Green tea EGCG, T cells, and T cell-mediated autoimmune diseases

Molecular Aspects of Medicine 33 (2012) 107–118

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Review

Green tea EGCG, T cells, and T cell-mediated autoimmune diseases q Dayong Wu a,⇑, Junpeng Wang a, Munkyong Pae a,1, Simin Nikbin Meydani a,b a b

Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, United States Department of Pathology, Sackler Graduate School of Biochemical Sciences, Tufts University, Boston, MA 02111, United States

a r t i c l e

i n f o

Article history: Available online 14 October 2011 Keywords: T cells Autoimmunity Multiple sclerosis Rheumatoid arthritis Green tea EGCG

a b s t r a c t One of the proposed health benefits of consuming green tea is its protective effect on autoimmune diseases. Research on the immunopathogenesis of autoimmune diseases has made significant progression in the past few years and several key concepts have been revised. T cells, particularly CD4+ T helper (Th) cells, play a key role in mediating many aspects of autoimmune diseases. Upon antigenic stimulation, naïve CD4+ T cells proliferate and differentiate into different effector subsets. Th1 and Th17 cells are the pro-inflammatory subsets of Th cells responsible for inducing autoimmunity whereas regulatory T cells (Treg) have an antagonistic effect. Green tea and its active ingredient, epigallocatechin-3gallate (EGCG), have been shown to improve symptoms and reduce the pathology in some animal models of autoimmune diseases. Whether or not EGCG’s effect is mediated through its impact on Th17 and Treg development has not been studied. We conducted a series of studies to investigate EGCG’s effect on CD4+ T cell proliferation and differentiation as well as its impact on the development of autoimmune disease. We first observed that EGCG inhibited CD4+ T cell expansion in response to either polyclonal or antigen specific stimulation. We then determined how EGCG affects naïve CD4+ T cell differentiation and found that it impeded Th1 and Th17 differentiation and prevented IL-6-induced inhibition on Treg development. We further demonstrated that EGCG inhibited Th1 and Th17 differentiation by downregulating their corresponding transcription factors (STAT1 and T-bet for Th1, and STAT3 and RORct for Th17). These effects provide further explanation for previous findings that administration of EGCG by gavage to experimental autoimmune encephalomyelitis (EAE) mice, an animal model for human multiple sclerosis (MS), reduced the clinical symptoms, brain pathology, and proliferation and TNF-a production of encephalitogenic T cells. Upon further investigating the working mechanisms for EGCG’s protective effect in the EAE model, we showed that dietary EGCG dose-dependently attenuated the disease’s severity. This protective effect of EGCG is associated with the suppressed proliferation of autoreactive T cells, reduced production of pro-inflammatory cytokines, decreased Th1 and Th17, and increased Treg populations in lymphoid tissues and central nervous system. EGCG-induced shifts in CD4+ T cell subsets in EAE mice are accompanied by the corresponding changes in their regulator molecules. Recent studies have also highlighted the critical role of Th17/Treg balance in the pathogenesis of rheumatoid arthritis (RA). EGCG has been shown to be anti-inflammatory and protective in several studies using animal models of inflammatory arthritis, but research, at the best, only to start looking into the mechanisms with a focus on T cells. Overall, future research should fully incorporate the current progress in autoimmunity into the study design to expand the

q Supported by USDA National Institute of Food and Agriculture grant 2010-65200-20360 (to D.W.) and USDA, Agriculture Research Service contract #58-1950-7-707 (to S.N.M.). ⇑ Corresponding author. Address: Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, United States. Tel.: +1 617 556 3368; fax: +1 617 556 3224. E-mail address: [email protected] (D. Wu). 1 Current address: Cellular and Molecular Physiology Section, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02115, United States.

0098-2997/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2011.10.001

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power of evaluating EGCG’s efficacy in treating autoimmune diseases. Data from human studies are essentially absent and thus are urgently needed. Ó 2011 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EGCG and T cell-mediated immune functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EGCG and T cell-mediated autoimmune diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. CD4+ T subsets: differentiation, function, and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Effect of EGCG on CD4+ T cell differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. T cells and autoimmune diseases: the case of experimental autoimmune encephalomyelitis (EAE) . . . . . . . . . . . . . . . . 3.4. Effect of EGCG on EAE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Role of T cells in pathogenesis of RA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Effect of EGCG on RA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Increasing evidence supports the beneficial effects of consuming green tea in several aspects of health and disease. The catechins present in high content in green tea are thought to be the major components responsible for green tea’s biological functions. These catechins include epicatechin, epigallocatechin, epicatechin-3-gallate, and epigallocatechin-3-gallate (EGCG). Among them, EGCG is the most biologically active and also the most abundant, accounting for 50–80% of the total catechins in green tea. Thus, EGCG is used in the majority of green tea studies. This review highlights our knowledge and current progress in the field of research on green tea and EGCG’s effect on immune cells, particularly T cells and the application of their immunomodulating properties in preventing and alleviating T cell-mediated autoimmune diseases.

