Epigallocatechin-3-Gallate Ameliorates Experimental Autoimmune Encephalomyelitis by Altering Balance among CD4+ T-Cell Subsets
2011; Elsevier BV; Volume: 180; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.09.007
ISSN1525-2191
AutoresJunpeng Wang, Zhihong Ren, Yanmei Xu, Sheng Xiao, Simin Nikbin Meydani, Dayong Wu,
Tópico(s)T-cell and B-cell Immunology
ResumoThe green tea component epigallocatechin-3-gallate (EGCG) may be beneficial in autoimmune diseases; however, the underlying mechanisms are not well understood. In this study, we determined the effect of EGCG on the development of experimental autoimmune encephalomyelitis, an animal model for human multiple sclerosis, and the underlying mechanisms. Female C57BL/6 mice were fed EGCG (0%, 0.15%, 0.3%, and 0.6% in diet) for 30 days and then immunized with specific antigen myelin oligodendrocyte glycoprotein 35-55. EGCG dose dependently attenuated clinical symptoms and pathological features (leukocyte infiltration and demyelination) in the central nervous system and inhibited antigen-specific T-cell proliferation and delayed-type hypersensitivity skin response. We further showed that EGCG reduced production of interferon-γ, IL-17, IL-6, IL-1β, and tumor necrosis factor-α; decreased types 1 and 17 helper T cells (Th1 and Th17, respectively); and increased regulatory T-cell populations in lymph nodes, the spleen, and the central nervous system. Moreover, EGCG inhibited expression of transcription factors T-box expressed in T cells and retinoid-related orphan receptor–γt, the specific transcription factor for Th1 and Th17 differentiation, respectively; the plasma levels of intercellular adhesion molecule 1; and CCR6 expression in CD4+ T cells. These results indicate that EGCG may attenuate experimental autoimmune encephalomyelitis autoimmune response by inhibiting immune cell infiltration and modulating the balance among pro- and anti-autoimmune CD4+ T-cell subsets. Thus, we identified a novel mechanism that underlies EGCG's beneficial effect in autoimmune disease. The green tea component epigallocatechin-3-gallate (EGCG) may be beneficial in autoimmune diseases; however, the underlying mechanisms are not well understood. In this study, we determined the effect of EGCG on the development of experimental autoimmune encephalomyelitis, an animal model for human multiple sclerosis, and the underlying mechanisms. Female C57BL/6 mice were fed EGCG (0%, 0.15%, 0.3%, and 0.6% in diet) for 30 days and then immunized with specific antigen myelin oligodendrocyte glycoprotein 35-55. EGCG dose dependently attenuated clinical symptoms and pathological features (leukocyte infiltration and demyelination) in the central nervous system and inhibited antigen-specific T-cell proliferation and delayed-type hypersensitivity skin response. We further showed that EGCG reduced production of interferon-γ, IL-17, IL-6, IL-1β, and tumor necrosis factor-α; decreased types 1 and 17 helper T cells (Th1 and Th17, respectively); and increased regulatory T-cell populations in lymph nodes, the spleen, and the central nervous system. Moreover, EGCG inhibited expression of transcription factors T-box expressed in T cells and retinoid-related orphan receptor–γt, the specific transcription factor for Th1 and Th17 differentiation, respectively; the plasma levels of intercellular adhesion molecule 1; and CCR6 expression in CD4+ T cells. These results indicate that EGCG may attenuate experimental autoimmune encephalomyelitis autoimmune response by inhibiting immune cell infiltration and modulating the balance among pro- and anti-autoimmune CD4+ T-cell subsets. Thus, we identified a novel mechanism that underlies EGCG's beneficial effect in autoimmune disease. Multiple sclerosis (MS) is a T-cell–mediated inflammatory autoimmune disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model for human MS, owing to the similarities in clinical, immunological, and neuropathological features between MS and EAE.1Gold R. Hartung H.P. Toyka K.V. Animal models for autoimmune demyelinating disorders of the nervous system.Mol Med Today. 