The epigenetic modification during the induction of Foxp3 with sodium butyrate

Abstract

Context: 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.

Objective: 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.

Materials and methods: 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.

Results: 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).

Conclusion: 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.

Keywords:

• Naive CD4 + T cell
• iTreg
• sodium butyrate
• epigenetic modification
• EZH2
• FOXP3
• EZH2 phosphorylation

Acknowledgements

Thanks all my lab mates for helping me to finish this article.

Disclosure statement

No potential conflict of interest was reported by the authors.