2. EGCG and T cell-mediated immune functions The immune-modulating effect of EGCG proposed by the early studies was largely based on the altered lymphocyte proliferation after in vitro supplementation with tea extracts or catechins. For example, Hu and colleagues showed that EGCG at dose of 24 lg/ml (55 lM) and higher inhibited both B and T cell proliferation, but a greater effect was observed in T cells (Hu et al., 1992). Similarly, other researchers reported that in vitro supplementation with green tea extracts inhibited lymphocyte proliferation in response to T cell mitogen concanavalin A (Con A) (Wilasrusmee et al., 2002) and allogeneic stimulator cells (Kim et al., 2007; Wilasrusmee et al., 2002). In support of these findings in murine lymphocytes, a study showed that green tea extracts also inhibited production of neopterin, a marker for activation of cellular immunity, by human peripheral blood mononuclear cells (PBMC) when stimulated with Con A (Zvetkova et al., 2001). While the data obtained from these in vitro studies have provided useful information for defining the nature of the immune-modulating activity of EGCG, in the majority of these studies, however, EGCG was administered at doses far exceeding physiologically relevant levels, i.e., achievable via oral intake, which makes the extrapolation of the obtained results for potential, clinical application questionable. Some recent studies have been conducted using the in vivo models, or the in vitro design that included using physiologically relevant concentrations of EGCG (<10 lM). For example, in an animal study, oral administration of 0.3% green tea polyphenol solution containing 50–60% of EGCG was effective at suppressing transplant-reactive T cell immunity as demonstrated by prolonged skin graft survival and decreased frequency of donor reactive interferon (IFN)-c-producing T cells during graft rejection (Bayer et al., 2004). Recently, we have shown that in vitro supplementation with EGCG (2.5– 10 lM) dose-dependently inhibits Con A-induced splenocyte proliferation, T cell division, and cell cycle progression of T cells (Wu et al., 2009a). This effect of EGCG is not due to apoptosis, and it is unrelated to the previously reported EGCG-induced H2O2 generation as no difference in H2O2 generation was observed with the addition of EGCG at this range of concentrations and furthermore, catalase did not prevent EGCG-induced inhibition (Wu et al., 2009a). Since suppressed T cell proliferation by EGCG was observed under conditions in which T cell response was induced and measured in total splenocytes, lymph node cells, or PBMC, all of which are present as a mixture of multiple cell types, we need to further determine whether this effect of EGCG represents its direct effect on T cells. Furthermore, the in vitro results need to be validated under in vivo conditions. In a subsequent study, we investigated: (1) the efficacy of in vivo supplementation with EGCG, (2) the direct effect of EGCG (2.5–15 lM) on T cell function using purified T cells to exclude any indirect effect through impacting accessory cells, and (3) the underlying mechanisms of EGCG-induced suppression on T cells (Pae et al., 2010a). The results showed that 0.3%

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EGCG for 6 wk was effective in reducing ex vivo T cell proliferation when stimulated with either Con A or anti-CD3/CD28. These results confirm that dietary EGCG supplementation could achieve in vivo levels that would be adequate to suppress T cells (Pae et al., 2010a). More definitive experiments with in vitro EGCG supplementation revealed that EGCG dose-dependently inhibited anti-CD3/CD28-stimulated cell division in T cells with a more prominent effect on CD4+ T cells than CD8+ T cells. Furthermore, in a model of antigen (Ag) (ovalbumin)-specific T cell proliferation in which purified T cells and antigenpresenting cells (APC) were incubated with or without EGCG (10 lM) separately, we demonstrated that EGCG markedly reduced T cell proliferation by affecting both T cells and APC; however, the direct effect of EGCG on T cells was predominant (Pae et al., 2010a). Another important feature of T cells is their ability to generate cytokines that support and regulate T cell expansion, differentiation, and