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 1

nlg-5-Aza targeted to CD4+ or CD8+ cells but not free 5-Aza ameliorates disease manifestations in lupus-prone MRL/lpr mice.

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nlg-5-Aza targeted to CD4+ or CD8+ cells but not free 5-Aza ameliorates ...
(A) 12-week-old MRL/lpr mice were treated i.v. with either anti-CD4 antibody– or anti-CD8 antibody–coated nanolipogel-ATTO590 (nlg-ATTO590) (a fluorescent dye derived from rhodamine), and isotype control antibody–coated nlg-ATTO590 was used as control. Mice were euthanized 30 minutes after nlg administration for analysis. n = 4 mice per group. (B–F) MRL/lpr mice were treated with either anti-CD4 antibody–coated nlg-5-Aza (15 μl nlg-5-Aza per mouse, a dose comparable to 5 μg 5-Aza per mouse) or anti-CD8 antibody–coated nlg-5-Aza (15 μl nlg-5-Aza per mouse) 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 quantitation of ATTO590 intensity in different T cell subsets from spleens of mice subjected to the indicated treatment (CD3+TCRβ+TCR-γδ–CD49b– gated). (B) Representative images of facial skin from mice subjected to the indicated treatment. (C) The ratio of urine albumin to creatinine from mice subjected to the indicated treatment. (D) Representative images of H&E staining of kidneys from mice with the indicated treatment and histopathologic scoring of kidneys from mice with the indicated treatment. Original magnification, ×4 (left); ×40 (right). Scale bar: 160 μm (left); 20 μm (right). (E) Representative images of PAS staining of kidneys from mice with the indicated treatment. Original magnification, ×40. Scale bar: 20 μm. (F) Representative images of Masson staining of kidneys from mice with the indicated treatment. Original magnification, ×40. Scale bar: 20 μm. Data represent the mean ± SEM. *P < 0.05, ***P < 0.005 vs. control; 2-tailed Student’s t test.