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J Mol Neurosci (2011) 44:31–40 DOI 10.1007/s12031-010-9483-1 Amelioration of Experimental Autoimmune Encephalomyelitis by β-elemene Treatment is Associated with Th17 and Treg Cell Balance Rongwei Zhang & Ayong Tian & Huimin Zhang & Zhike Zhou & Hongmei Yu & Lei Chen Received: 3 August 2010 / Accepted: 8 December 2010 / Published online: 19 January 2011 # Springer Science+Business Media, LLC 2011 Abstract Experimental autoimmune encephalomyelitis (EAE), an animal mode of multiple sclerosis (MS), was previously considered that is mediated by Th1 cells. However, a number of recent studies provided strong evidence that T helper cells that produce interleukin (IL)17 (Th17) and anti-inflammatory CD4+ Foxp3+ regulatory T cells (Tregs) play a dominant role in the pathogenesis of EAE. β-elemene is a natural antitumor plant drug with the role of multiple target, and it has been found to pass through the blood–brain barrier easily. It also has been strongly implicated as an immune modulatory agent, but R. Zhang : H. Zhang : Z. Zhou Department of Geriatrics, The First Affiliated Hospital of China Medical University, No.155 Nanjing Bei Street, Heping, 110001 Shenyang, Liaoning, People’s Republic of China A. Tian Department of Anesthesiology, The First Affiliated Hospital of China Medical University, No.155 Nanjing Bei Street, Heping, 110001 Shenyang, Liaoning, People’s Republic of China H. Yu Department of Neurology, The First Affiliated Hospital of China Medical University, No.155 Nanjing Bei Street, Heping, 110001 Shenyang, Liaoning, People’s Republic of China L. Chen (*) Department of Ophthalmology, The First Affiliated Hospital of China Medical University, No.155 Nanjing Bei Street, Heping, 110001 Shenyang, Liaoning, People’s Republic of China e-mail: [email protected] L. Chen e-mail: [email protected] the precise mechanisms of its action are largely unknown. In the present study, we mainly investigated the efficacy and mechanism of β-elemene against EAE in vivo and vitro. The treatment of C57 mice with β-elemene significantly delayed the onset of EAE, markedly suppressed MOG-specific T cell proliferation in a dose-dependent manner, dramatically reduced the IL-17, IL-6, IL-23, and RORγt production and induced the Foxp3 expression in both the periphery and the inflamed spinal cord. These findings indicated that β-elemene amelioration EAE was, to a large extent, due to inhibit differentiation and development of Th17 cells depends on down-regulating expression of IL-6, IL-23, RORγt signaling, and promoting expansion in Treg cells. Suggesting it is useful in the control of MS and other Th17 cell-mediated inflammatory diseases. Keywords β-elemene . Th17 . Treg . Cytokine Introduction Although multiple sclerosis (MS) is the most common inflammatory demyelization disease of the central nervous system (CNS), afflicting hundreds of thousands of people worldwide. Its pathogenesis is still only incompletely clear. There seems to be substantial heterogeneity in disease mechanisms. Some studies have shown that an autoimmune origin or at least a decisive autoimmune component is postulated. In the past, the role of the Th1 cells and Th2 cells for adaptive immune responses in MS had been addressed (Coffman 2006; Mosmann and Coffman 1989). But recent studies provided strong evidence that interleukin (IL)-17-producing T cells play a dominant role in the pathogenesis of MS and EAE. (Tzartos et al. 2007; Hofstetter et al. 2005; Hofstetter et al. 2007; Komiyama 32 et al. 2006). Another distinct T cell population called Tregs has also attracted attention recently because it plays an important role in maintenance of immune tolerance and in controlling the destructive self-reactive T cells found in autoimmune animal models of arthritis, multiple sclerosis, diabetes, and inflammatory bowel disease (Fontenot et al. 2003; Reddy et al. 2005; Kohm et al. 2002). Modulating the Treg population and other associated factors may prove to be an effective new therapy for autoimmune disease. Many clinical and pathologic features of EAE show close similarity to MS, therefore it has been used widely as an animal model to explore the mechanism of MS pathogenesis and to test the efficacy of potential therapeutic approaches for the treatment of MS. Naïve cluster of differentiatio (CD4)+ help T cells result in the development of Th1, Th2, Th17, or Treg depending on the cytokine milieu. It is well known that IL-12 (signaling through T-bet and STAT-4) regulates Th1 (Gately et al. 1998), while IL-4 (signaling through STAT-6 and GATA-3) regulates Th2 differentiation (Nelms et al. 1999), transforming growth factor beta (TGF-β) has usually been associated with the development of Treg cells (Chen et al. 2003), but it is interesting to note that in the presence of an inflammatory cytokine, such as IL-6, the inhibition of Treg cell development and differentiation of naïve CD4+ T cells into Th17 could be observed (Bettelli et al. 2006). It was shown in parallel by several groups that a combination of IL-6 and TGF-β is required for Th17 differentiation and that differentiated Th17 cells are maintained and expanded by IL-23, which is unable to drive Th17 differentiation of naïve T cells by itself (Bettelli et al. 2006; Stockinger and Veldhoen 2007). Another key transcription factor associated with the Th17 lineage, the orphan retinoid receptor—RORγt (Ivanov et al. 2006), inducing transcription of IL-17 in naïve CD4+ T cells, often activated by cytokine IL-6, RORγtdeficient CD4+ T cells produce no IL-17 in response to TGFβ and IL-6. Activation and proliferation of proinflammatory Th17 cells is reciprocally related to the immunosuppressive effects of Treg cells, both of which are contingent upon the cytokine environment, in particular IL-6, TGF-β, and IL-23 (Mangan et al. 2006; McGeachy et al. 2005). Elemene (1-methyl-1-vinyl-2, 4-diisopropenyl-cyclohexane) is a novel non-cytotoxic antitumor drug, which is isolated from the traditional Chinese medicinal herb Rhizoma zedoariae. The extract of elemene is a mixture of α-, β-,and δ-elemene, with β-elemene as the main component, accounting for 60∼72% of the three isoforms. When used as a single agent, elemene was shown to exhibit anticancer effects in human and murine tumor cells in vitro and in vivo and has substantial clinical activity against various tumors without severe side effects (Fu 1984; Tan et al. 2000; Wang et al. 1996) Another study had shown patient immunity was improved during the therapy with β-elemene (Wu et al. J Mol Neurosci (2011) 44:31–40 1999). We speculated that β-elemene can affect multiple target involved in the pathogenesis of immune disorder diseases including tumors and demyelinating diseases. In the present study, we examined the effects and mechanism of action of β-elemene on the pathogenesis of EAE. Our data indicated β-elemene can ameliorate the course of EAE mice immunized with myelin oligodendrocyte glycoprotein (MOG35–55) peptide by reducing IL-6 production, suppressing IL-6-activated RORγt and signal transducer and activator of transcription 3 (STAT3) pathway mediating the differentiation of Th17 cells and inducing Treg cells expansion in EAE. Our data suggest that β-elemene maybe is a good candidate in the treatment of MS and other Th17-mediated inflammatory diseases. Materials and Methods Animals C57BL/6 (8–10 weeks old) female mice were purchased from Shanghai SLAC Laboratories animal Co., Ltd. and were housed under a 12-h light/dark cycle in microisolater cages contained within a laminar flow system to maintain a pathogen-free environment. The studies were conducted in accordance with the Animal Component of Research Protocol guidelines at the China Medical University. Induction of Chronic EAE C57BL/6 mice were immunized subcutaneously at the two sites on the back with 100 ml (200 mg) of MOG35–55 peptide dissolved in distilled water and emulsified with an equal volume of complete Freund's adjuvant supplemented with 4 mg/ml mycobacterium tuberculosis H37Ra. All animals were also injected intraperitoneally on days 0 and 2 with 400 ng of Pertussis toxin. Control animals followed an identical immunization protocol (adjuvant+pertussis) without added MOG peptide. Animals were weighed daily and assessed for clinical signs of EAE by two independent observers. We used the same clinical EAE scoring system to assess neurological deficit in our mouse EAE model according to the following scale: score 0, no disease; score 1, loss of weight and tail weakness; score 2, weakness in hind limb; score 3, complete hind limb paralysis; score 4, hind limb paralysis with fore limb weakness or