effector function, and to regulate the functions of other immune cells, particularly MU and B cells. Studies have provided evidence suggesting that EGCG may modulate T cell-derived cytokines. For example, the in vitro EGCG treatment was shown to inhibit IL-2 production in response to allogenic stimulator cells (Kim et al., 2007), IL-2, TNF-a, and IFN-c production in Staphylococcus enterotoxin B-stimulated PBMC (Watson et al., 2005), and IFN-c production in Con A-stimulated splenocytes (Wu et al., 2009a), or anti-CD3/CD28-stimulated purified T cells or CD4+ T cells (our unpublished data). In contrast, other studies have reported that in vitro EGCG supplementation upregulates mRNA levels of Th1 cytokines (IL-2 and IFN-c) and Th2 cytokines (IL-5 and IL-13) in Jurkat cells (Wu et al., 2009b) and increases IL-2 production in response to PMA/ phytohaemagglutinin in human PBMC (Lyu and Park, 2005). We further investigated the effect of EGCG on IL-2 production and IL-2/IL-2R signaling in total T cells or naïve CD4+ T cells isolated from mice and found that IL-2 levels were unchanged in the cultures treated with EGCG for 24 h or shorter time but were elevated in the 48 h culture in a dose-dependent manner ((Pae et al., 2010a) and unpublished data). Combined with the unchanged IL-2 mRNA expression and decreased IL-2Ra expression in EGCG-treated T cells, we speculated that the higher levels of IL-2 may be associated with reduced IL-2 internalization and utilization due to inhibited IL-2R expression by EGCG (Pae et al., 2010a). We further found that expression of two other IL-2R subunits, b and c chains, were also inhibited by EGCG, and this reduced IL-2R expression was associated with impaired IL-2R downstream signaling as determined by reduced STAT5 phosphorylation (our unpublished data). T cell activation signaling through T cell receptor (TCR) is needed for IL-2 production and subsequent T cell expansion. Not surprisingly, several studies have indicated that EGCG may interfere with the early signaling events of T cell activation, including Zap70 kinase in Jurkat cells and thus, downstream signaling events such as linker for the activation of T cells, phospholipase Cc1, extracellular signaling-regulated kinase, and MAP kinase activity as well as AP-1 activation (Shim et al., 2008). Similarly, in vitro EGCG supplementation was shown to induce suppressor of cytokine signaling gene 1 expression in human PBMC, which may be mediated by EGCG-induced activation of STAT5, as suggested by authors based on their experiments using mouse splenic monocytes deficient in different STATs (Ripley et al., 2010). Since TCR activation through two sequential signals (TCR signal and IL-2/IL-2R signal) drives T cells to enter the cell cycle leading to their rapid proliferation and differentiation into effector cells, the inhibitory effect of EGCG on T cell signaling suggests that EGCG may interrupt the cell cycle progress. Indeed, we found that in vitro EGCG supplementation dose-dependently retarded the cell cycle progression of T cells as measured in either splenocytes (Wu et al., 2009a) or purified T cells (Pae et al., 2010a). Traverse of G0/ G1 and entry into S phase are known to be controlled by ordered activation of cyclin-dependent kinases (CDKs) (Sherr, 1996). CDK activity is up-regulated by cyclins and down-regulated by CDK inhibitors. p27kip1 is a powerful CDK inhibitor and a primary modulator of proliferative status. It functions to induce and maintain the cells in a quiescent state and its overexpression arrests cells in G1 phase (Toyoshima and Hunter, 1994). IL-2 plays a critical role in inducing cell cycle progression of T cells through down-regulation of p27kip1 (Firpo et al., 1994; Nourse et al., 1994) while EGCG has been shown to induce cell cycle arrest through increasing levels of p27kip1 in human NK and T cell lines (Lu et al., 2006; Nam et al., 2001). Therefore, EGCG may induce cell cycle arrest by affecting cell cycle related proteins through down-regulation of IL-2/IL-2R signaling or other mechanisms yet to be elucidated. Considering all these results, it appears that EGCG is in general suppressive toward T cell functions. This property of EGCG thus opens the door for its potential application in T cell-mediated autoimmune diseases.

