Anti-Inflammatory Effects of Ursolic Acid Figure 1. Ursolic acid inhibits lymphocyte proliferation in vitro. (A) For cell proliferation analysis CFSE labelled lymphocytes were treated with UA (0.25–5 mM, 4 h) and stimulated with the Con A (10 mg/ml) at 37uC for 72 h. Twenty thousand cells were acquired in a flowcytometer. Vehicle treated cells served as control. Percent daughter cells were calculated from decrease in mean fluorescence intensity. (B) Each bar represents percentage of daughter cells in each treatment group. (C & D) UA inhibits anti-CD3 induced T cell proliferation. CFSE labelled lymphocytes were treated with UA (1 & 5 mM, 4 h) and stimulated with coated anti-CD3mAb (1 mg/ml) and soluble anti CD28mAb (1 mg/ml) at 37uC for 72 h. Percent daughter cells were estimated by CFSE dye dilution. Representative histogram and corresponding bar diagram are shown in Fig. 1C and D respectively. (E) UA inhibits Con A induced cell cycle progression: Lymphocytes were treated with UA (0.25–5 mM, 4 h) and stimulated with the Con A (5 mg/ml) at 37uC for 72 h. The cells were stained with propidium iodide and twenty thousand cells were acquired in a flowcytometer. The hollow bars represent percentage of cells containing less than 2n DNA (sub-G1/apoptotic cells), light gray bars show cells containing 2n DNA (in G1 phase) and the dark gray bars indicate the cells containing more than 2n DNA (in S+G2/M phase). (F) Evaluation of the potential cytotoxicity of UA to lymphocytes. Lymphocytes were cultured with UA (5 mM) for 24 h and cytotoxicity was measured by PI staining. Vehicle treated cells served as control. Percentage apoptosis (pre-G1 peak) in lymphocytes was estimated and is shown in the histograms. (G) Each bar represents percentage of apoptotic in each treatment group. Data points represent mean6S.E.M. from three replicates and three such experiments were carried out. *p,0.01, as compared to vehicle treated cells and #p,0.01, as compared to Con A or antiCD3/CD28 stimulated cells. doi:10.1371/journal.pone.0031318.g001 Con A (5 mg/ml). Mitogen activated cells showed significantly higher expression of CD69, CD25, CD134 and CD28 as compared to that in control cells (Fig. 4A–D). Treatment of lymphocytes with UA prior to Con A stimulation completely inhibited the Con A induced upregulation of CD69, CD134, CD25 and CD28 expression on T cells (Fig. 4A–D). Interaction of antigen bound MHC and CD80 (B7.1) and CD86 (B7.2) on antigen presenting cells with TCR and CD28 on T cells respectively provide the two signals required for complete T cell activation. Lack of either MHC-TCR or CD28-CD80 interaction impairs the immune response. We studied effects of UA on LPS induced upregulation of CD19, CD80, CD86 and MHC II on activated B cells. As shown in Fig. 4E-H, treatment of cells with UA prior to LPS stimulation inhibited the upregulation of CD19, CD80, CD86 and MHC II on activated leukocytes. Modulation of intracellular redox status by UA Intracellular ROS and GSH levels are known to pay an important role in immune responses and several molecules have been shown to exhibit their immunomodulatory activity via a redox dependent manner [33]. Hence, we examined whether UA also acts in a similar manner via modulation of cellular redox status. Treatment of lymphocytes with UA significantly increased the DCF (Dichlorodihydrofluorescein) (Fig. 5A) fluorescence at 5 mM. To ascertain whether the observed increase in intracellular ROS is accompanied with a concomitant decrease in GSH levels we checked the levels of intracellular GSH in lymphocytes following UA treatment. We observed that 4 h after UA treatment there was a significant decrease in the levels of GSH in lymphocytes (Fig. 5B). To determine the role of redox in the observed anti-inflammatory effects of UA we checked whether antioxidants could abrogate the suppressive effects of UA. Fig. 5C– E shows the effect of thiol and non