Precision DNA demethylation ameliorates disease in lupus-prone mice
Hao Li, Maria G. Tsokos, Sean Bickerton, Amir Sharabi, Yi Li, Vaishali R. Moulton, Philip Kong, Tarek M. Fahmy, George C. Tsokos
Hao Li, Maria G. Tsokos, Sean Bickerton, Amir Sharabi, Yi Li, Vaishali R. Moulton, Philip Kong, Tarek M. Fahmy, George C. Tsokos
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Research Article

Precision DNA demethylation ameliorates disease in lupus-prone mice

Abstract

Defective DNA methylation in T cells leads to a series of T cell abnormalities in lupus; however, the full effect of T cell lineage–specific DNA methylation on disease expression has not been explored. Here, we show that 5-azacytidine, a DNA methyltransferase inhibitor, targeted to either CD4 or CD8 T cells in mice with established disease using a nanolipogel delivery system dramatically ameliorates lupus-related pathology through distinct mechanisms. In vivo targeted delivery of 5-azacytidine into CD4 T cells favors the expansion and function of Foxp3+ Tregs, whereas targeted delivery to CD8 T cells enhances the cytotoxicity and restrains the expansion of pathogenic TCR-αβ+CD4–CD8– double-negative T cells. Our results signify the importance of cell-specific inhibition of DNA methylation in the treatment of established lupus.

Authors

Hao Li, Maria G. Tsokos, Sean Bickerton, Amir Sharabi, Yi Li, Vaishali R. Moulton, Philip Kong, Tarek M. Fahmy, George C. Tsokos

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Figure 3

Specific targeting of nlg-5-Aza in CD4+ T cells suppresses autoimmunity by increasing Tregs.

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Specific targeting of nlg-5-Aza in CD4+ T cells suppresses autoimmunity ...
(A and B) MRL/lpr mice were treated i.v. with either anti-CD4 antibody–coated nlg-5-Aza (15 μl nlg-5-Aza/mouse) or anti-CD8 antibody–coated nlg-5-Aza (15 μl nlg-5-Aza/mouse). The treatment was provided every 10 days for 60 days, starting at 12 weeks of age. Free-5-Aza (5 μg/mouse) or empty-nlg was applied to 2 control groups separately. n= 5–6 mice per group in 2 independent experiments. (A) Flow cytometry analysis of the percentage of Foxp3+ cells in splenic CD4 T cells (Thy1.2+CD4+ gated) from mice subjected to the indicated treatment. (B) Quantitation of the absolute cell numbers of Foxp3+ Tregs (Thy1.2+CD4+gated) in the spleens of mice treated as indicated. (C) Heatmap analysis of DNA methylation index on indicated gene promoters or enhancers in splenic T lymphocytes from 12-week-old MRL/lpr mice. n= 2 mice per group in 2 independent experiments. (D) Quantitation of DNA methylation on indicated gene promoters or enhancers in indicated splenic T lymphocytes sorted from 12-week-old MRL/lpr mice 10 hours after anti-CD4 antibody–coated nlg-5-Aza (15 μl nlg-5-Aza/mouse) treatment. n= 4 mice per group. (E) Flow cytometry analysis shows the induction of Foxp3 in CD4 T cells polarized in vitro. Naive CD4 T cells from Foxp3-YFP-Tg mice were polarized under Treg-inducing conditions for 7 days with vehicle, 5-Aza (1 μM), or nlg-5-Aza (equivalent to 1 μM free 5-Aza) added 12 hours right before collection. n= 3–4 per group in 2 independent experiments. (F) Flow cytometry–based assay of in vitro Treg-mediated suppression (Thy1.1+CD4+ gated). Thy1.2+CD4+YFP+ T cells were sorted from Foxp3-YFP-Tg mice, treated with or without 5-Aza (1 μM) or nlg-5-Aza (equivalent to 1 μM free 5-Aza) for overnight, and then mixed with naive CFSE-labeled Thy1.1+CD4+ Tconv cells plus anti-CD3– and anti-CD28–stimulating antibodies for 72 hours at a 1:5 ratio. Proliferation of Tconv cells was determined by CFSE dilution. n= 3–4 for 2 independent experiments. Data represent the mean ± SEM. **P < 0.01, ***P < 0.005 vs. control; 2-tailed Student’s t test.