Effect of Homocysteine on the Differentiation of CD4⁺ T Cells into Th17 Cells

The hyperhomocysteinaemia (Hhcy) is a common phenomenon observed in patients with inflammatory bowel disease (IBD). Our previous study showed that Hhcy aggravated intestinal inflammation in an animal model of colitis. Increased levels of IL-17 and RORγt were also observed in this animal model of colitis with Hhcy. However, the direct effect of homocysteine on the differentiation of Th17 cells has never been studied. The aim of this study was to investigate the direct effect of Hhcy on the differentiation of CD4⁺ T cells into Th17 cells.

Lamina propria lymphocytes (LPLs) in colonic mucosa of Wistar rats were isolated and cultured under Th17-inducing (iTH17) environments. Different concentrations of the Hcy (0-100 μmol/ml) were added alone or combined with IL-23 (100 ng/ml) or folate (5 μmol/ml). The LPLs were divided into eight groups as follows: (1) Control group; (2) 10 μmol/ml Hcy group; (3) 25 μmol/ml Hcy group; (4) 50 μmol/ml Hcy group; (5) 100 μmol/ml Hcy group; (6) 100 ng/ml IL-23 group; (7) 50 μmol/ml Hcy + 100 ng/ml IL-23 group and (8) 50 μmol/ml Hcy + 100 ng/ml IL-23 + 5 μmol/ml folate group. The protein expression levels of IL-17, retinoid-related orphan nuclear receptor-γt (RORγt), p38 MAPK, phosphorylated p38 MAPK, cytosolic phospholipase A2 (cPLA2), phosphorylated-cPLA2 and cyclooxygenase 2 (COX2) were detected by immunoblot analysis. The protein level of prostaglandin E2 (PGE2) and IL-17 was detected by ELISA, and IL-17 and RORγt-positive CD4⁺ T cells were stained and analyzed by flow cytometry.

Hcy increased the protein levels of IL-17, RORγt, the ratio of phosphorylated p38 MAPK to p38 MAPK (p-p38/p38), the ratio of phosphorylated cPLA2 to cPLA2 (p-cPLA2/cPLA2) and COX2. The effect was concentration dependent to a certain degree; Hcy of 50 μmol/ml was the optimal concentration to increase the protein levels of those molecules. The level of IL-17 and PGE2 in the cell culture supernatants and the expression of IL-17 and RORγt in positive CD4⁺ T cells were also increased in the group of Hhcy. IL-23 showed a cooperative effect with Hcy on the differentiation of CD4⁺ Th cells into Th17 cells, whereas folate supplementation showed an inhibition action.

Homocysteine promoted the differentiation of CD4⁺ T cells into Th17 cells in a dose-dependant manner. This effect could be inhibited by folate.