thiol antioxidants on the suppressive effect of UA on Con A induced proliferation and cytokine secretion of lymphocytes. The suppression of Con A induced lymphocyte proliferation and cytokine secretion by UA could not be abrogated by thiol (glutathione (GSH), N-acetylcys- teine (NAC) and dithiothreitol (DTT)) or non-thiol antioxidant (trolox) suggesting that the effects of UA are independent of cellular redox status (Fig. 5C–E). UA suppressed mitogen induced MAPK, NF-kB, NF-AT and AP-1 activation in lymphocytes It is well known that following MHC-TCR engagement, a large number of proteins including MAPkinases, NFkB, AP1 and NF- AT are activated which co-ordinate with each other resulting in an immune response. To study the molecular mechanism of action of UA, we studied its ability to modulate signaling events that are involved in T cell activation. Fig. 6, shows the effect of UA on Con A induced MAPKinases (MEK, ERK and JNK), NF-kB, AP-1 and NF-AT activation in lymphocytes. Treatment of lymphocytes with UA inhibited Con A induced ERK and JNK phosphoryla- tion. The observed inhibition of ERK phosphorylation may be due to the inhibition of mitogen induced phosphorylation of c-raf and MEK which are upstream of ERK and are responsible for ERK phosphorylation upon activation (Fig. 6A). Lymphocytes treated with Con A (5 mg/ml) for 1 h showed degradation of IkB- a in the cytosolic fraction and activation of NF-kB, NFAT and AP-1 in the nuclear fraction as compared to that in vehicle treated control cells (Fig. 6B–D). However, cells treated with UA (5 mM) and then stimulated with Con A (5 mg/ml, 1 h) did not show IkB- a degradation (Fig. 6B). UA suppressed Con A mediated activation of all three important immunoregulatory transcription factors NF-kB, NFAT and AP-1 (Fig. 6B–D). The addition of excess unlabeled NF-kB (cold oligonucleotide, 100-fold) caused a complete disappearance of the band, whereas mutated oligonu- cleotide had no effect on DNA binding suggesting that the band belongs to NF-kB(Fig. 6B). Fig. 6E&F shows the effect of UA on Con A induced upregulation of NF-kB dependent genes in lymphocytes. Stimulation of lymphocytes with Con A (5 mg/ml) for 24 h resulted in significant upregulation of Bcl-2 and Bcl-xl protein levels (Fig. 6E&F). This increase in the levels of NF-kB dependent proteins (Bcl-2 and Bcl-xl) in Con A activated lymphocytes was inhibited by treatment with UA (Fig. 6E&F). UA delayed induction of graft-versus-host disease To study the in vivo efficacy of UA, we studied its ability to inhibit graft-versus-host disease (GVHD). Splenic lymphocytes from C57BL/6 mice were incubated with UA in vitro (5 mM, 4 h) and adoptively transferred to immunocompromised Balb/c mice. The mice that received untreated control cells developed GVHD that led to 100% death within 10 days (Fig. 7A) and demonstrated typical symptoms of GVHD, including alopecia, scleroderma, hunched posture, diarrhoea, and progressive weight loss. Howev- er, in mice that received UA treated cells showed 30% survival in better health for more than 30 days (Fig. 7A). Further, it was observed that mice receiving UA treated lymphocytes experienced inconspicuous weight loss as compared to control group (Fig. 7B). Fig. 7C–E show that on day 3 and day 5 post allo-transplantation, the levels of proinflammatory cytokines (IL-6, IFN-c and IL-2) were significantly higher in the serum of mice receiving vehicle treated allogenic lymphocytes as compared to those mice which received UA treated allogenic lymphocytes. This observation clearly shows potent anti-inflammatory activity of UA in vivo. UA exhibited therapeutic potential by acting post- mitogenic stimulation To explore its therapeutic potential, UA was added to lymphocytes post-mitogenic stimulation at different time points. It was observed that UA was able to completely suppress Con A PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31318