Green Tea Catechin Metabolites Exert Immunoregulatory Effects on

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Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4 T Cell and Natural Killer Cell Activity +

Yoon Hee Kim, Yeong-Seon Won, Xue Yang , Motofumi Kumazoe, Shuya Yamashita, Aya Hara, Akiko Takagaki, Keiichi Goto, Fumio Nanjo, and Hirofumi Tachibana J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01115 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 27, 2016

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Journal of Agricultural and Food Chemistry

Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4+ T Cell and Natural Killer Cell Activity

Yoon Hee Kim, †,‡,# Yeong-Seon Won, †,# Xue Yang, † Motofumi Kumazoe, † Shuya Yamashita, † Aya Hara, § Akiko Takagaki, § Keiichi Goto, § Fumio Nanjo, § and Hirofumi Tachibana *,†



Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology,

Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan ‡

Department of Food and Nutrition, College of Engineering, Daegu University, Gyeongsan

712-714, Korea §

Food Research Laboratories, Mitsui Norin Company, Ltd., 223-1 Miyabara, Fujieda-shi,

Shizuoka 426-0133, Japan

#

These authors contributed equally to this work.

*

Corresponding author

Hirofumi Tachibana 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Tel and Fax: (+81) (92) 642-3008 E-mail: [email protected]

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ABSTRACT: Tea catechins, such as (−)-epigallocatechin-3-O-gallate (EGCG), have

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been shown to effectively enhance immune activity and prevent cancer, although the

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underlying mechanism is unclear. Green tea catechins are instead converted to

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catechin metabolites in the intestine. Here we show that these green tea catechin

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metabolites enhance CD4+ T cell activity as well as natural killer (NK) cell activity.

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Our data suggest that the absence of a 4'-hydroxyl on this phenyl group (B-ring) is

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important for the effect on immune activity. In particular,

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5-(3′,5′-dihydroxyphenyl)-γ-valerolactone (EGC-M5), a major metabolite of EGCG,

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not only increased the activity of CD4+ T cells, but also enhanced the cytotoxic

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activity of NK cells in vivo. These data suggest that EGC-M5 might show the

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immunostimulatory activity.

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KEYWORDS: Tea catechin metabolites, 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone,

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EGCG, CD4+ T cells, NK cells cytotoxicity

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INTRODUCTION Green tea is particularly well known for its multiple beneficial effects. Several

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epidemiological studies have revealed a negative correlation between green tea

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consumption and the risk of cancer development.1-5 Green tea catechins are the major

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bioactive components of green tea that have been reported to exert protective effects

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against various types of cancer.6-9 However, according to previous reports, intact

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green tea catechins could not easily be absorbed by the intestines.

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Following the oral administration of decaffeinated green tea (200 mg/kg) to rats,

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the absorption rates of green tea catechins were 0.1−1.6% for

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(−)-epigallocatechin-3-O-gallate (EGCG), 13.7% for (−)-epigallocatechin (EGC), and

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31.2% for (−)-epicatechin (EC).10 Previously, we reported that EGCG is metabolized

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by intestinal bacteria. In particular, EGCG is hydrolyzed to compounds, such as gallic

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acid and EGC, by bacteria in the rat intestinal tract; subsequently, highly absorbable

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green tea catechin metabolites are generated from EGC by these intestinal bacteria.11

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We demonstrated that two metabolites, 5-(3′,4′,5′-trihydroxyphenyl)-γ-valerolactone

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and 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone, showed a hypotensive effect on

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angiotensin I-converting enzyme activity in spontaneously hypertensive rats.12

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However, the effect of green tea catechin metabolites on tumor immunity remains

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unknown.

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The Cylex ImmuKnow in vitro assay, which has been approved by the US Food

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and Drug Administration, provides a global assessment of cellular immune function

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that facilitates monitoring of the immune statuses of immunosuppressed patients. As

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most immune cell effector functions depend upon the cellular energy supply,13 this

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assay was designed to measure the amount of ATP produced by CD4+ cells after

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mitogenic, recall antigen, or allogeneic stimulation.14-17 Accordingly, this assay has

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received interest as a potential objective method for measuring overall immune

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function.18 Therefore, we evaluated the immune functions of green tea catechins and

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EGCG metabolites in CD4+ cells according to ATP levels.

