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. 2014 Jun 26;20(1):248-56.
doi: 10.2119/molmed.2013.00159.

Low-dose 5-aza-2'-deoxycytidine pretreatment inhibits experimental autoimmune encephalomyelitis by induction of regulatory T cells

Michael W Y Chan  1 , Chia-Bin Chang  1 , Chien-Hsueh Tung  2 , Justin Sun  1 , Jau-Ling Suen  3 , Shu-Fen Wu  1
Affiliations

Low-dose 5-aza-2'-deoxycytidine pretreatment inhibits experimental autoimmune encephalomyelitis by induction of regulatory T cells

Michael W Y Chan et al. Mol Med. .

Abstract

Forkhead box P3 (Foxp3) is the major transcription factor controlling the development and function of regulatory T (Treg) cells. Previous studies have indicated epigenetic regulation of Foxp3 expression. Here, we investigated whether the deoxyribonucleic acid (DNA) methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) applied peripherally could modulate central nervous system (CNS) inflammation, by using a mouse experimental autoimmune encephalomyelitis (EAE) model. We found that disease activity was inhibited in a myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE mouse briefly pretreated with low-dose (0.15 mg/kg) 5-Aza, ameliorating significant CNS inflammatory responses, as indicated by greatly decreased proinflammatory cytokines. On the contrary, control EAE mice expressed high levels of IFN-γ and interleukin (IL)-17. In addition, 5-Aza treatment in vitro increased GFP expression in CD4(+)GFP(-) T cells isolated from GFP knock-in Foxp3 transgenic mice. Importantly, 5-Aza treatment increased Treg cell numbers, in EAE mice, at both disease onset and peak. However, Treg inhibition assays showed 5-Aza treatment did not enhance per-cell Treg inhibitory function, but did maintain a lower activation threshold for effector cells in EAE mice. In conclusion, 5-Aza treatment prevented EAE development and suppressed CNS inflammation, by increasing the number of Treg cells and inhibiting effector cells in the periphery.