3. EGCG and T cell-mediated autoimmune diseases Autoimmunity is defined as a host’s dysregulated immune system that attacks its own tissues. A variety of autoimmune diseases are the manifestations of autoimmune disorders in different systems, organs, and tissues. Etiology of autoimmune diseases is not clear. For some of them, however, T cells clearly play a critical role in their pathogenesis. These diseases are thus referred to as T cell-mediated autoimmune diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (IBD), Sjogren’s syndrome, systemic lupus erythematosus, and psoriasis. There is no cure for most autoimmune diseases, and commonly used therapies have limited efficacy and often various side effects. In the development of autoimmune diseases, genetic predisposition is thought to be a key risk factor while environmental factors are also shown to play a role. Nutrition represents a modifiable environmental factor, which could potentially have a positive impact on autoimmune diseases. Results from a limited number of animal studies suggest that green tea and its active ingredient, EGCG, might be effective in improving the symptoms and pathological conditions of autoimmune diseases. However, the information is still very limited regarding this aspect. A great majority of the work in this field has been done using various animal models but little is known about clinical application in humans. Studies also take a cell-based,

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experimental approach, primarily as an initial screening step to identify potential candidates or for further mechanistic investigation following positive findings in the in vivo studies. The immunopathogenesis of T cell-mediated autoimmune diseases is best characterized in MS and its murine model experimental autoimmune encephalomyelitis (EAE). Our own work has focused on the effect of EGCG consumption on EAE as well as the underlying mechanisms. This review will thus devote much attention to EAE as a representative of autoimmune disease models to exemplify the role of T cells in the development of autoimmune diseases, the cellular and molecular regulation network involved, and the possible working mechanisms underlying the action of EGCG. Additionally, we will also discuss RA, another autoimmune disease, because RA is of the most common autoimmune diseases, and T cell involvement in its pathogenesis has clearly been identified. Further, the effect of green tea and EGCG has been studied in RA more than in any other autoimmune disease. Results from animal studies suggest a protective effect for green tea and EGCG. Inflammation is a central event in autoimmune diseases, either as a primary innate response or as a subsequent reaction to T cell activation. EGCG is widely viewed as an anti-inflammatory agent based on its inhibitory effect on inflammatory mediators as well as their regulators, which has, in fact, been the focus of a majority of studies reported thus far. However, the intention of this review is to discuss only those studies that demonstrate a T cell involvement. 3.1. CD4+ T subsets: differentiation, function, and regulation CD4+ T cells play a central role in the body’s defense system due to their great impact on the function of B cells and cells in the innate immune system. On the other hand, CD4+ T cells are also well-recognized as key drivers in the development of major T cell-mediated autoimmune diseases. As illustrated in Table 1 and Fig. 1, based on their unique cytokine profile and main regulatory function, CD4+ T cells can be further classified into at least four functionally distinct subsets, namely Th1, Th2, Th17, and regulatory T (Treg) cells (Abbas et al., 1996; Bettelli et al., 2006; Mangan et al., 2006; Mosmann et al., 1986; Mosmann and Coffman, 1989; Street and Mosmann, 1991; Veldhoen et al., 2006). Differentiation of naïve CD4+ T cells into these different effector cells is initiated by TCR engagement and co-stimulation in the presence of specific cytokines produced by the innate immune system upon encountering particular pathogens. Development of Th1 is promoted