paralysis; and score 5, moribund or deceased. Treatment Protocol β-elemene (0603091; 20 ml, 100 mg) was stored at 4°C. β-elemene treatment (20 mg/kg) by intraperitoneal injection started at day 1 after injection of MOG35–55 peptide. Injection of β-elemene q.o.d. continued until the end of the J Mol Neurosci (2011) 44:31–40 33 experiment (day 11, 15). Sham treated with an equal volume of saline. Histological Analysis To assess the degree of CNS inflammation, three mice from each group were sacrificed under anesthesia on day 15 (after the onset of EAE).The spinal cord was removed quickly. A portion of spinal cord sample was fixed in 10% neutral buffered formalin and then cut into 4-μm-thick sections. Tissues were stained with hematoxylin and eosin to assess tissue damage and inflammation, Bielschowsky silver impregnation for axonal damage. for 34 s and 72°C for 15 s. The data were analyzed using the standard curve method and the mRNA level of target gene for each sample was normalized against refercence gene GAPDH mRNA. Statistics The data are expressed as the mean±SD. Student's two-tailed t test was used to analyze the results. P values<0.05 or less was considered significant. All in vitro experimental data were representative of three independent experiments and represented the mean ratio of triplicate results in each experiment. T Cell Proliferation Assay Results Effects of β-elemene on proliferation T cells were analyzed by MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazo-lium bromide) assay. Draining lymph node (DLN) cells on days 11, 15 after immunization with MOG35–55 peptide were prepared, a total of 2×106 cells/well were stimulated for 72 h with or without specific Ag-MOG35–55 peptide and treated for 72 h with various concentrations of β-elemene (final concentration, 10 μg/mL MOG35–55; 2, 4, 6, 8, 10 μM β-elemene) or an equivalent amount of medium containing DMSO at 37°C in 95% humidity and 5% CO2 (three wells for each sample). The optical density was measured at 550 nm with a microplate reader (Bio-Rad 2550, Richmond, CA, USA). RNA Isolation and Quantitative Real-time RT-PCR Total RNA was extracted from treated DLN cells and spinal cord using an RNA isolation kit (Takara, Japan). Complementary DNA was prepared as recommended and used as the template for quantitative PCR. Levels of tissue and cellular mRNA for IL-17, IL-6, TGF-β, IL23p19, RORγt, Foxp3 from all groups were analyzed by real-time PCR. Real-time PCR was performed according to the manufacturer's instructions. Specific primers are shown in Table 1. The amplification conditions were 8 min at 95°C, followed by 45 cycles of 95°C for 5 s, 60°C Table 1 Premiers used in this study The Effect of β-elemene on C57BL/6 EAE Mice To test the use of β-elemene in the treatment of MS, we examined the protective effect of β-elemene on the pathogenesis of EAE mice. C57BL/6 mice were treated with β-elemene by intraperitoneal injection started at day 1 after injection of MOG35–55 peptide. Injection of β-elemene q.o.d. continued until the end of the experiment (day 15). The sham-treated mice were observed clinical manifestations on days 12–14, mean day of onset (12.03±0.83), and developed clinical paralysis for a mean maximum clinical score (MMCS) of 2.54 on day 15. In contrast, the mice treated with β-elemene mean day of onset (15.12±0.92), decreased the MMCS to 0 on day 15 (Fig. 1). The difference in the MMCS between β-elemene-treated and sham-treated groups was significant, with p<0.001 using the Student's t test. These results suggested that β-elemene delayed the onset of clinical neurologic signs in animals and decreased the clinical severity in EAE mice. β-elemene Decreases CNS Inflammatory Cells Infiltration and Axonal Damage in EAE To further study the effect of β-elemene on the pathogenesis of CNS inflammation, we examined the effect of β-elemene Cytokine Forward Reverse IL-17 Il-6 TGF-β IL-23p19 RORγt Foxp3 GAPDH GTTCTGCTGCCTGTCACATCATC CCACTTCACAAGTCGGAGGCTTA GTGTGGAGCAACATGTGGAACTCTA ACATGCACCAGCGGGACATA CAGTATGTGGTGGAGTTTGCCAAG CAGCTCTGCTGGCGAAAGTG TGTGTCCGTCGTGGATCTGA CGGGCAATTACTATCAGTTCCTGTC GCAAGTGCATCATCGTTGTTCATAC TTGGTTCAGCCACTGCCGTA CCTTGTGGGTCACAACCATCTTC TGTAGGCCCTGCACATTCTGAC TCGTCTGAAGGCAGAGTCAGGA CCTGCTTCACCACCTTCTTGA 34 J Mol Neurosci (2011) 44:31–40 treatment with β-elemene decreased obviously