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Figures

Figure 1
Figure 1
Treatment of mice with 5-Aza inhibits the development of experimental auto-immune encephalomyelitis (EAE). (A) Low-dose 5-Aza (0.15 mg/kg/d) or control DMSO treatments of C57BL/6 mice were administrated at 5 d before induction of EAE for a total of 10 d. Each line represents an independent experiment. Two independent experiments were performed (n = 5 mice/group). The time of disease onset and peak is also indicated. (B) Spinal cords of mice treated with 5-Aza or DMSO were stained with H&E at the peak of EAE pathogenesis (upper panels: 40×, lower panels: 100×); arrows show inflammatory cell infiltration. Histology was performed in three different mice for each treatment, and representative images are shown. (C) Luxol fast blue stain of spinal cords at the peak of EAE in mice pretreated with 5-Aza or DMSO (40×). 5-Aza-treated tissue showed intact myelin, whereas DMSO-treated mice showed attacked myelin. Histology was performed in three different mice for each treatment. Representative images are shown.
Figure 2
Figure 2
5-Aza treatment decreases the number of CNS-infiltrating lymphocytes in EAE mice. Brains and spinal cords of MOG-induced EAE mice treated with 5-aza or DMSO were harvested at EAE disease onset or peak, and CNS-infiltrating lymphocytes were isolated by Percoll density gradient. Three independent experiments were performed. (A) Total infiltrating cell numbers were analyzed after Percoll gradient isolation. Open bars indicate 5-Aza treatment and black bars show control DMSO-treated mice (each group contained five mice, *p < 0.05). (B) CNS-infiltrating cells stained with CD3, CD4 and CD25, and CD3+ cells were gated to count CD4+CD25+ T cells from 5-Aza-treated (5-Aza, left panel) or DMSO-treated (DMSO, right panel) control EAE mice during disease onset (upper panel) and disease peak (lower panel). (C) Flow cytometry analysis of the expression of inflammatory cytokines (IFN-γ: upper panel; IL-17: lower panel) in CNS-infiltrating CD4+ T cells. A representative profile is shown. Three independent experiments were performed.
Figure 3
Figure 3
Treatment of isolated splenocytes with 5-Aza increases the expression of Foxp3 and GFP. (A) Isolated murine splenocytes were activated by anti-CD3 (1 μg/mL), anti-CD28 (0.2 μg/mL) and mouse IL-2 (10 ng/mL). Cells were treated with 1 μmol/L 5-Aza (solid circles) or DMSO alone (open circles) every day for 4 d. Foxp3 mRNA expression was measured by quantitative real-time PCR with normalization to β-actin (**p < 0.001). (B, C) CD3+GFP− T cells were harvested from Foxp3-EGFP C57BL/6 transgenic mice (C.Cg-FOXP3tm2Tch/J) and isolated by a BD FACSAria III cell sorter. The isolated CD3+GFP−T cells were activated as above and then treated with 1 μmol/L 5-Aza or DMSO alone. Flow cytometry was used to monitor GFP expression. A representative flow cytometry result is shown (B). Results are summarized from d 0 to d 4, with 5-Aza (solid circles) and DMSO alone (open circles) (C) (*p < 0.05). All data are representative of three independent experiments with triplicate culture wells.
Figure 4
Figure 4
Treatment of mice with 5-Aza induces distinct changes in spleen morphology and spenocyte populations during EAE progression. Mice pretreated with 5-Aza or DMSO were MOG-induced for EAE, as described in Figure 1 (n = 3–5 mice/group, two independent experiments were performed). (A) Spleens were harvested from 5-Aza-treated (left panel) or DMSO-treated (right panel) EAE mice at d 6 and 10 and disease peak after MOG induction. Each interval between bars represents 1 cm. (B) Total cell numbers of splenocytes were counted at the indicated time points (*p < 0.05, **p < 0.01; white bar: 5-Aza, black bar: DMSO). (C) Splenocytes stained with CD3, CD4 and CD25, and CD3+ cells, were gated to analyze the percentage of CD4+CD25+ T cells from 5-Aza-treated (white bars) or DMSO-treated (black bars) EAE mice at the indicated time points (left panel). Representative results from flow cytometry are also shown (right panel) (*p < 0.05). (D) The total number of CD4+CD25+ T cells in splenocytes from 5-Aza-treated (white bars) or DMSO-treated (black bars) EAE mice were counted at the indicated time points (*p < 0.05, **p < 0.01).
Figure 5
Figure 5
Regulatory T cells have similar suppressive function, but effector T cells have a lower activation threshold in 5-Aza-treated mice (A) Effector T cells isolated from naive B6 mice were cocultured with Treg cells isolated from 5-Aza-treated (5-Aza Treg) or DMSO-treated (DMSO Treg) EAE mice at disease onset and peak. Treg inhibition assays were then performed by CFSE-labeling. (B) Effector T cells isolated from EAE mice treated with DMSO (DMSO, left panel) or 5-Aza (5-Aza, right panel) were cocultured with Treg cells isolated from 5-Aza-treated mice for Treg inhibition assay (upper panel). For control experiments, effector T cells were stimulated with anti-CD3 antibody only (lower panel). (C) CFSE-labeled CD4+CD25− effector T cells isolated from DMSO-treated (Effector T-DMSO) or 5-Aza-treated (Effector T-Aza) EAE mice were cocultured with regulatory T cells isolated from 5-Aza-treated (Treg-Aza) or DMSO-treated (Treg-DMSO) EAE mice for 5 d, and CFSE signals were detected by flow cytometry. A representative CFSE profile is shown. Three independent experiments were performed with similar trends. (D) CD4+CD25− T cells isolated from DMSO- or 5-Aza-treated EAE mice were stained with CD69 activation marker before (upper panel) or after (lower panel) stimulation with anti-CD3. Data are representative of three independent experiments with similar results.
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References

    1. Goverman JM. Immune tolerance in multiple sclerosis. Immunol Rev. 2011;241:228–40. - PMC - PubMed
    1. Pachner AR. Experimental models of multiple sclerosis. Curr Opin Neurol. 2011;24:291–99. - PubMed
    1. Simmons SB, Pierson ER, Lee SY, Goverman JM. Modeling the heterogeneity of multiple sclerosis in animals. Trends Immunol. 2013;34:410–22. - PMC - PubMed
    1. Rangachari M, Kuchroo VK. Using EAE to better understand principles of immune function and autoimmune pathology. J Autoimmun. 2013;45:31–9. - PMC - PubMed
    1. Kroenke MA, Segal BM. Th17 and Th1 responses directed against the immunizing epi-tope, as opposed to secondary epitopes, dominate the autoimmune repertoire during relapses of experimental autoimmune encephalomyelitis. J Neurosci Res. 2007;85:1685–93. - PubMed

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