Table 1 Features of CD4+ T cell subsets. CD4+ T cell subset

Priming cytokine

Transcription factor

Effector cytokine

Function

Role in immunopathology

Th1 Th2 Th17

IL-12 IL-4 TGF-b/IL-6

T-bet, STAT1, STAT4 GATA3, GATA6 RORct, STAT3

Intracellular pathogens Extracellular pathogens Extracellular bacteria and fungi

Treg

TGF-b

Foxp3

IFN-c, IL-2, TNFa IL-4, IL-5, IL-13 IL-17A/F, IL-21, IL-22, IL-26 TGF-b, IL-10

Autoimmunity Allergy, asthma Autoimmune inflammation Immune tolerance

Immune regulation

Fig. 1. CD4+ T cell differentiation and its regulation network. Activated naïve CD4+ T cells can differentiate into different subsets under appropriate cytokine milieu. Each subset of CD4+ T cells is characterized by their specific cytokine profile. The differentiation process is controlled by the corresponding regulation network mainly composed of involved signal transducers and downstream transcription factors. Interaction exists among different CD4+ T cell subsets at their development stage as well as in their function to regulate autoimmune response.

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by IL-12 and IL-18, Th2 by IL-4, Th17 by TGF-b plus IL-6 or IL-21, and Treg by TGF-b. Hallmark cytokines for Th1, Th2, and Th17 are IFN-c, IL-4, and IL-17, respectively, and Treg cells are often identified as CD4+CD25+Foxp3+ cells. Th1 cells are mainly responsible for immunity to intracellular pathogens while Th2 cells target extracellular pathogens. The recently identified Th17 cells are believed to play a critical role in the clearance of extracellular bacteria and fungi, particularly those not well defended by Th1 or Th2 immunity (Dong, 2006; Weaver et al., 2006, 2007). Treg cells function mainly to maintain selftolerance and regulate immune responses (Sakaguchi, 2004). Abnormal activation of Th1 and Th17 cells and reduced function of Treg cells are associated with organ-specific autoimmune diseases while Th2 cells contribute primarily to allergy and asthma. As mentioned above, naïve CD4+ T cells differentiate into distinct subsets. Th1 polarization is mainly driven by IL-12 and synergized by IL-18 (Robinson et al., 1997; Robinson and O’Garra, 2002), and the resulting increase in production of its signature cytokine IFN-c gives further positive feedback. IL-4 is both a signature cytokine for and a driving factor in Th2 cell differentiation, which requires the involvement of IL-2 (Cote-Sierra et al., 2004; Le Gros et al., 1990). Th17 differentiation is initiated by TGF-b and IL-6, and it is amplified by IL-21. Th17 cell proliferation and stabilization are supported by IL-23 (Zhu and Paul, 2008). Treg can be both constitutively present and induced under appropriate conditions, and thus it is further distinguished as natural (n)Treg and induced (i)Treg, respectively. TGF-b is important for nTreg development (Liu et al., 2008), and it plays a key role in iTreg differentiation (Chen et al., 2003). Given the duplicity of TGF-b in driving naïve Th cells to both Th17 and Treg, the presence of IL-6 appears to be the decisive factor in differentiation of these two subsets, i.e., TGF-b for Treg and TGF-b plus IL-6 for Th17 (Bettelli et al., 2007). In addition to transcription factors common to T cell activation, some transcription factors have a specific effect in determining the fate of Th cells (see Fig. 1). Signal transducers and activators of transcription (STAT) can regulate Th cell differentiation through the induction of these lineage-specific transcription factors. T-box protein expressed in T cells (T-bet) is the master regulator for Th1 differentiation (Szabo et al., 2000). STAT1 is a transducer for IFN-c-induced T-bet activation (Lighvani et al., 2001), and STAT4 is a transducer for IL-12 to amplify Th1 response (Thierfelder et al., 1996). GATA-3 is the master regulator for Th2 differentiation, and STAT6 is the key transducer for IL-4-induced GATA-3 activation (Kurata et al., 1999; Zhu et al., 2001). Retinoic acid receptor-related orphan receptor (ROR)ct is the master regulator for Th17 differentiation, and STAT3 is the key transducer that mediates action of IL-6, IL-21, and IL-23 (Nurieva et al., 2007; Yang et al., 2007). Foxp3 is a master transcription factor found in Treg cells, and thus it is used as the signature marker for Treg differentiation. Cytokine TGF-b is key factor for Treg cell development and function. Several transducer molecules have been shown to mediate the effect of TGF-b on Foxp3 expression (Bommireddy and Doetschman, 2007; Rubtsov and Rudensky, 2007; Zhou et al., 2008). SMAD proteins are intracellular TGF-b signal transducers, and SMAD3 mediates most actions of TGF-b (Derynck et al., 1998; Feng and Derynck, 2005). TGF-b binds to its receptors (TGFbRII and I), which in turn phosphorylate SMAD3. Phosphorylated SMAD3 forms a complex with SMAD4 (co-mediator SMAD) and translocates into the nucleus, where they cooperate with sequence-specific transcription factors to regulate gene expression. Inhibitory SMAD (SMAD7) inhibits TGF-b signaling by inducing TGFbR degradation (Di Guglielmo et al., 2003), and SMAD7 overexpression in T cells prohibits induction of Foxp3 (Dominitzki et al., 2007). There is a cross inhibition among different CD4+ T cell lineages in their functions and during their differentiation. This cross regulation is present at multiple layers, i.e., cytokines, transcription regulators, and master genes (Bettelli et al., 2006; Dominitzki et al., 2007; Weaver et al., 2007; Zhu and Paul, 2008). In particular, the decision of antigen-stimulated cells to differentiate into either Th17 or Treg cells mainly depends on the cytokine-regulated balance of RORct and Foxp3. It is widely accepted that Th1 and Th17 cells promote and Treg cells mitigate development of autoimmune diseases (see below for more details). Thus, development of naïve CD4+ T cells into these subsets has become a target for drug therapy in autoimmune diseases. Based on our results to be discussed later, green tea EGCG has the potential to decrease pathogenesis of autoimmune diseases by influencing CD4+ T cell differentiation. 3.2. Effect of EGCG on CD4+ T cell differentiation In our previous studies, we found that EGCG inhibited CD4+ T cell proliferation and IFN-c production but that it did not affect IL-4 and IL-10 production (Pae et al., 2010a). These results indicate that EGCG may specifically suppress Th1 response, which may explain its reported protective effect in some T cell-mediated autoimmune diseases. Given the increasingly emphasized importance of Th17 and Treg in the pathogenesis of autoimmune diseases and the absence of such information in the published studies showing a protective effect of EGCG in autoimmune diseases, we tested if EGCG could affect CD4+ T cell differentiation in a manner that would explain its effect on autoimmune diseases. All the results reviewed below are unpublished data, some of which were published in the conference abstract (Pae et al., 2010b). In our experiments, we activated purified naïve CD4+ T cells in the presence of 10 lM EGCG and under the appropriate conditions that drive Th1, Th17, and Treg polarizations, respectively. We found that induced differentiation of naïve CD4+ T cells into Th1 was reduced by half (60% vs. 31%) in the presence of EGCG. In accordance with this result, the expression of main signal transducer p-STAT1 and master regulator T-bet were also reduced by EGCG. Likewise, naïve CD4+ T cell differentiation into Th17 cells was reduced by about 60% (18% vs. 7%) in the presence of EGCG, which coincided with a reduction in their main signal transducer p-STAT3 and master regulator RORct. On the other hand, EGCG did not affect nTreg cell population but slightly increased the frequency of iTreg cells. More interestingly, addition of IL-6 reduced iTreg differentiation, and this reduction was greatly prevented by EGCG. Based on these results, we conclude that EGCG downregulates Th1 and Th17 differentiation and

