C6-ceramide in combination with transforming growth factor-β enhances Treg cell differentiation and stable FoxP3 expression in vitro and in vivo

Ceramides, sphingosine-based lipid molecules, are generated mainly from the hydrolysis of sphingomyelin and play pivotal roles in biological processes including cell growth, differentiation, and inflammation. In this study, we investigated the effect of exogenous ceramides on the differentiation of regulatory T (Treg) cells and expression of FoxP3 gene in Treg cells. A cell-permeable C6-ceramide (C6) was capable of upregulating Treg cell differentiation when acting together with transforming growth factor-beta (TGF-β), and this induction was independent of T-cell receptor (TCR) and CD28 strength. Additionally, TGF-β/C6 treatment sustained the expression of FoxP3 gene in Treg cells, as the percentages of FoxP3⁺ Treg cells in the TGF-β/C6-treated group remained high for prolonged periods compared to those in the group treated with TGF-β alone. Furthermore, C8-ceramide was also capable of sustaining Treg cell populations and FoxP3 expression, whereas C2-, C16-, and C24-ceramides did not. Importantly, adoptive transfer of the TGF-β/C6-induced Treg cells into syngenic mice showed that TGF-β/C6-induced Treg cells maintained their FoxP3 expression in vivo significantly longer periods than the TGF-β-induced Treg cells. Taken together, our findings indicate that C6 can be utilized to increase Treg cell populations and also to sustain their FoxP3 expression in the treatment of autoimmune diseases or graft rejection.