inflammatory cells and axonal loss in spinal cord. β-elemene Inhibits Ag-specific Lymphocytes Responses and IL-17 mRNA Expression Fig. 1 Effect of β-elemene on progression of EAE in C57BL/6 mice. Clinical assessment of EAE was performed daily and mice were scored for disease according to the same criteria. The results are expressed as the mean daily clinical score of each experimental group. β-elemene therapy (20 mg/kg) delayed onset of clinical neurologic signs in animals and reduced the magnitude of clinical deficit in β-elemene-treated mice compared to sham-treated mice on EAE in vivo. Mice from sham-treated and 20 mg/kg β-elemene-treated groups were sacrificed on days 15 (after the onset of EAE). Spinal cord sections from mice were stained with hematoxylin and eosin and Bielschowsky silver impregnation to assess tissue inflammation and axonal injury. As shown in Fig. 2, the sham-treated mice showed profound inflammatory cells infiltration and reduced density of apparently normal axons in spinal cord. Conversely, Fig. 2 β-elemene inhibited the inflammatory cells infiltration and improved axonal loss in the spinal cord of the EAE mice. H & E and Bielshowsky staining demonstrated areas of inflammation and loss of axon in the sham-treated mice (a, c). In contrast, β-elemene-treated mice show very mild inflammation with axon integrity (b, d). a, b c, d: original magnification ×400 To define the mechanisms Ag-specific lymphocytes or Ag-nonspecific lymphocytes responsible for EAE, DLN cells, isolated from EAE mice on days 11, 15 after immunization with MOG35–55 peptide were cultured for 72 h with or without specific Ag-MOG35–55 peptide. Local DLN cells activated with specific Ag-peptide resulted in a robust proliferation response, whereas DLN cells activated without specific Ag-peptide were relatively weakly in proliferation (Fig. 3a). To define the mechanisms involved in the inhibition of CNS inflammation by β-elemene, we have chosen to further examine the effect of β-elemene on Ag-specific DLN cells responses in vitro. As shown in Fig. 3b, MOG35–55-immunized DLN cells displayed a strong proliferative response to specific Ag in vitro and addition with β-elemene resulted in a dose-dependent decrease in proliferation. To address the proliferating DLN cells, we have further assayed the mRNA expression of IL-17 from specific Ag-induced DLN cells by real-time RT-PCR at different time points after MOG35–55 immunization (Fig. 4a). Data showed that higher level mRNA of the J Mol Neurosci (2011) 44:31–40 35 Fig. 3 β-elemene inhibited specific lymphocytes proliferation in vitro. a MOG35–55 peptide-immunized-DLN cells, isolated on days 11,15 after immunization, were re-stimulated with 10 μg/ml specific Ag-MOG35– 55 peptide or without for 72 h. Ag-specific lymphocytes proliferation was confirmed by MTT assay. b MOG35–55 peptide-immunized-DLN cells were restimulated in vitro with same Ag peptide in the presence of different concentration of β-elemene. Ag-specific lymphocytes proliferation was dose-dependently inhibited by β-elemene. Data are representative of three independent experiments and indicate the mean ratio of triplicate results in each experiment IL-17 was expressed in the DLN cells cultured with 10 μg/ml MOG35–55 peptide only for 72 h when compared with treatment of cells with same peptide in the presence of 10 μM β-elemenen during the early stage of EAE. Further- more, we also checked the IL-17 mRNA expression levels in the spinal cord tissue both 20 mg/kg β-elemene-treated and sham-treated mice by real-time RT-PCR at different time points (Fig. 5a). Results indicated that the mRNA expression of Fig. 4 β-elemene suppressed IL-17 and its differentiation related genes mRNA expression in vitro. MOG35–55 peptideimmunized DLN cells obtained at two different time points (day 11, 15) were re-stimulated in the presence of 10 μg/ml same peptide with 0 or 10 μM β-elemene for 72 h. The mRNA levels of cytokines were quantified by real time PCR. The expression levels of IL-17, IL-6, IL-23, RORγt except TGF-β were suppressed in specific Ag-induced DLN cells with 10 μM β-elemene (a–e). Data are representative of three independent experiments and indicate the mean ratio of triplicate results in each experiment. Data represents mean±SD for six individual mice. Single asterisk, p<0.05; double