2000; 6: 88-91Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar The EAE model, which has been widely used to study MS, is also one of the most favored tools for the study of T-cell–mediated autoimmunity. The key pathogenesis of MS involves a process in which myelin-reactive CD4+ T cells enter the CNS and orchestrate their effector function on the myelin sheath, resulting in tissue destruction and consequent loss of function.2Ando D.G. Clayton J. Kono D. Urban J.L. Sercarz E.E. 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Sodium benzoate, a food additive and a metabolite of cinnamon, modifies T cells at multiple steps and inhibits adoptive transfer of experimental allergic encephalomyelitis.J Immunol. 2007; 179: 275-283PubMed Google Scholar, 6Miyake M. Sasaki K. Ide K. Matsukura Y. Shijima K. Fujiwara D. Highly oligomeric procyanidins ameliorate experimental autoimmune encephalomyelitis via suppression of Th1 immunity.J Immunol. 2006; 176: 5797-5804PubMed Google Scholar, 7O'Connor R.A. Prendergast C.T. Sabatos C.A. Lau C.W. Leech M.D. Wraith D.C. Anderton S.M. Cutting edge: Th1 cells facilitate the entry of Th17 cells to the central nervous system during experimental autoimmune encephalomyelitis.J Immunol. 2008; 181: 3750-3754PubMed Google Scholar, 8Ren Y. Lu L. Guo T.B. Qiu J. Yang Y. Liu A. Zhang J.Z. 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Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category.J Exp Med. 2008; 205: 799-810Crossref PubMed Scopus (594) Google Scholar Another important T-cell type involved in regulation of EAE development is regulatory T cells (Tregs), characterized by their CD4+CD25+ phenotype, together with expression of their master transcription factor forkhead box3 (Foxp3). Tregs reduce the inflammatory responses and clinical symptoms in mice with EAE.11Reddy J. Illes Z. Zhang X. Encinas J. Pyrdol J. Nicholson L. Sobel R.A. Wucherpfennig K.W. Kuchroo V.K. Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis.Proc Natl Acad Sci U S A. 2004; 101: 15434-15439Crossref PubMed Scopus (168) Google Scholar, 12Vila J. Isaacs J.D. Anderson A.E. Regulatory T cells and autoimmunity.Curr Opin Hematol. 2009; 16: 274-279Crossref PubMed Scopus (52) Google Scholar, 13Zhang X. Koldzic D.N. Izikson L. Reddy J. Nazareno R.F. Sakaguchi S. Kuchroo V.K. Weiner H.L. IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells.Int Immunol. 2004; 16: 249-256Crossref PubMed Scopus (285) Google Scholar The etiology of most autoimmune diseases, including MS, is not clearly understood. Both genetic predisposition (a key risk factor) and environmental factors are involved. Current therapies, which mainly focus on the use of immune-suppressant drugs, have limited efficacy and various adverse effects. Nutrition represents an alternative and complementary approach that could potentially improve autoimmune disorders. Green tea may be such a nutrition factor. Catechins in green tea are thought to be the major components responsible for the tea's biological effects. These catechins include epicatechin, epigallocatechin, epicatechin-3-gallate, and epigallocatechin-3-gallate (EGCG), among which EGCG is the most biologically active and most abundant (accounting for 50% to 80% of the total tea catechins).14Yang C.S. Maliakal P. Meng X. Inhibition of carcinogenesis by tea.Annu Rev Pharmacol Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (834) Google Scholar Although green tea or EGCG can quench several different reactive oxygen species and its health benefits have been partially attributed to its antioxidant properties, our previous study15Wu D. Guo Z. Ren Z. Guo W. Meydani S.N. Green tea EGCG suppresses T cell proliferation through impairment of IL-2/IL-2 receptor signaling.Free Radic Biol Med. 2009; 47: 636-643Crossref PubMed Scopus (68) Google Scholar indicates that the T-cell–suppressive effect of EGCG is not related to its effect on oxidative stress. Results from a few animal studies suggest that green tea EGCG might be effective in improving the symptoms and pathological conditions associated with autoimmune inflammatory diseases in several animal models.16Abboud P.A. Hake P.W. Burroughs T.J. Odoms K. O'Connor M. Mangeshkar P. Wong H.R. Zingarelli B. Therapeutic effect of epigallocatechin-3-gallate in a mouse model of colitis.Eur J Pharmacol. 