Anti-Inflammatory Effects of Ursolic Acid Figure 2. Ursolic acid inhibits mitogen induced proliferation of CD4+ T cells, CD8+ T cells and B cell. CFSE labelled purified CD4+T cells (A) or CD8+Tcells (C) were pre-treated with ursolic acid (5 mM, 4 h) before stimulation with coated anti-CD3mAb (1 mg/ml) and soluble anti CD28mAb (1 mg/ml) for 72 h at 37uC. Percent daughter cells were calculated from decrease in mean fluorescence intensity. (B&D) Each bar represents percentage of daughter cells in different treatment groups. (E,F) UA inhibits LPS induced proliferation of B cells. CFSE labeled lymphocytes were treated with UA (5 mM, 4 h) and stimulated with the LPS (50 mg/ml) at 37uC for 72 h. Vehicle treated cells served as control. Percent daughter cells were calculated from decrease in mean fluorescence intensity. Each bar represents mean6S.E.M. from three replicates and two such independent experiments were carried out. *p,0.01, as compared to vehicle treated cells and #p,0.01, as compared to Con A or LPS or anti-CD3/anti-CD28 stimulated cells. doi:10.1371/journal.pone.0031318.g002 induced cytokine secretion in murine lymphocytes even when it was added up to 4 h after mitogenic stimulation (Fig. 7F). Since UA inhibited the expression of co-stimulatory molecules on activated T cells and B cells, we studied whether UA renders these cells anergic and hence incapable to respond to any further stimuli. We observed that splenic lymphocytes treated with UA for 4 h did not respond to mitogenic stimuli when activated with Con A even though they were washed and rested for 48 h following UA treatment. This shows that UA treated cells are hyporesponsive to antigenic stimulation which might be due to a possible induction of anergy (Fig. 7G). Discussion Triterpenoids form natural components of human diets. An average of 250 mg per day of triterpenes, largely derived from vegetable oils, cereals, fruits and vegetables is consumed [34]. Ursolic acid (UA) is a part of traditional medicine and has been shown to possess many biological activities, such as antioxidative, anti-inflammatory, anticancer and hepato-protective activities [23]. Recent reports indicated that the anticarcinogenic, anti- inflammatory, and proapoptotic effects of ursolic acid were due to its ability to inhibit immunoregulatory transcription factor NFkB in response to a wide variety of carcinogens and inflammatory agents [27]. However, there are no detailed reports on the immunosuppressive effects of UA in T cells which are the primary cell type involved during an adaptive immune response. To explore the immunomodulatory properties of UA, we studied its effect on T cell activation, proliferation and effector responses to different activating stimuli. We observed that UA was able to inhibit T cell proliferation in response to both polyclonal and antigen specific activation in a dose dependent manner PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31318