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upregulates iTreg differentiation. These findings may help us understand the protective effect of EGCG observed in several T cell-mediated, autoimmune disease animal models. 3.3. T cells and autoimmune diseases: the case of experimental autoimmune encephalomyelitis (EAE) Altered T cell responses play a key role in autoimmune pathologies. Typical disease manifestation is exemplified in MS and RA. MS is a T cell-dependent, autoimmune inflammatory disease and EAE is a well-characterized rodent model for human MS. EAE is a highly reproducible and widely-used tool for studying T cell-mediated autoimmunity. EAE is typically induced by immunizing susceptible strains with a component of myelin such as basic myelin protein (BMP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG), or a peptide derived from one of these proteins such as PLP peptide139–151 and MOG peptide35–55, emulsified in an adjuvant to generate strong T cell response. Development of EAE is characterized as an ascending paralysis that begins from the tail, followed by manifesting in rear legs, and eventually progressing into forearms. MS pathogenesis involves a process in which autoreactive myelin-specific CD4+ T cells orchestrate their effector function toward myelin sheath in the central nervous system (CNS) resulting in tissue destruction and thus, the loss of function (Ando et al., 1989; Kuchroo et al., 2002; Merrill et al., 1992). Tissue inflammation precedes the clinical signs. EAE has long been considered the prototypic Th1-mediated autoimmune disorder, which is associated with increased production and activity of proinflammatory cytokines, predominantly IFN-c and other Th1 cytokines, by autoreactive T cells activated systemically or at specific sites. IFN-c was found to be present in EAE lesions during active disease and its levels were reduced during remission (Baker et al., 1991; Khoury et al., 1992). Th1 cells were able to transfer the disease while mice deficient in the transcription factors important for Th1 differentiation were resistant to EAE development (Dardalhon et al., 2008). In the past few years, rapid progress has been made in this field. In particular, compelling evidence has highlighted the importance of newly defined Th17 cells, which may play a primary role in this model as well as in some other autoimmune diseases. Some of the autoimmune responses formerly attributed to Th1 cells are now believed to be mediated by Th17 cells. Although the Th1 centered theory in EAE and other autoimmune diseases has been challenged, the importance of Th1 cells in EAE continues to be emphasized by the investigators. For example, myelin-reactive Th1 devoid of contaminating Th17 cells were shown to be highly pathogenic after transfer to normal recipient animals, and Th1 cells were highly capable of homing to the CNS and orchestrating inflammation and cellular recruitment, which facilitated the entry of Th17 cells (O’Connor et al., 2008). Additionally, some intervention agents ameliorate EAE symptoms and pathology through suppressing Th1 response (Brahmachari and Pahan, 2007; Miyake et al., 2006; Ren et al., 2008). A recent study showed that adoptive transfer of either Th1 or Th17 cells induced mouse EAE with similar clinical symptoms but distinct infiltration patterns of cells of innate immunity, i.e., macrophages were dominant with adoptive Th1 transfer and neutrophils were dominant with Th17 and their corresponding chemokine expression patterns (Kroenke et al., 2008). In a more recent study, adoptive transfer of either pathologic Th1 or Th17 cells could induce EAE, but with distinguishable characteristics in pathology and symptoms (Domingues et al., 2010). Altogether, regardless of their relative importance and distinct roles (to be further elucidated), it is likely that both Th1 and Th17 cells are main drivers in T cell-mediated autoimmune diseases. Another important type of T cells involved in regulation of T cell-mediated autoimmune diseases is Treg cells, wellsummarized in a recent review (Buckner, 2010). The severity and mortality of actively-induced EAE in non-transgenic mice were enhanced by treatment with anti-CD25 antibody following immunization while transfer of CD4+CD25+ Treg cells from naïve mice decreased the severity of active EAE (Zhang et al., 2004). Similarly, EAE-resistant B10.S mice had a higher proportion of PLP139–151-reactive CD4+CD25+ Treg cells, and depletion of CD4+CD25+ Treg cells facilitated the expansion of PLP139–151-reactive cells with production of Th1 cytokines in EAE-resistant B10.S mice (Reddy et al., 2004). Taken together, these results suggest that a dysregulated balance in T cell subpopulations Th1, Th17, and Treg contributes to initiation of EAE and thus, an agent that could impact this balance favorably might impede EAE development. After learning that EGCG suppresses CD4+ T cell expansion and differentiation toward downregulating pro-inflammatory Th1 and Th17 and upregulating anti-inflammatory Treg cells, we tested whether oral administration of EGCG could provide protection in an autoimmune disease animal model. 3.4. Effect of EGCG on EAE In the only published study on this topic, Aktas et al. (2004) found that administration of EGCG to EAE mice by gavage (300 lg/mouse twice daily from the day of immunization on) reduced clinical symptoms, brain inflammation, and neuronal damage. Draining lymph node (LN) cells from EGCG-treated mice had a lower proliferative response to the in vitro rechallenge with immunization antigen PLP139–151 and a lower production of TNF-a, but EGCG had no effect on IL-4 and IFN-c production. The authors reported no effect of EGCG on Treg cells, defined as CD4+CD25+ cells. However, given the fact that activated CD4+ T cells also express CD25 (IL-2Ra chain) and that the authors did not use the specific marker Foxp3 to identify Treg cells, it is likely that the change in activated T cells might have masked real changes in Treg population. This study demonstrated the efficacy of EGCG in improving EAE symptoms and provided evidence suggesting that the effect of EGCG was mediated through suppression of T cell activity. Published prior to the introduction of Th17/Treg concept into immunopathogenesis, the study understandably provided limited mechanistic information. Taking advantage of the recent developments in understanding pathogenesis of autoimmune immunity and using a multi-dose dietary supplementation design, we

