BJ-3105, a 6-Alkoxypyridin-3-ol Analog, Impairs T Cell Differentiation and Prevents Experimental Autoimmune Encephalomyelitis Disease Progression
Fig 5
BJ-3105 controls T cell differentiation through inhibiting phosphorylation of JAK-STAT signaling pathway.
Naïve CD4+ T cells from spleens and draining lymph nodes were isolated and cells were cultured in Th1 and Th17 cells differentiation conditions with BJ-3105. Drug and cytokine untreated groups were used as control. (A) Naïve CD4+ T cells were polarized to Th1 cells with anti-CD3 (2 μg/mL), anti CD28 (1 μg/mL) and IL-12 (10 ng/mL) plus anti-IL4 Ab (5 μg/mL) for 72 h of culture. Th17 cells were differentiated with anti-CD3 (2 μg/mL), anti CD28 (1 μg/mL), TGF-β (1 ng/mL), IL-6 (10 ng/mL) plus anti-IL4 Ab (5 μg/mL) and anti-IFN-γ Ab (5 μg/mL) for 72 h of culture. Phosphorylated STAT1 and STAT4 from Th1 cells and STAT3 from Th17 cells were analyzed by flow cytometer. (B) Phosphorylated and total JAK1/2, STAT1 and STAT4 were detected by immuno-blotting under Th1 polarizing condition. (C) Phosphorylated and total JAK1/2 and STAT3 were detected by immuno-blotting under Th17 polarizing condition. (D) Western blot analysis of phosphorylated and total JAK1, JAK2, STAT1, STAT3 and STAT4 proteins in splenocytes and CNS infiltrated mononuclear cells from EAE induced mice. β-actin as loading control were detected by immune-blotting. Representative results of three experiments are shown.