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Natural killer (NK) cells also play a considerable role in immunoregulation. NK

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cells were identified as innate immune cells that form an intrinsic defense system,

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which is characterized by cytolytic function.19 NK cells are activated in response to

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interleukin (IL)-2 and interferon (IFN)-γ, which are secreted from CD4+ T cells.20

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Recent reports have described a close relationship between an increasing incidence of

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cancer and reduced NK cell activity.21,22 Indeed, elevated NK cell activity is a

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favorable prognostic factor in cancer patients.23-25 In this report, we investigated the

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effect of green tea catechin metabolites on immunoregulatory activity against CD4+ T

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cell and NK cell.

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MATERIALS AND METHODS

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Green Tea Catechins and Metabolites. The following green tea catechins were

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purchased from Sigma-Aldrich (St. Louis, MO, USA): (−)-epicatechin (EC),

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(−)-catechin (C), (−)-epigallocatechin-3-O-gallate (EGCG),

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(−)-gallocatechin-3-O-gallate (GCG), (−)-epicatechin-3-O-gallate (ECG),

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(−)-catechin-3-O-gallate (CG), (−)-gallocatechin (GC), and (−)-epigallocatechin

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(EGC). The following green tea catechin metabolites were obtained from Mitsui

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Norin (Mitsui Norin Company, Ltd., Shizuoka, Japan):

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1-(3′,4′,5′-trihydroxyphenyl)-3-(2′′,4′′,6′′-trihydroxyphenyl)propan-2-ol (EGC-M1),

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4′-dehydroxylated EGC (EGC-M2),

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1-(3′,5′-dihydroxyphenyl)-3-(2′′,4′′,6′′-trihydroxyphenyl)propan-2-ol (EGC-M3),

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4-hydroxy-5-(3′,5′-dihydroxyphenyl)valeric acid (EGC-M4),

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5-(3′,5′-dihydroxyphenyl)-γ-valerolactone (EGC-M5),

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4-hydroxy-5-(3′,4′,5′-trihydroxyphenyl)valeric acid (EGC-M6),

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5-(3′,4′,5′-trihydroxyphenyl)-γ-valerolactone (EGC-M7), 3-(3′,5′-dihydroxyphenyl)

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propionic acid (EGC-M8), 5-(3′,5′-dihydroxyphenyl)valeric acid (EGC-M9),

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5-(3′,4′,5′-trihydroxyphenyl)valeric acid (EGC-M10), and

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5-(3′-hydroxyphenyl)valeric acid (EGC-M11). We obtained these metabolites as

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previously descrived.11,26 The purities of green tea catechins and its metabolites were

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confirmed by HPLC analysis (at least 95%).

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Animal and Oral Administration. BALB/c mice (male, 8–10 weeks old) were

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purchased from Kyudo (Saga, Japan), housed in a temperature- and

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humidity-controlled room, and fed a commercial diet and water ad libitum. The

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animal experimental component of this study was authorized and conducted according

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to the guidelines for animal experiments of the Animal Care and Use Committee of

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Kyushu University, Fukuoka, Japan. Animal studies were conducted according to the

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approval of law number 105 and notification number 6 of the Japanese government

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regarding the welfare of experimental animals. Mice were divided into three

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experimental groups. EGC-M5 and EGC were dissolved in an aqueous sterile saline

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solution containing 2.5% dimethyl sulfoxide (DMSO). For the experiment, 200 µL of

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EGC-M5 or EGC solution at a dosage of 10 mg/kg body weights were orally

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administered to the mice once daily for 14 days. Control mice were given an

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equivalent volume of sterile saline containing 2.5% DMSO.

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Single Cell Preparation and CD4+ Positive Cell Isolation. To isolate CD4+ cells,

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spleens were collected from 8–10 week old BALB/c mice and mechanically disrupted

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by grinding with a syringe plunger on a 40 µM nylon mesh cell strainer to yield single

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cells. Red blood cells were lysed with a hypotonic RBC lysis buffer, and a single-cell

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suspension was subsequently prepared in Roswell Park Memorial Institute (RPMI)

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1640 medium supplemented with 100 U/mL of penicillin, 100 µg/mL of

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streptomycin, and 5% fetal bovine serum (FBS). CD4+ T cells were purified from

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splenocyte suspensions using an EasySep® Mouse CD4 Positive Selection Kit

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(StemCell Technologies Inc., Vancouver, BC, Canada). Isolated CD4+ cells routinely

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had a purity of >92% and viability of >95% (Supplementary Figure 1).