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revisited this issue. In our recent study (Wang et al., 2011), we fed C57BL/6 mice EGCG (0%, 0.15%, 0.3%, and 0.6% in diet) for 30 d and then immunized them with specific antigen MOG35–55. EGCG dose-dependently attenuated the clinical symptoms and pathology (leukocyte infiltration and demyelination) in the CNS and inhibited Ag-specific T cell proliferation and delayed-type hypersensitivity skin response induced by autoantigen MOG35–55. We further showed that EGCG reduced production of pro-inflammatory cytokines IFN-c, IL-17, IL-6, IL-1b, IL-12, IL-23, and TNF-a, decreased Th1 and Th17, and increased Treg populations in LN, spleen, and CNS. Moreover, EGCG inhibited expression of T-bet & RORct, master regulators for Th1 and Th17 differentiation, respectively, which is accordance with the reduced populations of both subsets in EGCG-treated mice. The observed reduction in IL-6, IL-1b, IL-12, IL-23, and TNF-a production by EGCG treatment are also assumed to contribute to both the alleviation of EAE and the change in composition of CD4+ T cell subsets based on the information provided below. IL-6 is a decisive factor in switching differentiation toward Th17 cells away from Treg conversion (Bettelli et al., 2006, 2007; Korn et al., 2008). IL-1b and IL-6 (Acosta-Rodriguez et al., 2007) or IL-23 and IL-1b (Wilson et al., 2007) are essential for Th17 development; IL-12 is the master cytokine in driving Th1 differentiation (Hsieh et al., 1993; Seder et al., 1993); TNF-a, a Th1 cytokine, promotes EAE symptoms and pathology (Begolka et al., 1998; Issazadeh et al., 1995, 1998; Kuroda and Shimamoto, 1991). All these cytokines are upregulated in human MS and murine EAE (Acosta-Rodriguez et al., 2007; Imitola et al., 2005; Sospedra and Martin, 2005; Wilson et al., 2007). This study also found that EGCG treatment reduced plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1), and CD4+ T cells expressing C-C chemokine receptor 6 (CCR6) in EAE mice. These results may explain the reduced T cell infiltration into the CNS in EGCG-treated EAE mice because (1) adhesion molecules play an important role in the migration of immune cells into the CNS, (2) MS patients have increased serum levels of soluble ICAM-1 (Rieckmann et al., 1993; Sharief et al., 1993), (3) ICAM-1 is more important than other adhesion molecules in promoting pathological cell infiltration into the CNS and development of EAE (Bullard et al., 2007; Kebir et al., 2009), and (4). CCR6 is essential for the first wave of autoreactive Th17 migrating into the uninflamed CNS by interacting with its only ligand CCL20, constitutively expressed in the epithelial cells of the choroid plexus (Reboldi et al., 2009). CCL20 is probably not responsible for reduced cell infiltration by EGCG because CCL20 expression in the CNS was not changed by EGCG treatment. It appears that the effect of EGCG on EAE is more therapeutic rather than preventive in nature because starting treatment 30 d before immunization delayed onset and reduced severity of disease but did not reduce the disease incidence. Starting EGCG supplementation during the induction phase (day 7 post-immunization) and effector phase (day 12 post-immunization) also similarly attenuated the symptoms except that EGCG no longer delayed onset when the treatment started during the effector phase. These findings support the proposed beneficial effect of EGCG on autoimmune diseases and more importantly, they shed light on the key mechanisms underlying this effect of EGCG. 3.5. Role of T cells in pathogenesis of RA RA is a chronic inflammatory disease characterized by the activation of synovial tissue and its invasion into cartilage and bone resulting in the progressive destruction of joints. It is likely that RA is not simply a single disease but rather, it may represent a spectrum of distinct clinical entities (Cope, 2008; Imboden, 2009). Although RA is considered an autoimmune disease, as yet there is no consistent evidence defining the responsible autoantigens. RA is widely accepted as a T cellmediated autoimmune disorder given that increasing evidence supports the notion that T cells participate in the etiology and pathogenesis of RA. Large numbers of T cells in diffused or clustered form are present in synovial biopsies of RA patients (Duke et al., 1982). The animal models for studying RA, such as collagen-induced or adjuvant-induced arthritis, are clearly T cell-dependent. As discussed earlier in this review, recent progress in the research of CD4+ T cell subsets has greatly revised our understanding of the immunopathology of autoimmune disease development. Similar to our discussion of the current view on the pathogenesis of MS/EAE, the long standing Th1/Th2 paradigm has been challenged and the roles of Th17 and Treg in RA are now being increasingly emphasized as new evidence emerges. For example, IL-17 is found to be increased in blood and synovial fluid of RA patients (Chabaud et al., 1999; Hwang and Kim, 2005; Ziolkowska et al., 2000). IL-17, whose pathogenic role has been characterized in studies on animal models of inflammatory arthritis, is shown to be responsible for priming collagen-specific T cells and collagen-specific IgG2a production. Collagen-induced arthritis (CIA) was markedly suppressed in IL-17 deficient mice (Nakae et al., 2003), whereas blocking IL-17 with neutralizing Ab (Lubberts et al., 2004) or soluble IL-17 receptor (Bush et al., 2002) was able to alleviate the disease. IL-17 can also induce production of inflammatory cytokines (IL-1b, TNF-a, IL-6, IL-23), chemokines (CXC-1, -2, -5, -8, CCL2, and CCL20), matrix metalloproteinases, nitric oxide, and receptor activator of NF-jB (RANK)/RANK ligand, thus promoting inflammatory infiltration and cartilage damage (Sarkar and Fox, 2010). The importance of IL-17 in human RA is supported by some but not all studies, suggesting that IL-17 and Th17 cells are proinflammatory in at least a subset of RA patients (Imboden, 2009). As mentioned earlier, Treg cells maintain immune tolerance and play a critical role in preventing autoimmunity. In RA, however, results appear paradoxical as RA patients have larger numbers of Treg cells in their synovial fluid (Cao et al., 2006; Mottonen et al., 2005) or in some cases their peripheral blood (Han et al., 2008); some other studies also reported seeing more Treg cells in synovial fluid than in peripheral blood (Cao et al., 2004; van Amelsfort et al., 2004). Thus it appears that synovial inflammation persists despite the presence of Treg cells. One explanation for this phenomenon is that identification of Treg cells based on CD25 expression might have limitation. For example, it may have included the activated CD4+ T cells that express CD25 as IL-2Ra chain. Another explanation is that Treg cells are enriched in synovial fluid as a result of preferential trafficking to the joints because Treg cells express appropriate chemokine receptors (Oh et al., 2010). The lack of quantitative abnormality of Treg cells in RA patients has led people to the question whether these cells are functionally

