Ursolic acid suppresses interleukin-17 (IL-17) production by selectively antagonizing the function of RORgamma t protein

Abstract

Th17 cells have recently emerged as a major player in inflammatory and autoimmune diseases via the production of pro-inflammatory cytokines IL-17, IL-17F, and IL-22. The differentiation of Th17 cells and the associated cytokine production is directly controlled by RORγt. Here we show that ursolic acid (UA), a small molecule present in herbal medicine, selectively and effectively inhibits the function of RORγt, resulting in greatly decreased IL-17 expression in both developing and differentiated Th17 cells. In addition, treatment with UA ameliorated experimental autoimmune encephalomyelitis. The results thus suggest UA as a valuable drug candidate or leading compound for developing treatments of Th17-mediated inflammatory diseases and cancer.

FIGURE 1.

UA dose-dependently inhibits Th17 differentiation. A, the effect of UA on human Th17 differentiation. Left, intracellular staining. Right, real-time RT-PCR (normalized to GAPDH). B, the effect of UA on mouse Th17 cell differentiation. Left, intracellular staining. Right, real-time RT-PCR (normalized to β-actin). The in vitro differentiation was repeated >4 times, and real-time RT-PCR was repeated 2 times with consistent results.

FIGURE 2.

UA selectively blocks the function of RORγt but not RORα. A, mouse naive CD4⁺ T cells were differentiated under neutral condition and infected with RORα, RORγt, or control pMIG viruses on day 1. 2 μm UA or DMSO was added 6 h after viral infection. The cells were restimulated on day 4 for intracellular staining (gated on hCD2⁺ cells). B, 293T cells were transfected with the RORE reporter together with RORα, RORγt, or control plasmids. UA or DMSO was added after transfection, and the cells were harvested next day for Dual-Luciferase activity assays. The data were normalized to an internal control Renilla luciferase. C, the dose-dependent inhibitory results of UA on RORγt/RORα binding to its co-activator peptide or on Th17 differentiation were fitted to a sigmoidal dose-response curve to determine the corresponding IC₅₀ values. x axis, log concentration (nm) of UA. y axis, relative binding of RORγt/RORα to its co-activator peptide or relative percentage of IL-17⁺ cells. All the assays were repeated at least 2 times with consistent results.

FIGURE 3.

UA suppresses IL-17 production in mature Th17 cells. After being preincubated with UA, the mature human Th17 cells or mouse Th17 cells generated in vitro were then restimulated with anti-CD3 overnight for secreted cytokine analysis in the presence of the indicated amounts of UA. Both human and mouse experiments were repeated 2–3 times with consistent results.

FIGURE 4.

UA treatment ameliorated EAE disease in mice. For EAE induction, mice were given either DMSO or UA by i.p. injection every other day after the first MOG immunization and monitored daily for clinical symptom development after the second MOG immunization. Results are the combination of two independent EAE experiments, and the disease onset date of DMSO-treated mice was set to day 1 for statistical analysis. A, the percentage of mice that developed EAE disease (n = 10 for DMSO group and n = 8 for UA group). B, the clinical scores of diseased mice (n = 7 for the DMSO group and n = 6 for the UA group). C, the total number of CD4⁺, IL-17⁺, and IFN-γ⁺ cells in the central nervous system of EAE mice. D, the effect of UA on MOG-specific IL-17 production in the spleens of EAE mice.