2008; 579: 411-417Crossref PubMed Scopus (89) Google Scholar, 17Aktas O. Prozorovski T. Smorodchenko A. Savaskan N.E. Lauster R. Kloetzel P.M. Infante-Duarte C. Brocke S. Zipp F. Green tea epigallocatechin-3-gallate mediates T cellular NF-kappa B inhibition and exerts neuroprotection in autoimmune encephalomyelitis.J Immunol. 2004; 173: 5794-5800PubMed Google Scholar, 18Gillespie K. Kodani I. Dickinson D.P. Ogbureke K.U. Camba A.M. Wu M. Looney S. Chu T.C. Qin H. Bisch F. Sharawy M. Schuster G.S. Hsu S.D. Effects of oral consumption of the green tea polyphenol EGCG in a murine model for human Sjogren's syndrome, an autoimmune disease.Life Sci. 2008; 83: 581-588Crossref PubMed Scopus (46) Google Scholar, 19Haqqi T.M. Anthony D.D. Gupta S. Ahmad N. Lee M.S. Kumar G.K. Mukhtar H. Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea.Proc Natl Acad Sci U S A. 1999; 96: 4524-4529Crossref PubMed Scopus (308) Google Scholar, 20Morinobu A. Biao W. Tanaka S. Horiuchi M. Jun L. Tsuji G. Sakai Y. Kurosaka M. Kumagai S. (-)-Epigallocatechin-3-gallate suppresses osteoclast differentiation and ameliorates experimental arthritis in mice.Arthritis Rheum. 2008; 58: 2012-2018Crossref PubMed Scopus (87) Google Scholar, 21Ran Z.H. Chen C. Xiao S.D. Epigallocatechin-3-gallate ameliorates rats colitis induced by acetic acid.Biomed Pharmacother. 2008; 62: 189-196Crossref PubMed Scopus (72) Google Scholar Preliminary evidence17Aktas O. Prozorovski T. Smorodchenko A. Savaskan N.E. Lauster R. Kloetzel P.M. Infante-Duarte C. Brocke S. Zipp F. Green tea epigallocatechin-3-gallate mediates T cellular NF-kappa B inhibition and exerts neuroprotection in autoimmune encephalomyelitis.J Immunol. 2004; 173: 5794-5800PubMed Google Scholar, 19Haqqi T.M. Anthony D.D. Gupta S. Ahmad N. Lee M.S. Kumar G.K. Mukhtar H. Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea.Proc Natl Acad Sci U S A. 1999; 96: 4524-4529Crossref PubMed Scopus (308) Google Scholar, 21Ran Z.H. Chen C. Xiao S.D. Epigallocatechin-3-gallate ameliorates rats colitis induced by acetic acid.Biomed Pharmacother. 2008; 62: 189-196Crossref PubMed Scopus (72) Google Scholar has linked this effect of EGCG to altered T-cell function, including production of cytokines, such as IFN-γ and tumor necrosis factor (TNF)-α. Although these results are encouraging, further studies are needed to address several important issues. First, studies to determine a clear, definitive effect of EGCG on autoimmune diseases are inconsistent. Second, little is known about the dose-response relationship because of a lack of appropriately designed dietary supplementation studies, which would have a great relevance to its use as a food component, supplement, or therapeutic agent. Finally, and most important, the mechanisms for the proposed beneficial effect of EGCG in autoimmunity are not well elucidated. In particular, for the most part, the studies thus far have not considered the previously mentioned, recently developed theory on the immunopathogenesis of autoimmune inflammatory diseases (eg, reciprocal action of Th17 and Treg). To address these issues, we conducted a study using a mouse EAE model to determine how dietary supplementation with EGCG at different doses affects the disease's symptoms and pathological characteristics, as well as the related immune responses as the mechanisms by which EGCG exerts its effect. Specific pathogen-free C57BL/6 female