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

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⁺CD8 ^-Foxp3⁺ Treg cells or CD4⁺CD8 ^-CD25⁺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⁺CD8 ^-CD25⁺ cells predominantly. Furthermore, CD4 ⁺CD8^-CD25⁺ 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.