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Measurement of ATP in CD4+ T Cells. CD4+ T cells were suspended in RPMI 1640

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medium with 5% FBS at a density of 1.5 × 105 cells/mL and subsequently incubated

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with green tea catechins (EC, C, EGCG, GCG, ECG, CG, GC, and EGC), 11 types of

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green tea catechin metabolites (EGC-M1–M11), or phytohemagglutinin (PHA) as a

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positive control (2 µg/mL, Supplementary Figure 2) for 72 h. After incubation, 50 µL

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aliquots of each cell suspension were collected for ATP measurement. The cell

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activation value was determined by adding 50 µL of ATPlite solution (ATPlite 1-step

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kit; PerkinElmer®, Waltham, MA, USA). The ATP level in the control group was set

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at 100%.

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Cytokine Detection by Enzyme-Linked Immunosorbent Assay (ELISA).

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Splenocytes (density: 1 × 106 cells/mL) were cultured in the presence or absence of 5,

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10, 25, or 50 µM EGC or EGC-M5, or 2 µg/mL PHA, and incubated in 96-well

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culture plates for 72 h in a humidified atmosphere of 5% CO2 at 37°C. The mouse

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PHA-stimulated IFN-γ and IL-2 in spleen were determined using an ELISA kit

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according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). The

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levels of mouse IFN-γ and IL-2 in the diluted splenocyte medium were detected at

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450 nm using a microplate reader (PerkinElmer, Waltham, MA, USA).

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Cell Culture and Evaluation of NK Cell Cytotoxicity. Murine lymphoma YAC-1

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cells were obtained from the Cell Resource Center for Biomedical Research (Tohoku

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University, Sendai, Japan). YAC-1 cells were cultured in RPMI 1640 medium

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supplemented with 10% FBS (Intergen, Purchase, NY) in a humidified atmosphere of

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5% CO2 at 37°C. Splenocytes isolated from mice in each group were used as effector

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cells. YAC-1 cells that had been sensitized to NK cell cytotoxicity were used as target

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cells. Target cell killing was determined using a lactate dehydrogenase (LDH) release

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assay (LDH-Cytotoxicity Assay Kit II; Biovision, Inc., Milpitas, CA, USA). YAC-1

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cells (density: 1 × 105 cells/mL) were cocultured with splenocytes at a final

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effector/target (E:T) ratio of 50:1 in 24-well culture plates for 6 h in an incubator. Ten

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microliters of supernatant were transferred from each well into a 96-well plate,

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followed by centrifugation for 4 min at 500 × g. Subsequently, 100 µL of LDH

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substrate was added, and the plate was incubated at room temperature. After 20 min,

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10 µL of stop solution were added immediately to each well, and the optical density

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(OD) of each reaction was measured on a microplate reader at 450 nm. NK cell

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cytotoxicity was calculated using the following formula: cytotoxicity % =

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(experimental OD − effector cell spontaneous OD − target cell spontaneous

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OD)/(target cell lysis OD − target cell spontaneous OD) × 100.24.

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Staining of Immune Cells for Flow Cytometric Analysis. Single-cell splenocyte

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suspensions were stained with the following antibodies: anti-mouse CD335

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(NKp46)-Brilliant Violet 421TM, anti-mouse CD49b-APC, anti-mouse CD11b-PE,

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and anti-mouse CCR4-APC (BioLegend, San Diego, CA, USA); anti-mouse

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granzyme B-PE, anti-mouse perforin-FITC, and anti-mouse CD3-FITC (eBioscience);

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anti-mouse CD8-PE, anti-mouse CD11c-V450, anti-mouse F4/80-PE, anti-mouse

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IFN-γ-PE, anti-mouse CXCR3-APC, anti-mouse T-bet-Alexa Fluor® 488, anti-mouse

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GATA3-Alexa Fluor® 488, anti-mouse STAT3-Alexa Fluor® 488, anti-mouse

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CD25-APC, anti-mouse CD4-PE, and anti-mouse FoxP3-Alexa Fluor® 488 (BD

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Biosciences; San Diego, CA, USA); and anti-mouse CD45RA (B220)-BE

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(Invitrogen; Carlsbad, CA, USA). Stained cells were analyzed on a BD FACSVerseTM

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flow cytometer (BD Biosciences). Data were analyzed using FlowJo 7/8 software

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(TreeStar, Ashland, OR, USA).