asterisk, p<0.01 36 J Mol Neurosci (2011) 44:31–40 Fig. 5 β-elemene suppressed IL-17 and its differentiation related genes mRNA expression in vivo. Mice from β-elemenetreated (20 mg/kg) and shamtreated control groups were sacrificed on days 11, 15 after immunized with MOG35–55 peptide. The mRNA levels of cytokines in the spinal cord were quantified by real time PCR. The expression levels of IL-17, IL-6, IL-23, RORγt except TGF-β was significantly reduced in β-elemene-treated mice compared with the shamtreated mice (a–e). Data represents mean±SD for six individual mice. Single asterisk, p<0.05; double asterisk, p<0.01 IL-17 was reduced in spinal cord in β-elemene-treated mice, whereas higher expression of IL-17 was seen in spinal cord in sham-treated mice similar during the early stage of EAE. These results suggested that β-elemene inhibits EAE by inhibiting Th17 cell proliferation in spinal cord of the EAE mice. β-elemene Suppresses Gene Expressions Associated with the Differentiation of Th17 Cells The development of clinical EAE has been associated with the production of various inflammatory cytokines and signal transducers associated with the Th17 phenotype, including TGF-β, IL-6, IL-23, RORγt. To determine the mechanisms involved in β-elemene regulation of Agspecific Th17 responses in EAE, the mRNA levels of the products have been identified and quantified by real time PCR in vivo and in vitro. As data have shown that expression of IL-6, IL-23, RORγt were significantly reduced in the spinal cord in 20 mg/kg β-elemene-treated mice compare with the sham-treated mice at different time points after MOG35–55-immunization (Fig. 5b–e). Similar results have been presented between specific Ag MOG35– 55 peptide only induced local DLN cells and treatment of cells with 10 μM β-elemene (Fig. 4b–e). The higher levels of IL-6, IL-23, RORγt were expressed in specific Ag only induced local DLN cells compare with the treatment of cells with 10 μM β-elemene. The expression of TGF-β neither in vitro nor in vivo has significance between shamtreated and β-elemene-treated group. These results suggested that β-elemene can suppress the expression of inflammatory cytokines and signal transducers associated with differentiation and production of Ag-specificTh17 cells. β-elemene Induces Treg Cell Expansion The effect of β-elemene on expression of mRNA for Foxp3 in both the periphery and the inflamed spinal cord from EAE-induced C57BL/6 mice was determined by real time J Mol Neurosci (2011) 44:31–40 37 EAE is a commonly studied animal model of central nervous system inflammatory demyelinating disease, and it is believed that autoreactive T cells and proinflammatory cytokines play key roles in the early inductive phase and later paralytic stages of EAE pathogenesis (Steinman. 2001; Goverman et al. 2005; Huseby et al. 2001). β-elemene, an extract from the ginger plant R. zedoariae, is a promising novel anticancer drug (Zhou et al. 2001; Zhou et al. 2003; Yang et al. 1999). Although β-elemene has been dramatic effective for the treatment of human tumors, the mechanisms underlying its action are not understood. Especially it has been found to have the capacity to pass through the blood–brain barrier easily. Some studies have previously demonstrated that β-elemene therapy is of substantial clinical benefit in central nervous system diseases (Wei and Chuanzhen. 2008). In view of the pathological mechanisms of MS/EAE, which may be involved with the immune disorder similar to tumors in this study, we utilized in vitro and in vivo methods to examine the effects and mechanism of action of β-elemene on the pathogenesis of EAE. We found in vitro that exposure of enriched MOG35– 55-immunized DLN cells cultures to β-elemene significantly downregulated proliferative response to specific Ag. Most importantly, β-elemene treatment also resulted in a marked expansion in the Treg cell population and a corresponding reduction in Th17 cells and MOG-specific T cells within both peripheral lymphoid tissue and diseased EAE spinal cords. By modulating multiple functional immune cell types and their corresponding cytokines, β-elemene therapy transformed a significantly destabilized autoimmune process in EAE animals into one of relatively normal immune status. As a consequence, less severe neurologic deficits and less neuropathologic