Base pair

CY

Cyclophosphamide

NOD

Non-obese diabetes

Supplementary material

109_2009_530_MOESM1_ESM.doc (1024 kb)
Supplemental figure 1The raw data of gels for Fig. 2e. (DOC 1023 kb)
109_2009_530_MOESM2_ESM.doc (41 kb)
Supplemental figure 2No significant change of the percentage of CD4+CD25+ Foxp3+ Treg cells in the peripheral lymphoid tissues of DAC-treated mice. Spleen, and lymph nodes (including cervical, inguinal, and axillary LNs) were harvested from the control and DAC-treated mice in the indicated time, cells were stained with PE-Cy5-labeled anti-CD4 mAb, FITC-labeled anti-CD25 mAb, and PE-labeled anti-Foxp3 mAb and analyzed by FCM. The percentage and total number of CD4+CD25+ cells, the percentage of Foxp3+ cells in CD4+CD25+ cells of spleen and lymph nodes are summarized in the control and DAC-treated mice. (DOC 41 kb)
109_2009_530_MOESM3_ESM.doc (352 kb)
Supplemental figure 3The raw data of gels for Fig. 4d and e. (DOC 352 kb)
109_2009_530_MOESM4_ESM.doc (34 kb)
Supplemental figure 4Methylation status of Foxp3 promoter and H19 gene. (A) Schematic view of mouse Foxp3 gene and the selected sequence in Foxp3 promoter. Distribution and position of individual CpG motifs within the selected sequence are shown below. (B) Bisulfite sequencing of the selected sequence in the Foxp3 promoter in CD25CD4SP cells and CD25+CD4SP cells in DMSO and DAC-treated mice. (C) Bisulfite sequencing of the selected CpG island in the H19 promoter in CD25CD4SP cells and CD25+CD4SP cells in DMSO and DAC-treated mice. filled circle methylated CpG, empty circle nonmethylated CpG. (DOC 33 kb)
109_2009_530_MOESM5_ESM.doc (67 kb)
Supplemental figure 5No significant difference of Foxp3 induction by titrated amounts of DAC between splenic CD4+CD25 cells and CD25CD4 SP thymocytes during the in vitro activation. Splenic CD4+CD25 cells and CD25CD4 SP thymocytes were selected from C57BL/6 mice, cells were performed in vitro cultures (anti-CD3 mAb, 2 μg/ml; anti-CD28 mAb, 2 μg/ml; IL-2, 10 ng/ml) for 4 days in the different concentrations of DAC, Foxp3 expression was analyzed. A) One representative for Foxp3 staining in gated CD4+CD25 and CD4+CD25+cells after sorted thymic or splenic CD4+ T cells cultured with 2 μM DAC for 4 days. B) Mean percentages of Foxp3+ cells in gated CD4+CD25+ cells after sorted thymic or splenic CD4+ T cells cultured with different doses of DAC for 4 days. Data are shown as mean ± SD. (DOC 67 kb)

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Transplantation Biology Research Division, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of ZoologyChinese Academy of SciencesBeijingChina
  2. 2.China-U.S. Research Center for Life SciencesChinese Academy of SciencesBeijingChina
  3. 3.Department of Hematology, Dongzhimen HospitalBeijing University of Traditional Chinese MedicineBeijingChina
  4. 4.Key Laboratory of Biotechnology Pharmaceutical Engineering of Zhejiang Province, School of Pharmaceutical ScienceWenzhou Medical CollegeWenzhouChina
  5. 5.Central LabThe First Affiliated Hospital of Soochow UniversitySuzhouChina