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. 2017 May 22;8(34):56802-56815.
doi: 10.18632/oncotarget.18063. eCollection 2017 Aug 22.

Decitabine inhibits T cell proliferation via a novel TET2-dependent mechanism and exerts potent protective effect in mouse auto- and allo-immunity models

Xue Wang #  1 , Jun Wang #  2 , Yong Yu  3 , Tonghui Ma  4 , Ping Chen  4   5 , Bing Zhou  4   6 , Ran Tao  4   7
Affiliations

Decitabine inhibits T cell proliferation via a novel TET2-dependent mechanism and exerts potent protective effect in mouse auto- and allo-immunity models

Xue Wang et al. Oncotarget. .

Abstract

Multiple sclerosis (MS) is an autoimmune disease characterized by the dysregulated immune response including innate and adaptive immune responses. Increasing evidence has proven the importance of epigenetic modification in the progression of MS. Recent studies revealed that low-dose decitabine (Dec, 5-Aza-2'-deoxycytidine), which incorporates into replicating DNA and inhibits DNA methylation, could prevent experimental autoimmune encephalomyelitis (EAE) development by increasing the number of regulatory T cells (Tregs). Here, we showed that higher-dose decitabine relative to previous studies could also distinctly protect mice from EAE and allogeneic cardiac transplantation. Mechanistic studies revealed decitabine suppressed innate responses in EAE mice through inhibiting the activation of microglia and monocyte-derived macrophages that contributed to reduce the severity of EAE. Furthermore, differentiation of naïve CD4+ T cells into Th1 and Th17 cells was significantly suppressed by decitabine in vivo and in vitro. Though in vitro studies showed decitabine could induce Treg differentiation, there was no obvious change in the percentage of Tregs in Dec-treated EAE mice. Most importantly, we found that T cell proliferation was potently inhibited in vivo and in vitro by higher-dose decitabine through increased gene expression of the DNA dioxygenase TET2 which facilitated the expression of several cell cycle inhibitors. Collectively, our study provides novel mechanistic insights of using the epigenetic modifying agents in the management of both allo- and auto-immune responses.

Keywords: T cell proliferation; TET2; cardiac transplantation; decitabine; experimental autoimmune encephalomyelitis.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Systemic administration of decitabine is exquisitely protective against EAE
Clinical scores of EAE mice received either vehicle or decitabine (0.25 mg/kg/day, intraperitoneal injection) treatment for 14 days with the preventive (A) or treatment (C) protocols (n=5 mice per group). Data were expressed as mean±S.E.M. Spinal cord sections were stained with H&E or LFB at day 18 for prevention protocol (B) or day 30 for treatment protocol (D) after immunization (original magnification, ×40). *P < 0.05.
Figure 2
Figure 2. Decitabine inhibits proinflammatory response of CNS in EAE mice
CNS (brain and spinal cord) mononuclear cells from EAE mice treated with vehicle or decitabine at day18 after immunization were assessed by flow cytometry (A). The expression of MHC-II on the CD11b+ population was analyzed. Results were presented as representative plot (B), and histogram with MFI level (C). Quantification of mRNA expression of proinflammatory cytokines and chemokines in spinal cords of EAE mice treated with vehicle or decitabine (n=5 mice per group) at day 18 after immunization (D, E, F). Data (C, D, E, F) were expressed as mean±S.E.M. *P < 0.05, **P < 0.01.
Figure 3
Figure 3. Decitabine inhibits cardiac allograft rejection and donor-specific T cell response
(A) Kaplan-Meier survival analysis of cardiac allografts with or without decitabine treatment (0.25 or 0.5 mg/kg/day, intraperitoneal injection). (B) Allogeneic T cell immunity in cardiac transplantation mice treated with vehicle or decitabine was evaluated by monitoring the alloantigen-specific IFN-γ production via ELISPOT assay. Data (A, B) were expressed as mean±S.E.M. ***P < 0.001.
Figure 4
Figure 4. Decitabine modulates T cell differentiation in vivo and in vitro
Splenocytes were harvested from EAE mice treated with vehicle or decitabine at day18 after immunization. The frequency of Th1 and Th17 in splenocytes was analyzed by FACS (A, B). Splenocytes were restimulated with MOG35–55 peptide (25 μg/ml), and culture supernatants were collected at 72 h for cytokine measurement by ELISA (C). The frequency of Treg population in splenocytes was analyzed by FACS (D, E). The effect of decitabine on Th1, Th17, Treg differentiation in vitro was analyzed by FACS (F). Data (B, C, E) were expressed as mean±S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. Decitabine inhibits naïve T proliferation in vitro and modulated MOG-reactive CD4+T cell response in vivo
The effect of decitabine on the proliferation of T cell subsets stimulated with anti-CD3 plus anti-CD28 mAbs at different concentrations was examined by CFSE-based assay (A). Splenocytes from EAE mice treated with or without decitabine were restimulated ex vivo with indicated concentrations of the MOG35–55 peptide, and cell proliferation was assessed by [3H] thymidine incorporation assay (B). All data were expressed as mean±S.E.M. ***P < 0.001.
Figure 6
Figure 6. The inhibition of decitabine on T cell proliferation is associated with increased expression of several cell cycle inhibitors and TET2
Decitabine (10 μM) promoted the mRNA expression of several cell cycle inhibitors (p15, p16, p21, p27) upon T cell activation (A). The effect of decitabine (10 μM) on the cell cycle progression of naïve CD4+ T cell stimulated with anti-CD3 plus anti-CD28 mAbs at different concentrations was analyzed by western blot (B) and real-time PCR (C). Decitabine (10 μM) promoted the expression of TET2 and TET3 but reduced TET1 expression in naïve CD4+ T cells stimulated with anti-CD3 plus anti-CD28 mAbs in vitro (D). After knocking down the expression of TET2 in naïve CD4+ T cells, these cells were treated with vehicle or decitabine (10 μM). The different impacts on the transcriptional level of cell cycle inhibitors (p15, p16, p21, p27) (E) and the rate of cell proliferation (G) were shown. (F) ChIP assays of naïve CD4+ T cells, using an anti-TET2 antibody and PCR primers specific for promoters of p15, p16, p21 and p27. IgG antibody was used as negative control and input DNA was used as internal control. Data (A, C, D, E, F, G) were expressed as mean±S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.
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