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 4

Specific targeting of nlg-5-Aza in CD8+ T cells significantly reduces DN T cells.

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Specific targeting of nlg-5-Aza in CD8+ T cells significantly reduces DN...
(A and B) MRL/lpr mice were treated i.v. with anti-CD8 antibody–coated nlg-5-Aza (15 μl nlg-5-Aza/mouse) every 10 days for 60 days, starting at 12 weeks of age. Free-5-Aza (5 μg/mouse) or empty-nlg was applied in 2 separated control groups. (E and H) OT-I TCR-Tg CD8 T cells from CD45.1 OT-I TCR-Tg Rag1–/– B6 mice were transferred i.v. (5 × 106 per mouse) into mOVA-Tg recipient mice. 12 hours after the transfer, recipient mice were administrated i.v. with either control nlg or anti-CD45.1–coated 5-Aza-nlg (15 μl nlg-5-Aza/mouse) and sacrificed with an addition 60 hours. (A) FACS analysis of T cell subsets (Thy1.2+TCR-γδ–TCRβ+CD49b– gated) in the spleens and kidneys from mice subjected to the indicated treatments. (B) Quantitation of the absolute cell numbers of CD4–CD8– DN T cells from the spleens or kidneys of mice subjected to the indicated treatment. (C) Quantitation of DNA methylation in indicated gene promoters or enhancers from splenic T lymphocytes sorted from 12-week-old MRL/lpr mice 10 hours after anti-CD8 antibody–coated nlg-5-Aza (15 μl nlg-5-Aza/mouse) treatment. n = 4 mice per group. (D) Flow cytometry analysis of cell surface CD8 downregulation on OT-I TCR-Tg T cells from CD45.1 OT-I TCR-Tg Rag1–/– B6 mice (CD45.1+TCRβ+ gated) cocultured with OVA257–264–loaded antigen-presenting cells for 12 hours with or without addition of 5-Aza (1 μM) and representative dot plots relative to the mean of fluorescence intensity (MFI) of CD8 expression. (E) Flow cytometry analysis of cell surface CD8 downregulation on transferred OT-I TCR-Tg T cells and representative dot plots relative to MFI of CD8 expression. (F) Flow cytometry analysis of intracellular perforin expression in splenic CD4, CD8, DN T cells from female MRL/lpr mice at 16 weeks of age. (G) Flow cytometry analysis of intracellular perforin expression in CD8+ or CD8– OT-I TCR-Tg T cells (CD45.1+TCRβ+ gated) cocultured with OVA257–264–loaded antigen-presenting cells for 12 hours with or without addition of 5-Aza (1 μM). (H) Flow cytometry analysis of intracellular perforin expression in CD8+ or CD8– OT-I TCR-Tg T cells. Data represent the mean ± SEM. n= 3–6 mice per group in 2 independent experiments for (A, B, and D–G). *P < 0.05, **P < 0.01, ***P < 0.005 vs. control; 2-tailed Student’s t test.