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abnormal. Although still a topic for debate, some evidence supports a reduced functionality of Treg cells in RA patients. For example, Treg cells isolated from RA patients exhibit a compromised suppressor function (Ehrenstein et al., 2004; Valencia et al., 2006). Compared to healthy controls, RA Treg cells had similar (Ehrenstein et al., 2004) or reduced (Valencia et al., 2006) function in suppressing CD4+CD25 T cell proliferation, but they were unable to inhibit TNF-a and IFN-c production by CD4+CD25 T cells. Th17 and Treg cells are mutually antagonistic in autoimmunity and their differentiations are also reciprocally regulated. Further, studies have shown that Treg cells can be converted to Th17 cells (Radhakrishnan et al., 2008; Xu et al., 2007). IL-6 is a clearly defined, key factor in tipping the balance toward Th17 and away from Treg as discussed earlier. In addition to the observation that TNF-a downregulates Treg generation and reduces their suppressive functions (as mentioned above), TNF-a is both up and down stream to IL-17 regulation and can synergize with IL-17 to induce synovial inflammation and tissue destruction (Lubberts et al., 2005). Thus, IL-6 and TNF-a have become therapeutic targets for controlling RA and other autoimmune diseases (Feldmann et al., 2005; KEYSTONE and WARE, 2010; Nishimoto, 2006; Nishimoto et al., 2004). As shown in the previous studies, given its suppressive effect on inflammatory cytokines including IL-6 and TNF-a, green tea or EGCG is presumed to possess therapeutic value in RA. However, as can be seen below, only a limited number of studies on this subject exist, and they barely addressed the involvement of Th17/Treg cells. 3.6. Effect of EGCG on RA In line with the T cell-suppressive and anti-inflammatory properties of EGCG, animal studies using the experimental arthritis model suggest that green tea/EGCG may have a protective effect on RA. Haqqi et al. were the first to report that green tea polyphenols (the largest part being EGCG) given in drinking water to mice reduced the incidence of CIA together with a reduced expression of IFN-c, TNF-a, and cyclooxygenase(COX)-2, as well as total IgG and type II collagen-specific IgG (Ab) in arthritic joints (Haqqi et al., 1999). In a more recent study (Kim et al., 2008), Kim and colleagues fed Lewis rats GTP extract in drinking water (8 or 12 g/L) for 1–3 wk before injecting them with heat-killed Mycobacterium tuberculosis H37Ra to generate adjuvant-induced arthritis (AA). The authors found that the rats in both 8 and 12 g/L GTP diet groups for 2 or 3 wk had significantly reduced severity of arthritis, but this effect was greater in 8 g/L group than in 12 g/L group. This improvement in symptoms was accompanied by decreased serum levels of Ab against the disease-related antigen Bhsp65 as well as increased IL-10 production and decreased IL-17 mRNA expression in lymph node cells after in vitro challenge with Bhsp65. T cell proliferation and production of IFN-c and IL-4 in response to Bhsp65 were not different between GTP and control rats. The increased IL-10, decreased IL-17, and decreased anti-Bhsp65 Ab were suggested by the authors to be the main mechanisms for the protective effect of green tea polyphenols against AA. Given the very limited outcome measurements presented in this study, the mechanisms’ picture is far from complete. Since this study did not analyze the subsets of CD4+ T cells, it is difficult to assess if the effect of GTP is mediated through favorably altered CD4+ T cell differentiation. The best extrapolation is that the observed reduction in IL-17 mRNA to certain degree might have reflected a reduced number of Th17 cells, similarly but with even lesser certainty, the increased IL-10 might be partly attributed to an upregulated Treg population. The observation that GPT failed to suppress expansion of pathologic T cells is in contradiction to what has been seen in the case of EAE. The protective effect of green tea in arthritis is also studied in green tea’s effect on chemokines and their receptors, the factors that promote inflammation and joint destruction in RA development. Green tea extract administration (200 mg/kg/day p.o.) was shown to ameliorate AA in rats. The authors found a decrease in MCP-1/CCL2 and GROa/CXCL1 levels and an increase in CCR-1, -2, -5, and CXCR1 receptor expression in the joints of green tea extractadministered rats (Marotte et al., 2010). Altered production of inflammatory cytokine IL-6 may be another factor that mediated the protective effect of EGCG on RA. The pathologic role of IL-6 in RA development has been well documented (Nishimoto, 2006). RA patients have higher levels of IL-6 in their serum and synovial fluid, and this elevated IL-6 is correlated with disease activity (Houssiau et al., 1988). IL-6 has become a pharmacological target for controlling RA (Nishimoto, 2006; Nishimoto et al., 2004). As mentioned above, IL-6 drives Th17 differentiation and inhibits Treg differentiation and thus, modulating IL-6 activity is expected to affect autoimmune diseases such as MS and RA. Along this line, Ahmed et al. reported that EGCG administration (100 mg/kg, i.p. daily) to mice during the onset (day 7–17 after induction) of AA reduced IL-6 levels in serum and joints, with concomitant amelioration of rat AA. EGCG treatment was also found to decrease expression of membrane-bound gp130 protein (a component of IL-6 receptors for transmitting IL-6 signal) in the joint homogenates and to enhance synthesis of soluble gp130, an endogenous inhibitor of IL-6 signaling and trans-signaling. Furthermore, EGCG treatment inhibited IL-6/soluble IL-6 receptor-induced matrix metalloproteinase-2 activity in RA synovial fibroblasts and in joint homogenates via upregulated soluble gp130, which seems to indicate a mechanism for arthritis inhibited by EGCG treatment. 4. Concluding remarks Evidence supporting health benefits of green tea consumption in multiple body systems and a variety of diseases has been accumulating including its applications for prevention and/or alleviation of autoimmune diseases. Information along this line is almost exclusively obtained from studies that utilize several established animal models that share a varied degree of similarity to the represented human diseases in their pathogeneses and/or clinical manifestations. The results thus far

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are promising but not without controversy. In this regard, there is a definite paucity of mechanistic insight concerning the effect of EGCG. Although research in the immunopathology of autoimmune diseases has progressed rapidly recently and novel concepts continue to emerge publications on the role of EGCG in autoimmunity have not kept pace with the new progress in autoimmunity research. Based on the current views of the pathogenesis of autoimmune disease and together with what we have learned about the possible cellular and molecular targets for EGCG action, we are convinced that EGCG has strong therapeutic potential in treating autoimmune diseases. It is anticipated that new studies are under way and that we will soon see a significant update in the field. Finally, it is important to mention that there is an obvious lack of human study data. 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