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Statistical Analysis. Data are presented as means ± standard errors of the means

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(SEM). Statistically significant differences between the means values were

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determined using Dunnett’s test and Tukey’s test of variance where appropriate, and

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the analyses were conducted using Prism software (GraphPad, La Jolla, CA, USA).

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RESULTS AND DISCUSSION During the past decade, many studies demonstrated the cancer preventive effects of

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green tea catechins.6-9,27 However, according to previous reports, intact green tea

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catechins, notably EGCG, could not easily be absorbed by the intestines. Recently, the

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catabolism of green tea catechins, mediated by intestinal bacteria, has been

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recognized as an important metabolic activity.28-30 We previously reported the

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biotransformation of green tea catechins by intestinal flora, as well as the EGCG

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metabolic pathway in the rat intestinal tract.11,26 EGC and gallic acid, which are

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degradation products of EGCG, are hydrolyzed to create 11 metabolites

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(EGC-M1–M11).11,26 In this report, we investigated the effects of green tea catechins

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and resulting metabolites on immunomodulatory in mice.

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In immune cells, most effector functions depend upon the cellular energy supply; 13

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accordingly, an assay was designed to measure the amount of ATP in CD4+ T cells

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after mitogenic, recall antigen, or allogeneic stimulation.14-17 This assay has received

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interest as a potential objective method for measuring overall immune function.18

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Therefore, we assessed the immune functions of green tea catechins and their

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metabolites in CD4+ cells based on ATP levels. The relative ATP levels produced by

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CD4+ T cells in response to green tea catechins and EGCG metabolites are indicated

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in Figure 1. The metabolites without the 4′-hydroxyphenyl group on the phenyl group

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(B-ring), namely EGC-M2, M3, M4, M5, M8, M9, and M11 (Figure 1A, B), induced

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increased CD4+ T cell activity. Conversely, EGCG metabolites with the

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4′-hydroxyphenyl group as well as intact catechins including EGCG, EGC, ECG, EC,

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C, CG, GC and GCG, did not increase the relative ATP levels in CD4+ cells (Figures

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1B, C). The predicted biological availability of green tea catechins was 39% of the

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total ingested amount of catechins resulting from green tea intake, and EGC-M5 is the

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most predominant metabolite detected in human and rat urine.31-36 Previous studies

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reported that EGC-M5 could be absorbed in any amount into both the human and rat

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intestine.11, 36-38 Our data revealed that CD4+ T cell activity increased to a much

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greater extent following treatment with highly absorbable green tea catechin

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metabolites that did not include the 4′-hydroxyphenyl group on the phenyl group

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(B-ring) compared with that following treatment with either the metabolites with the

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4′-hydroxyphenyl group or poorly absorbable green tea catechins.

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IFN-γ and IL-2 are known as T cell growth factors and effectors of activated CD4+

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T cells.39,40 Moreover, IFN-γ, which promotes the differentiation of activated CD4+ T

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cells into type 1 T helper (Th1) cells, can inhibit cancer cells by inducing cytotoxic

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activity in NK cells. The IFN-γ level has an important role on the anti-cancer immune

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functions of NK cells.41 As shown in Figure 2, EGC-M5 upregulated PHA-induced

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IFN-γ production in splenocytes. IFN-γ production was induced to a significantly

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greater extent in splenocytes treated with 10-, 25-, and 50-µM EGC-M5 than in those

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treated with the same concentrations of EGC (Figure 2A). On the other hand,

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EGC-M5 showed no significant effect on IL-2 level (Figure 2B).

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Various epidemiological studies have demonstrated that green tea ingestion can

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inhibit various types of cancers and infectious diseases.4,5,42-44 We formerly reported

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the biological effects of EGCG, the most significant polyphenol in green tea, on

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molecular biological mechanisms.8,9,45,46 However, the effects of green tea catechin

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metabolites on antitumor immunity remained unknown.