mice (aged 6 to 8 weeks) were obtained from Charles River (Wilmington, MA). These mice were maintained at a constant temperature and humidity, with a 12-hour light-dark cycle. Water and a nutritionally adequate, nonpurified mouse diet (Teklad 7012; Harlan Teklad, Madison, WI) were provided ad libitum. In the feeding study, mice were pair fed the experimental diets, as described later. All mice were observed daily for general health and clinical signs of disease. At the end of the study, mice were euthanized by CO2 asphyxiation, followed by exsanguination, and tissues were collected postmortem. All conditions and handling of the animals were approved by the Animal Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University (Boston, MA) and conducted according to the NIH Guidelines for the Care and Use of Laboratory Animals. In the experiment to determine effect of EGCG at different doses on EAE development, mice were randomly divided into four groups and pair fed the AIN 93M diet (Research Diet) supplemented with 0%, 0.15%, 0.3%, or 0.6% EGCG (w/w) for 30 days. EGCG (TEAVIGO, containing 95% EGCG) was provided by DSM Nutritional Products (Kaiseraugst, Switzerland). After 30 days of feeding, mice were immunized to induce EAE, as described later, while they continued to receive their experimental diets. In the experiment to determine whether EGCG is still effective at treating EAE after disease initiation, mice were divided into three groups and fed the control diet for 1 week before being immunized to induce EAE. While continuing to feed the control group the control diet throughout the entire study, we switched one group at day 7 and the other group at day 12 after immunization to the diet containing 0.6% EGCG. Mice were maintained on these diets until day 30 after immunization. Mice were immunized s.c. in the flanks with 200 μg of myelin oligodendrocyte glycoprotein (MOG)35-55 peptide (synthesized by the Tufts University Core Facility Laboratory) in a 200-μL emulsion of complete freund's adjuvant (CFA) containing 5 mg/mL heat-killed Mycobacterium tuberculosis H37Ra extract (Sigma-Aldrich, St. Louis, MO) on day 0 and i.p. injected with 200 ng per mouse of pertussis toxin (List Biological Laboratories, Campbell, CA) on days 0 and 2. The clinical symptoms were scored daily from day 0 to 30 after immunization, as follows: 0, no signs; 0.5, partial tail paralysis; 1, limp tail or tail paralysis; 2, complete loss of tail tonicity or abnormal gait; 3, partial hind limb paralysis; 4, complete hind limb paralysis; and 5, moribund. Mice were euthanized on day 30 after immunization and perfused by intracardiac infusion with 10% paraformaldehyde. The brain and spinal cord were removed and fixed in 10% paraformaldehyde. Fixed samples were embedded in paraffin, and cross sections were stained with H&E or Luxol fast blue for evaluation of inflammation or demyelination, respectively. For immunohistochemistry (IHC), the sections were deparaffinized with xylene, rehydrated through a series with ethanol, and incubated in 0.3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. Immunostaining was conducted using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Nonspecific binding was blocked by incubation with normal rat serum for 30 minutes. The sections were then incubated with the following primary antibodies at indicated dilutions at 4°C overnight: anti-CD3 (1:100) and anti-F4/80 (1:50) from AbD SeroTec (Oxford, UK) and anti-CD45R/B220 (1:25) and anti-Ly-6G and Ly-6C (1:25) from BD Pharmingen (San Diego, CA). Sections were then incubated with a biotinylated secondary antibody (anti-rat IgG) for 30 minutes, washed, and incubated for another 30 minutes with ABC (avidin and biotinylated enzyme complex) reagent. Color was developed by adding peroxidase substrate diaminobenzidine. Sections were counterstained with Mayer's hematoxylin (Sigma-Aldrich) and, finally, mounting solution and coverslips were