a School of Basic Medicine and Clinical Pharmacy , China Pharmaceutical University , Nanjing , China.
Immunopharmacol Immunotoxicol. 2018 Aug;40(4):309-318. doi: 10.1080/08923973.2018.1480631. Epub 2018 Jul 13.
CD4 + CD25+ regulatory T (Treg) lymphocytes are critical for immune homeostasis. Foxp3 (Forkhead Box protein P3) is always considered as a marker of function and identities determination of Treg cells because of special occurring in Treg cell. People who lack Treg cells or have a low expression of Foxp3 gene will suffer fatal autoimmunity. Scientists are trying to use Treg cells as a treatment for autoimmune disease, such as systemic lupus erythematosus.
Our objective was to induce Foxp3 + CD4+ T cells from naïve CD4 + T cells isolated from C57 mice spleen in vitro using stimuli that include the short chain fatty acid sodium butyrate. Furthermore, to explore the relationship between Foxp3+ T cells induction and epigenetic modification, by observing the changes of Foxp3, Ezh2 (Enhancer of Zeste Homolog 2) and phosphorylated Ezh2 in the induced Treg cells.
The naïve CD4+ T cells were separated from C57 mice spleen by immunomagnetic separation. Anti-CD28, anti-CD3, IL-2, TGF-β1, and sodium butyrate were added with proper concentration to induce Foxp3 expression during 72 hours. Then, we observed the effect of GSK126 (Ezh2 inhibitor) on the induction within the same over 72 hours duration. Then, western blot and Q-PCR were used to see the changes in gene/protein expression of Foxp3, Ezh2, and phosphorylated Ezh2.
According to our results, group 3 that received full stimulus had a significant higher level of Foxp3 and Ezh2 expression (p < .05, comparing with group 1,2) and adding 5 mM sodium butyrate to the full stimulus (group 5) increased significantly the induction of Foxp3 and Ezh2 than control group and higher concentration group (p < .05, comparing with group 3,4, 6). The gene and protein expression of Foxp3 and Ezh2 both were enhanced in group 5 (p < .05 comparing with group 3). However, phosphorylated Ezh2 decreased in group 5 (p < .05 comparing with group3). Sodium butyrate removed part inhibition of GSK126, result in Foxp3 and Ezh2 expression (p < .05, p < .01, comparing with group7).
In this study, we were able to transform CD4 + T cells into CD4 + Foxp3 + T cell by using stimulus like antibodies (anti-CD28, anti-CD3) and cytokines (IL-2, TGF-β1). Sodium butyrate contributes to CD4 + Foxp3 + T cell induction in vitro and at an optimum concentration of 5 mM. Sodium butyrate promotes expression of Ezh2 and Fxop3 of T cells in vitro; in addition, to lowering relative expression of phosphorylated Ezh2 probably be influencing some pathways like PI3K-Akt. Epigenetic modification is also thought to take essential part into the upregulation of Foxp3 from naïve CD4 + Tcells.
CD4+CD25+调节性 T（Treg）细胞对于免疫稳态至关重要。叉头框蛋白 P3（Foxp3）通常被认为是 Treg 细胞功能和身份确定的标志物，因为它在 Treg 细胞中特异性出现。缺乏 Treg 细胞或 Foxp3 基因表达水平低的人会患上致命的自身免疫性疾病。科学家们正试图将 Treg 细胞作为治疗自身免疫性疾病的一种方法，如系统性红斑狼疮。
本研究旨在使用包括短链脂肪酸丁酸钠在内的刺激物，从 C57 小鼠脾脏中分离的幼稚 CD4+T 细胞体外诱导 Foxp3+CD4+T 细胞。此外，通过观察诱导 Treg 细胞中 Foxp3、Ezh2（增强子结合蛋白 2 同源物 2）和磷酸化 Ezh2 的变化，探讨 Foxp3+T 细胞诱导与表观遗传修饰之间的关系。
采用免疫磁珠分离法从 C57 小鼠脾脏中分离幼稚 CD4+T 细胞。在 72 小时内，用适当浓度的抗 CD28、抗 CD3、IL-2、TGF-β1 和丁酸钠诱导 Foxp3 表达。然后，我们观察了在相同的 72 小时时间内 GSK126（Ezh2 抑制剂）对诱导的影响。然后，采用 Western blot 和 Q-PCR 检测 Foxp3、Ezh2 和磷酸化 Ezh2 的基因/蛋白表达变化。
根据我们的结果，第 3 组（接受全刺激）Foxp3 和 Ezh2 的表达水平显著升高（p<0.05，与第 1、2 组相比），在全刺激中加入 5 mM 丁酸钠（第 5 组）比对照组和较高浓度组（p<0.05，与第 3、4、6 组相比）显著增加了 Foxp3 和 Ezh2 的诱导。第 5 组 Foxp3 和 Ezh2 的基因和蛋白表达均增强（p<0.05，与第 3 组相比）。然而，第 5 组磷酸化 Ezh2 减少（p<0.05，与第 3 组相比）。丁酸钠部分去除了 GSK126 的抑制作用，导致 Foxp3 和 Ezh2 的表达（p<0.05，p<0.01，与第 7 组相比）。
在这项研究中，我们能够通过使用抗体（抗 CD28、抗 CD3）和细胞因子（IL-2、TGF-β1）等刺激物将 CD4+T 细胞转化为 CD4+Foxp3+T 细胞。丁酸钠有助于体外 CD4+Foxp3+T 细胞的诱导，最佳浓度为 5 mM。丁酸钠在体外促进 Ezh2 和 Fxop3 的表达；此外，降低相对表达的磷酸化 Ezh2 可能会影响 PI3K-Akt 等途径。表观遗传修饰也被认为在从幼稚 CD4+T 细胞上调 Foxp3 中起着重要作用。