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Next, we demonstrated NK cell cytotoxic activity against YAC-1 target cells in

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mouse splenocytes treated with EGC-M5 in vivo. Splenic NK cell cytotoxic activity

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against YAC-1 cells increased considerably compared with that observed in the EGC

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intake and control groups (Figure 3A, B). In this study, we for the first time

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investigated the effect of EGC-M5 on NK cell-mediated cytotoxicity and found that

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treatment with this metabolite increased cell death in YAC-1 lymphoma cells.

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NK cells produce perforin and granzyme B, which triggers apoptosis and necrosis

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in target cells. These enzymes create holes in the target cell membrane, thus

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facilitating destruction.45 We investigated the induction of granzyme B+ NK cells,

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perforin+ NK cells, IFN-γ+ NK cells, and IFN-γ+ CD8+ cells in spleens from

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EGC-M5-fed mice by flow cytometry. Granzyme B+ NK cells were increased by the

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oral administration of EGC-M5 (Figure 3C). However, EGC-M5 did not affect the

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populations of perforin+ NK cells, IFN-γ+ NK cells, and IFN-γ+ CD8+ cells (Figure

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3D–F). These results suggest that EGC-M5-induced granzyme upregulation might

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partially contribute to the EGC-M5-induced upregulation of NK cell activity.

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We also assessed the effect of EGC-M5 on the following populations of

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splenocytes: helper T cells (CD3+CD4+), cytotoxic T cells (CD3+CD8+), B cells

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(B220+), total dendritic cells (DCs; CD45RA+CD11c+ + CD45RA−CD11c+),

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macrophages (CD11c+F4/80+), and NK cells (NKp46+CD49b+CD11b+) (Table 1). In

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addition, we evaluated splenic Th1 cells (CD4+CXCR3+T-bet+), Th2 cells

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(CD4+CCR4+GATA3+), Th17 cells (CD4+CCR4+STAT3+), and Treg cells

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(CD4+CD25+FoxP3+) (Table 2). However, we found that EGC-M5 intake did not

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affect splenic helper T cells, cytotoxic T cells, B cells, DCs, macrophages, NK cells,

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Th1 cells, Th2 cells, or Th17 cells. EGC-M5 might inhibit YAC-1 cells via CD4+ T

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cell activity and NK cell cytotoxicity, and thus respectively promote IFN-γ production

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and granzyme B+ NK cell proliferation.

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Our data showed the EGC-M5-glucuronide significantly increased CD4+ cell activity as same as EGC-M5 (Supplementary Figure 3). A previous epidemiological study reported the lack of a relationship between green

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tea intake and the risk of viral infection.48 As components of the innate immune

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system, NK cells play critical roles against viral infection. The T cell-dependent

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production of IFN-γ by NK cells elicits a strong protective effect against influenza A

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viral infection.49 EGCG extracted from green tea was found to induce an

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immunoregulatory effect via Treg cells in vivo with respect to NK cell cytolysis,

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antigen-specific proliferation, and the secretion of cytokines, such as IL-2 and IFN-γ.50

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However, few details of the underlying mechanisms are known. In the present study,

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for the first time it was found that EGC-M5 increase NK activity, which was

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accompanied by upregulated IFN-γ production. These results suggest that EGC-M5

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has a protective effect against viral infection via an immune response that includes

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NK cell cytolysis and IFN-γ production.

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In conclusion, we suggest that green tea catechin metabolites induce immune

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activation mediated by CD4+ T cell activity and NK cell cytotoxicity. In particular,

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EGCG metabolites lacking a 4′-hydroxyphenyl group were found to increase CD4+ T

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cell activity, and EGC-M5 induced anti-cancer effects mediated by NK cells

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cytotoxicity. Therefore, these results are likely to contribute to cancer prevention and

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treatment, as well as to the studies of the biological activity of intestinal metabolites

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in the body.

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ABBREVIATIONS USED

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ATP: adenosine triphosphate

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C: catechin

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CG: catechin-3-O-gallate

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DMSO: dimethyl sulfoxide

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EC: epicatechin

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ECG: epicatechin-3-O-gallate

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ECL: enhanced chemiluminescence

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EGC: epigallocatechin

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EGCG: epigallocatechin-3-O-gallate

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EGC-M1: 1-(3′,4′,5′-trihydroxyphenyl)-3-(2′′,4′′,6′′-trihydroxyphenyl)propan-2-ol

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EGC-M2: 4′-dehydroxylated EGC