added. On day 27 after immunization, mice were injected with MOG35-55 peptide in the right footpad and saline in the left footpad as control. The footpad thickness was measured 24, 48, and 72 hours after the injection using a micrometer (QUICKmini; Mitutoyo, Kawasaki, Japan). Specific responses were calculated by subtracting the thickness of the left footpad (saline) from that of the right footpad (MOG). After mice were euthanized, the draining lymph nodes (LNs) were aseptically removed and single-cell suspensions were prepared. LN cells were placed in sterile RPMI 1640 medium (Biowhittaker, Walkerville, MD), supplemented with 25 mmol/L HEPES, 2 mmol/L glutamine, 100 kU/L penicillin, and 100 mg/L streptomycin (all from Invitrogen, Carlsbad, CA). Cells suspended in RPMI 1640 medium containing 5% heat-inactivated fetal bovine serum (Invitrogen) were plated in triplicate in 96-well round-bottom cell culture plates and stimulated with 0, 1, 10, or 100 μg/mL MOG35-55 peptide for 72 hours. Cultures were pulsed with 1 μCi/well [3H]-thymidine (Perkin Elmer; Life Sciences, Boston, MA) during the final 4 hours of incubation. The cells were harvested onto glass-fiber filter mats (Wallac, Gaithersburg, MD) by a Tomtec harvester (Wallac), and cell proliferation was quantified as the amount of [3H]-thymidine incorporation into DNA, as determined by liquid scintillation counting in a 1205 Betaplate counter (Wallac). Results are expressed as cpm. The LNs and spleen were collected, and single-cell suspensions in RPMI 1640 medium and 5% fetal bovine serum were prepared. The LNs and spleen cells were separately cultured in 24-well culture plates in the presence of 100 μg/mL MOG35-55 peptide for 72 hours. Supernatants were then collected and measured with an enzyme-linked immunosorbent assay (ELISA) for IL-17 (kit from eBiosciences, San Diego, CA), transforming growth factor (TGF)-β (kit from R&D Systems, Minneapolis, MN), and IFN-γ, IL-6, and TNF-α (all from BD Pharmingen). Intracellular cytokine levels were determined using a flow cytometry, as described later. Heparinized plasma samples were collected and centrifuged to obtain plasma. Plasma concentrations of intercellular adhesion molecule 1 (ICAM-1) and IL-1β were measured using the Duoset ELISA kit (R&D Systems), and IL-12/IL-23 p40 was measured using the ELISA Ready-SET-Go! kit (eBiosciences), following the manufacturer's instructions. Mice were euthanized on day 12 after immunization and perfused through the left ventricle with cold PBS. Brain and spinal cord were removed, and the tissues from two mice were pooled into one CNS sample. Mononuclear cells were isolated from the CNS samples using the Neural Tissue Dissociation Kits (Mitenyi Biotec, Auburn, CA). The isolated cells were either directly analyzed for surface markers or stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL ionomycin (both from Sigma-Aldrich) in the presence of monensin (Golgi Stop; BD Pharmingen) for 4 hours for measurement of intracellular markers. The expression of cell surface and intracellular markers was determined by flow cytometry, as described later. To determine the cell phenotype and expression of CCRs, cells were surface stained by appropriate antibodies labeled with fluorochromes. For intracellular cytokine measurements, cells were restimulated with 50 ng/mL PMA and 500 ng/mL ionomycin (both from Sigma-Aldrich) in the presence of monensin (BD Pharmingen) for 4 hours. Cells were blocked by anti-CD16/32 (Fc block; BD Pharmingen), fixed and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen), and stained with fluorochrome-labeled antibodies for each cytokine tested. The expression of transcription factors was analyzed after intracellular staining using a similar procedure. Foxp3 staining was performed using the Mouse Foxp3 Buffer Set (BD Pharmingen). The antibodies used for flow cytometry were as follows: CD3 (145-2C11), anti-CD4 (GK1.5), anti-IFN-γ (XMG1.2), and