Anti-Inflammatory Effects of Ursolic Acid 24 h at 37uC. The concentration of cytokines in the supernatant was estimated using ELISA. (D) UA inhibits LPS induced cytokine production from macrophage. Splenic adherent macrophage were pre-treated with UA (5 mM, 4 h) and then stimulated with LPS (50 mg/ml) for 24 or 48 h. The concentration of cytokines in the supernatant was estimated using ELISA. 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. (E) Quantative real time RT-PCR analysis of differential genes expression in CD4+ T cells activated with antiCD3/CD28 in the presence or absence of Ursolic acid. Purified CD4+T cells were pre-treated with UA for 4 h before stimulation with anti-CD3/anti-CD28 mAb for 24 h at 37uC. The relative expression ratio was calculated and plotted as mean 6 SEM. *p,0.01, as compared to vehicle treated cells and #p,0.01, as compared to anti-CD3/anti-CD28 stimulated cells. doi:10.1371/journal.pone.0031318.g003 (Fig. 1A–D). These anti-proliferative effects of UA were not due to increased apoptosis in T cells but due to its ability to induce cell cycle arrest in G1 phase (Fig. 1E–G). The immunosuppressive effects of UA were not limited to CD4+ and CD8+ T cells (Fig. 2A–D), but it also suppressed LPS induced proliferation of B cells (Fig. 2E&F) suggesting a common mechanism its of action in these lymphocyte subsets. Cytokines secreted by different cells participating in the immune response are known to play a critical role in successful pathogen clearance. Any alteration in this highly regulated network of cytokines by external or internal factors may result in undesirable consequences. IL-2, TNF-a, and IFN-c are secreted by Th1 type cells and can activate macrophages and promotes cell-mediated immune responses against invasive intracellular pathogens. Th2 (IL-4, IL-5, IL-6, IL-10, and IL-13) cytokines promote humoral immune responses against extracellu- lar pathogens [35]. UA suppressed both Th1 and Th2 cytokines secreted by activated lymphocytes in response to both polyclonal and antigen specific stimuli in vitro (Fig. 3A–C). Interestingly, UA also mitigated LPS induced secretion of IL-1b, TNF-a and IL-6 in splenic adherent macrophages (Fig. 3D). Quantitative real time RT-PCR for 6 genes which were known to be involved in activation and proliferation of T cells was performed and UA also suppressed the expression of these genes (Fig. 3E). These results suggested that UA acts on several cell types in exhibiting anti- inflammatory activity. Recent investigations suggest cellular redox status may play key role in the regulation of immune responses . Addition of antioxidants has been shown to modulate T cell responses as measured in terms of proliferation and cytokine secretion implicating the importance of ROS in antigen mediated T cell activation [16]. Our group has also recently shown that perturbation of cellular redox status can lead to immunosuppres- sion [33]. Even though UA was observed to increase the basal ROS levels in lymphocytes, the addition of thiol or non-thiol antioxidants could not abrogate the suppressive effects of UA on lymphocyte proliferation and cytokine secretion suggesting a redox independent mechanism of action in lymphocytes (Fig. 5A–D). Mechanisic studies of anti-inflammatory effects of UA revealed that it acts not only by inhibiting early events in T cell activation but also had potent suppressive effects on the co-stimulatory molecules. It was observed that lymphocytes treated with UA failed to upregulate T cell activation markers CD69, CD25 and CD134 and co-stimulatory marker CD28 upon mitogenic stimulation (Fig. 4A–D). The role of CD28 as the most important co-stimulatory receptor is well established. Engagement of the T cell receptor (TCR) in the absence of CD28 costimulation results in a long-term hyporesponsive state in T cells called clonal anergy and represents one mechanism of peripheral tolerance [36]. Also, the interaction of CD134 with CD134L is known to be involved in T cell activation and also synergize with CD28-B7 co-stimulatory pathway to generate an efficient T cell effector responses [37,38]. Experiments showing the inhibitory effects of UA on activation and co-stimulatory markers on B cells (CD19, CD80 and CD86) also emphasize its ability to block the co-stimulatory pathway (Fig. 4E–G). The inhibition of LPS induced MHCII on APCs by UA may also aid in its potent anti-inflammatory properties by blocking antigen presentation to T cells (Fig. 4H). Since many of these lymphocyte activation markers, costimu- latory molecules and cytokine genes are under NF-kB and AP-1 regulation, experiments were performed to examine the effect of UA on these transcription factors and MAPKinases. Both ERK and JNK signaling pathways are vital mediators of a number of cellular processes including growth, proliferation, and survival of T cells [39,40]. We observed that UA was able to inhibit mitogen induced phosphorylation of ERK and JNK and their upstream kinases, MEK and c-raf (Fig. 6A). Further, we also observed that UA was able to inhibit mitogen induced increase in nuclear levels of NF-kB, NF-AT and AP-1 in lymphocytes (Fig. 6B–D). Shishodia et al, have earlier shown suppression of TNF-a induced NF-kB by UA in different tumor cell lines [27]. However, this report shows the effect of UA on mitogen induced NF-kB and its regulated proteins in normal lymphocytes (Fig. 6. B,E&F). Signal 2 mediated by CD28-B7 interaction is required for the induction of NF-kB and AP-1 in Ag-stimulated T cells [41] . This CD28 signaling is provided by the costimulatory molecules B7-1/B7-2 present at the cell surface of APCs [42]. The inhibition of NFkB and AP-1 exhibited by UA might be a consequence of its ability to block the co-stimulatory pathway by down regulating the levels of CD28 and B7. For complete T cell activation and cytokine secretion, the cooperative binding of NF-AT and AP-1 to composite NF-AT/AP-1 binding sites is necessary [43,44,45]. The inhibition of NF-AT by UA may lead to inhibition of IL-2 secretion and the observed anti-inflammatory effects. Thus, UA inhibits the activation of the transcription factors NF-AT, NF-kB, and AP-1 which are required to function in a co-ordinated manner to regulate antigen induced immune response. More importantly, we demonstrated the immunosuppressant activity of UA in vivo. We studied the in vivo anti-inflammatory potential of UA using a mouse model of graft-versus-host-disease which is a frequent complication of allogenic bone marrow transplant in which the engrafted donor T cells attack the recepients’ organs and tissues. Clinically, cyclosporine A and tacrolimus have been used in organ transplantation to prevent allograft rejection [46] . However, these drugs are reported to show undesirable side effects that needs to be overcome before they can be used in other inflammatory disorders and autoimmune disease [20,47]. We observed that treatment of donor lymphocytes with UA prior to allogenic transplantation significantly improved symptoms associated with acute GVHD, delayed GVHD associated mortality and morbidity in recipient mice (Fig. 7A&B) and also decreased the levels of proinflammatory cytokines in the serum (Fig. 7C–E). Interestingly, UA was able to inhibit Con A induced cytokine secretion in murine lymphocytes even when added up to 4 h post-stimulation (Fig. 7F). Consistent with previous results, UA treatment induced a state of hypo- responsiveness in lymphocytes. Further, lymphocytes treated with UA, washed and rested for 48 h showed decreased secretion of IL- 2 in response to Con A stimulation (Fig. 7G). PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31318