Cells were sorted into CD4 +CCR6 − and CD4 +CCR6 + subsets using a BD Influx (BD Biosciences) and routinely >95% purity was achieved. 7-AAD (Biolegend, USA) was used to discriminate living cells. Antibodies used for human CCR6 T cell FACS sorts were anti-CD4 AF700 (BD Biosciences), anti-CD3 BV510 (BD Biosciences) and CCR6 PE (BD Biosciences). When zebularine was used, the indicated doses of drug were added at the beginning of the culture.įor FACS-sorted human cells, pre-enriched CD4 + T cells were incubated with a combination of primary antibodies against cell surface markers at 4☌ for 30 min and then washed using PBS (v/v 10% FCS and 1mM EDTA).
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Total CD4 + T cells were then isolated with the CD4 (元T4) T cell isolation kit using the autoMACS Pro Separator (Miltenyi, Germany) and stimulated by plate-bound of anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) antibodies, as well as mIL-12 10 ng/mL, mIL-2 10 ng/mL, anti-IL-4 10 μg/mL (Th1) mIL-6 20 ng/mL, rhTGF-β1 1 ng/mL, anti-IL-4 10 μg/mL, anti-IFN-γ 10 μg/mL (Th17) and mIL-2 50 ng/mL, rhTGF-β1 2 ng/mL (Treg) for 3 days.
For murine CD4 + T cells, spleen and lymph node cells were filtered through a 40-μm cell strainer, followed by the red blood cell lysis using ammonium-chloride-potassium (ACK) buffer. Zebularine was also added at the indicated concentrations at the beginning of cultures. Human peripheral CD4 +CCR6 − and CD4 +CCR6 + cells ( N = 4 3 female and 1 male average age of 42) were FACS-sorted from isolated CD4 + T cells, resuspended to 2 × 10 6 cells/mL in RPMI-1640 supplemented with 10% FCS, L-glutamine, and penicillin/streptomycin (all Thermofisher, stimulated with plate-bound anti-CD3 (5 μg/mL clone UCHT1) and anti-CD28 (5 μg/mL clone CD28.2) antibodies (eBioscience) (CCR6 − cells) or with plate-bound antibodies and a polarizing cytokine mixture of 20 ng/mL IL-6, 10 ng/mL IL-23, 10 ng/mL IL-1β (all from R&D Systems), 100 ng/mL anti-IFN-γ, 100 ng/mL anti-IL-4 (eBioscience) (CCR6 + cells) for 5 days. In addition, the immunosuppressive effects of zebularine in vivo were also evaluated using the murine experimental autoimmune uveitis model. Here, to elucidate whether zebularine is able to control the intraocular inflammation through regulating the expression of inflammatory cytokines in CD4 + T cells, we explored the changes of IFN-γ, IL-17, and Foxp3 ( 17) in response to zebularine treatment in Th1, Th17, and Treg cells. We therefore hypothesize that another DNA methylation inhibitor, zebularine, which has relatively low cellular toxicity and a longer half-life ( 16), has the potential to suppress uveitis. In addition, an anti-inflammatory effect of these two drugs has also been observed in murine models of asthma ( 14) and experimental autoimmune encephalomyelitis ( 15). Among these small-molecule inhibitors, two DNA methyltransferase (DNMT) inhibitor pro-drugs, 5-azacytidineĪnd 2′-deoxy-5-azacytidine have been approved for the treatment of myelodysplastic syndrome and acute myeloid leukemia ( 13).
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Two key epigenetic mechanisms-DNA methylation and histone modifications-regulate chromatin accessibility, and have been the targets of a series of compounds developed in recent years for the treatment of cancers and immune-mediated diseases ( 12). Therefore, modulating the epigenetic program that controls Th1, Th17, and Treg functions may serve as a new way to control the intraocular inflammation in uveitis. And many genome-wide and locus-specific epigenetic changes have been found in patients with immune-mediated diseases such as systemic lupus erythematosus and rheumatoid arthritis ( 12). The expression of signature cytokines and master transcription factors, as well as Th functions, are under tight control of coordinated epigenetic alterations ( 10, 11). Modulating Th cell differentiation and function has been proposed as a therapeutic strategy for uveitis ( 9). Consequently there is a need to develop improved approaches to achieve control of intraocular inflammation.Ībnormal activation of the T helper (Th) cells and imbalance between inflammatory Th1/Th17 and regulatory T (Treg) cells play a key role in the pathogenesis of autoimmune uveitis ( 6– 8).
Affected patients are at risk of visual impairment or blindness ( 2), in particular those who are resistant or intolerant to conventional immunosuppressive therapies ( 3– 5). Autoimmune uveitis is a heterogeneous collection of diseases characterized by intraocular inflammation ( 1).
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