anti-IL-10 (JES5-16E3) were from eBiosciences; anti-transcription factors T-box expressed in T cells (T-bet; O4-46), anti-Foxp3 (MF23), anti-IL-4 (11B11), and anti-IL-17 (TC11-18H10) were from BD Pharmingen; and anti-CCR6 (140706) and anti-retinoid-related orphan receptor (ROR)–γt (600380) were from R&D Systems. All flow cytometric measurements were conducted using an Accuri C6 flow cytometer (BD Accuri Cytometers, Ann Arbor, MI), and acquired data were analyzed with FlowJo 7.6 software (Treestar Inc., Ashland, OR). To determine mRNA levels of selected analytes, total RNA was extracted from brain, spinal cord, and splenocyte samples using TRIzol reagent (Invitrogen) and was then reverse transcribed into cDNA using a SuperScript VILO cDNA synthesis kit (Invitrogen), following the manufacturer's instructions. Real-time PCR was performed in triplicate using SYBR Green Master Mix (Qiagen, Valencia, CA) in an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA). The levels of tested genes were normalized to β-actin gene as an internal control. The sequences of the primers used are as follows: CCL20, 5′-CGACTGTTGCCTCTCGTACA-3′ (forward) and 5′-AGCCCTTTTCACCCAGTTCT-3′ (reverse); T-bet, 5′-GCCAGGGAACCGCTTATATGTC-3′ (forward) and 5′-CTGTGAGATCATATCCTTGGGCTG-3′ (reverse); RORγt, 5′-TGCAAGACTCATCGACAAGG-3′ (forward) and 5′-AGGGGATTCAACATCAGTGC-3′ (reverse); IL-23p19, 5′-GACTCAGCCAACTCCTCCAG-3′ (forward) and 5′-GGCACTAAGGGCTCAGTCAG-3′ (reverse); IL-12p40, 5′-AGGTCACACTGGACCAAAGG-3′ (forward) and 5′-TGGTTTGATGATGTCCCTGA-3′ (reverse); IL-12p35, 5′-CATCGATGAGCTGATGCAGT-3′ (forward) and 5′-CAGATAGCCCATCACCCTGT-3′ (reverse); IL-27p28, 5′-CTCTGCTTCCTCGCTACCAC-3′ (forward) and 5′-GGGGCAGCTTCTTTTCTTCT-3′ (reverse); EBi3, 5′-CGGTGCCCTACATGCTAAAT-3′ (forward) and 5′-GCGGAGTCGGTACTTGAGAG-3′ (reverse); and β-actin, 5′-TGTTACCAACTGGGACGACA-3′ (forward) and 5′-GGGGTGTTGAAGGTCTCAAA-3′ (reverse). All results are expressed as mean ± SEM. Statistical analysis was conducted using Systat 12 statistical software (Systat Software, Chicago, IL). Differences were determined using one-way analysis of variance, followed by Tukey's honestly significant difference post hoc test for multiple comparisons, or a nonpaired Student's t-test. Significance was set at P < 0.05. Mice were fed different doses of EGCG for 30 days and then immunized with MOG35-55 peptide to induce EAE while continuing their EGCG consumption. Almost all of the mice developed EAE after immunization, but the time of onset varied to some extent among individual mice. Nevertheless, compared with the control group, mice fed 0.6% EGCG had a delayed average time of onset (14 ± 0.7 versus 11.5 ± 0.8 days; P < 0.05), whereas no significant difference was observed in the mice fed 0.15% or 0.3% EGCG (Figure 1 and Table 1). Furthermore, the cumulative disease index (mean sum of clinical scores over the entire observation period) showed a dose-dependent decrease in EGCG-fed mice, which lasted until the completion of the study (ie, 30 days after immunization) (Figure 1 and Table 1). The improvement in symptoms was not statistically significant in the 0.15% EGCG group.Table 1Effect of Dietary EGCG on EAE SymptomsGroupDay of onsetCDI⁎Sum of clinical scores over the entire observation period.Area of curve†Values are given as number (percentage) of 60.50.Control11.5 ± 0.7662.18 ± 1.1060.50 (100)EGCG (%) 0.1512.0 ± 0.8054.82 ± 1.9154.31 (89.8) 0.312.5 ± 0.6549.68 ± 4.88‡P < 0.05 versus the control.49.41 (81.7) 0.614.0 ± 0.70‡P < 0.05 versus the control.45.41 ± 3.84§P < 0.01 versus the control.42.78 (70.7)Values are given as the mean ± SEM (n = 12/group) unless otherwise indicated.CDI, cumulative disease index. Sum of clinical scores over the entire observation period.† Values are given as number (percentage) of 60.50.‡ P < 0.05 versus the control.