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EGC-M3: 1-(3′,5′-dihydroxyphenyl)-3-(2′′,4′′,6′′-trihydroxyphenyl)propan-2-ol

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EGC-M4: 4-hydroxy-5-(3′,5′-dihydroxyphenyl)valeric acid

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EGC-M5: 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone

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EGC-M6: 4-hydroxy-5-(3′,4′,5′-trihydroxyphenyl)valeric acid

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EGC-M7: 5-(3′,4′,5′-trihydroxyphenyl)-γ-valerolactone

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EGC-M8: 3-(3′,5′-dihydroxyphenyl) propionic acid

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EGC-M9: 5-(3′,5′-dihydroxyphenyl)valeric acid

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EGC-M10: 5-(3′,4′,5′-trihydroxyphenyl)valeric acid

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EGC-M11: 5-(3′-hydroxyphenyl)valeric acid

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ELISA: enzyme-linked immunosorbent assay

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FBS: fetal bovine serum

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GC: gallocatechin

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GCG: gallocatechin-3-O-gallate

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IFN: interferon

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IgE: immunoglobulin E

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IL: interleukin

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LDH: lactate dehydrogenase

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NK cell: natural killer cell

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OD: optical density

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PHA: phytohemagglutinin

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RPMI: Roswell park memorial institute medium

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SEM: standard errors of the means

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Th1: type 1 T helper

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CONFLICT OF INTEREST The authors declare no conflict of interest.

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ACKNOWLEDGMENTS

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This work was kindly supported in part by Grants-in-Aid for Scientific Research

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(KAKENHI) from the Japan Society for the Promotion of Science to H. Tachibana.

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(Grant Number 22228002 and 15H02448).

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SUPPORTING INFORMATION

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Supporting Information is available free of charge on the ACS Publications website.

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The initial population comprised total mouse splenocytes; after isolation, the CD4+

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cell content is approximately 92% (Supplementary Figure 1). The effect of PHA on activated CD4+ cells isolated from mouse splenocytes. The

401

CD4+ cells were incubated in the presence of PHA as a positive control for 72 h

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(Supplementary Figure 2).

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The effect of 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone-3′-O-glucuronide

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(EGC-M5-glucuronide) on the activation of splenic CD4+ cells (Supplementary

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Figure 3).

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REFERENCES (1) Tavakol, H. S.; Akram, R.; Azam, S.; Nahid, Z. Protective effects of green tea

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on antioxidative biomarkers in chemical laboratory workers. Toxicol. Ind. Health

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2015, 31, 862−867.

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FIGURE CAPTIONS Figure 1. The effects of green tea catechins and metabolites on the activation of splenic CD4+ cells. (A) Proposed pathway of EGCG metabolism by intestinal bacteria. (B) CD4+ cells were incubated in the presence of EGCG, EGC, or metabolites (10 µM) for 72 h. (C) CD4+ cells were incubated in the presence of green tea catechins (10 µM) for 72 h. The results are presented as means ± standard errors of the means (n = 4–6). *P < 0.05, **P < 0.01 vs. control, Dunnett’s test. Gray circles indicate no effect; red circles indicate activity in the pathway of EGCG metabolism.

Figure 2. The effect of EGC-M5 on the levels of IFN-γ and IL-2 produced by splenocytes. (A) Splenocytes were incubated in the presence or absence of EGC-M5 or EGC (0, 5, 10, 25, and 50 µM) for 72 h; IFN-γ level determined by ELISA. (B) Splenocytes were incubated in the presence or absence of EGC-M5 (0, 5, 10, 25, and 50 µM) for 72 h; IL-2 level determined by ELISA. The results are presented as means ± standard errors of the means (n = 3–4). *P < 0.05, **P < 0.01, ***P < 0.001, and ns (not significant) vs. control, Dunnett’s test. PHA, phytohemagglutinin.

Figure 3. The effect of EGC-M5 on splenocyte-mediated cytotoxicity against YAC-1 cells from mice treated with EGC or EGC-M5. (A, B) Splenocyte cytotoxicity towards YAC-1 cells at effector splenocyte to YAC-1 target ratios of 20:1 and 40:1. (C–F) YAC-1 cell-induced granzyme, perforin, and IFN-γ production were measured by flow cytometry in YAC-1 cells-induced splenocytes. The results are presented as means ± standard errors of the means (n = 10). Superscript letters indicate differences with P values of