abnormalities were found in β-elemene-treated EAE animals compared to sham-treated controls. The pathogenesis of MS/EAE is a complex process. β-elemene appears to act at multiple steps to play a role in the inflammation of MS. In the present study, we used the EAE model of MS to test the potential therapeutic effects of β-elemene in the treatment of MS. Our data showed the treatment of EAE mice with β-elemene delayed the onset and reduced obviously clinical severity of EAE in vivo. The inhibition of clinical paralysis by β-elemene was associated with a decrease in inflammation and axonal loss in the CNS. More than two decades after coining of the Th1/Th2 paradigm (Coffman 2006; Mosmann and Coffman 1989), Th17, a new player has entered the field of autoimmunity. Recent researches showed defective Th17 differentiation do not develop EAE or EAU (Liu et al. 2008). Th17 cells, like the Ag-specific T cells, reside mainly in the peripheral lymphatics during the EAE induction phase after immunization. Later, when increased numbers of infiltrating inflammatory cells invade the CNS, Th17 cells, together with MOG-specific T effector cells, were easily detected in diseased spinal cords. β-elemene treatment abrogates the proliferation of peripheral Th17 cells during the early induction phase as well as at onset stages of EAE, and Fig. 6 β-elemene treatment induced Treg cell marker Foxp3 mRNA expression in vitro and vivo. Mice from β-elemene-treated (20 mg/kg) and sham-treated mice were sacrificed on days 11, 15 after immunized with MOG35–55 peptide. Meantime, MOG35–55 peptide-immunized DLN cells obtained at the same time points (day 11, 15) were re-stimulated in the presence of 10 μg/ml same peptide with 0 or 10 μM β-elemene for 72 h. The mRNA levels of Foxp3 in the spinal cord and DLN cells were quantified by real time PCR. β-elemene therapy induced a substantial increase in Foxp3 mRNA expression in EAE spinal cords (a) and DLN (b). The β-elemene induced Foxp3 mRNA expression in the CNS and peripheral lymphatic system became even more evident in onset stages of the disease and correlated with less severe neurologic deficit. Data represents mean±SD for six individual mice. Single asterisk, p<0.05; double asterisk, p<0.01 PCR similarly at different time points after MOG35–55immunization. We observed that sustained reductions in Foxp3 mRNA expression during the early EAE induction period in sham-treated EAE animals compared to controls (p< 0.01). β-elemene treatment induced a significant expansion of Foxp3 mRNA expression within the spinal cord that could be detected at a very early stage (Fig. 6a). The increased Foxp3 mRNA expression was even more prominent 15 days after MOG35–55-immunization, (p<0.001). Similar results had been presented between specific Ag MOG35–55 peptide only induced local DLN cells and treatment of cells with 10 μM β-elemene (Fig. 6b). Discussion 38 β-elemene treatment inhibits Th17 cell formation within the spinal cord during the acute phases of EAE. These data indicated that β-elemene ameliorate EAE by inhibiting the Ag-specific Th17 cell proliferation. However, it is still unknown by what mechanism βelemene suppresses expansion of the Th17 cell subset in EAE animals, since pro-inflammatory cytokines determine T cell differentiation and the final outcome of MS/EAE. To further address the mechanisms involved in the inhibition of Th17 cell by β-elemene, we examined the crucial cytokines involved in differentiation and production of Th17 cells. studies have shown Th17 cells differentiation is independent of IL-23 and is induced by TGF-β plus IL-6 (Yang et al. 2008; Nurieva et al. 2007), but maintenances of pro-inflammatory Th17 cells requires the presence of IL-23 (Mangan et al. 2006; Langrish et al. 2005; Rachitskaya et al. 2008). Both in vitro and in vivo differentiation of Th17 cell lineage is required in the activation of STAT3, which is activated by IL-6, and then up-regulation of RORγt (Zhou et al. 2007; Yang et al. 2008a) that is served as the master switch of differentiation of Th17 cell (Wei et al. 2007; Ichiyama et al. 2008; Manel et al. 2008; Zhou et al. 2008). Our data discovered that β-elemene obviously inhibits RORγt, IL-6, IL-23 production in vivo and in vitro, suggesting that the inhibition of RORγt, IL-6, and IL-23 is a mechanism of regulation of EAE by