§ P < 0.01 versus the control. Open table in a new tab Values are given as the mean ± SEM (n = 12/group) unless otherwise indicated. CDI, cumulative disease index. T cells play a key role in the development of EAE through their antigen (Ag)–specific effector response. Clonal expansion of Ag-specific T cells on antigen encounter is a prerequisite for the initiation and development of T-cell–mediated immunopathological characteristics. We, thus, determined whether EGCG attenuates EAE development via its impact on Ag-specific T-cell response. We used two approaches to address this question: an ex vivo proliferation assay to determine Ag-specific proliferation and an in vivo delayed-type hypersensitivity (DTH) assay to assess T-cell–mediated inflammatory response after rechallenge with the immunization autoantigen. In the assay of ex vivo T-cell proliferation, we rechallenged LN cells with MOG35-55 peptide or ovalbumin as a control after these cells were isolated from mice with EAE fed different doses of EGCG. The addition of MOG35-55 (1 to 100 μg/mL) stimulated T-cell proliferation in a concentration-dependent manner, which was dose dependently inhibited by dietary EGCG supplementation (Figure 2A). Nonspecific Ag ovalbumin did not induce proliferative response (data not shown). In concordance with the results for symptoms, this effect was not significant in the 0.15% EGCG group. Consistent with the results of an ex vivo T-cell proliferation assay, EGCG also dose dependently reduced the DTH response, as determined by the Ag rechallenge-induced footpad thickness, and a significant reduction was observed even in the 0.15% EGCG group (Figure 2B). Because mice fed 0.6% EGCG had the greatest protective effect, and they tolerated this level well, in the subsequent experiments we used this dose to further determine the working mechanisms of EGCG and to compare its efficacy when administered at different times during EAE development. In the pathogenesis of EAE, autoreactive T cells and other associated immune cells cross the blood-brain barrier (BBB), infiltrating the CNS, where these cells and their products orchestrate inflammatory cascades, ultimately causing damage to the myelin sheath. Because we found that EGCG ameliorated EAE symptoms and inhibited the Ag-specific T-cell response, we intended to learn if this protective effect of EGCG would be reflected in the corresponding changes in tissue inflammation and damage. Figure 3A shows the pathological characteristics of spinal cord samples collected from mice 30 days after immunization. Mice with EAE fed the control diet showed extensive inflammatory cell infiltration into the white matter of spinal cords, and this change was greatly reduced by EGCG treatment. We further found that EAE-induced tissue inflammation and the protective effect of EGCG were well reflected in the levels of tissue destruction (ie, demyelination) (Figure 3B). To identify the populations of immune cells that infiltrated into the CNS of mice with EAE and to determine how they are affected by EGCG supplementation, we conducted IHC and found that EGCG treatment reduced the frequency of neutrophils (Gr-1), MΦ (F4/80), and T cells (CD3), but not B cells (CD45R, B220), in the spinal cords on day 12 after immunization (estimated peak time for inflammatory infiltration) (Figure 3C). We also isolated mononuclear cells from spinal cords and brains on day 12 after immunization and conducted a quantitative analysis using flow cytometry. In agreement with the IHC results, EGCG treatment reduced total infiltrating cells, neutrophils, MΦ, total T cells, and CD4+ T cells; there was also a trend for reduced B cells (P = 0.09) (Figure 3D). However, EGCG treatment did not significantly change the composition of infiltrating cells, except for a reduced percentage of neutrophils (data not shown). These results indicate that EGCG reduces infiltration of most cell types to a similar extent, without substantial preference. In EAE pathogenesis, a key step after effe
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