β-elemene. Since IL-6 has been reported to promote differentiation of naïve CD4+ T cells into Th17 cells in vitro, the level of IL-6 secretion by inflammatory cells could be playing a crucial role for in vivo development and expansion of Th17 cells (Hirota et al. 2007). Our in vitro and vivo studies demonstrated significantly reduced IL-6 levels in β-elemenetreated mice, suggesting that down-regulation of Th17 by β-elemene could be a consequence of reduced IL-6 secretion, a crucial acute protein, and an initiator of Th17 cell differentiation, but not a result of the up- or downregulation of TGF-β. A less severe inflammatory environment in peripheral lymphoid tissue as well as in the CNS after β-elemene therapy may prevent significant Th17 migration into the CNS, thereby delaying disease onset and inducing less severe CNS pathology. In addition, the β-elemene mediated non-specific and specific immune modulating effects, on peripheral lymphoid tissues and CNS in β-elemene-treated EAE animals, likely contributes to their less severe clinical course. Antiinflammatory regulatory T cells have been found to be an important CD4 cell subpopulation for controlling the development of autoimmune disease (Fernandez-Martin et al. 2006; Sakaguchi 2005; Reddy et al. 2004; Viglietta et al. 2004). Adoptive transfer of Treg cells has been shown to be of therapeutic efficacy in several animal autoimmune disease models. Since Foxp3 is a marker for Treg cells (Kasper et al. 2007), our study, by examining J Mol Neurosci (2011) 44:31–40 Foxp3 mRNA expression, shows that β-elemene therapy in MOG-immunized mice is associated with rapid expansion of the Treg cell population in peripheral lymphoid tissue and in CNS, this β-elemene-induced expansion of Treg cells was sustained over 2 weeks. In fact, β-elemene-treated EAE animals that manifested delayed onset of clinical disease and less neurologic damage were found to have higher numbers of Treg cells within both DLNs and spinal cord. The β-elemeneinduced, higher Treg population may play an important role in reducing the peripheral immune response to MOG autoantigen, and may be the major source of Tregs migrating into CNS to minimize inflammation within CNS white matter. A recent study showed that anti-inflammatory vasoactive intestinal peptide also prevents neurologic progression in MOG-EAE by inducing Treg cell expansion in peripheral lymphoid tissue and the CNS (Yang et al. 2008). As reported, TGF-β directed the differentiation of Treg and proinflamatory Th17 cells, by facilitating IL-17 production early after exposure to IL-6 and in retarding further Th17 differentiation, but only higher TGF-β directed the differentiation of Treg (Yang et al. 2008b). RT-PCR analysis on TGF-β from DLN cells and spinal cord demonstrated that there was a rapid marked increase on EAE mice; meantime, a trend toward diminished RNA expression of TGF-β cytokines by β-elemene treatment had been seen, but it did not reach a statistical significance. So as reported, the differentiation of a CD4+ effector cell to either a Th17 inflammatory cell or a Foxp3 regulatory cell is dependent upon the cytokine environment as well as other intrinsically expressed molecules (Bettelli. 2007). β-elemene may have a direct effect on disease progression by either mediating inhibition of the Th17 pathway, which would reduce proinflammatory mediators and ameliorate disease activity, or a direct enhancement only at higher level of TGF-β and resulting a corresponding increase in Treg function that may indirectly reduce IL-17expression and polarization. In conclusion, we investigated the mechanism behind the neuroprotective effects of β-elemene in classic autoimmune EAE animals and found that β-elemene has striking pleiotropic immunomodulatory effects on the peripheral lymphatic tissues as well as on the CNS target organ. β-elemene appears to restrain the overwhelming autoimmune response in MOG-immunized EAE animals and restores it to a more balanced immune status. Our studies suggest that multiple alterations including a reduction in total autoreactive T cells, as well as an alteration in the balance between Th17 cells and expanded Treg cell populations within peripheral lymphoid tissue and CNS all contribute to β-elemenemediated neuroprotection. 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