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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ACS Paragon Plus Environment


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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

331 332 333 334 335 336 337 338 339 340 341 342 343 344 345



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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+

399 400

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

422

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

423

2015, 31, 862−867.

424

(2) Xu, Y.; Zhang, M.; Wu, T.; Dai, S.; Xu, J.; Zhou, Z. The anti-obesity effect of

425

green tea polysaccharides, polyphenols and caffeine in rats fed with a high-fat diet.

426

Food Funct. 2015, 6, 297−304.

427

(3) Jaffri, J. M.; Mohamed, S.; Rohimi, N.; Ahmad, I. N.; Noordin, M. M.; Manap,

428

Y. A. Antihypertensive and cardiovascular effects of catechin-rich oil palm (Elaeis

429

guineensis) leaf extract in nitric oxide-deficient rats. J. Med. Food 2011, 14, 775−783.

430

(4) Tang, J.; Zheng, J. S.; Fang, L.; Jin, Y.; Cai, W.; Li, D. Tea consumption and

431

mortality of all cancers, CVD and all causes: a meta-analysis of eighteen prospective

432

cohort studies. Br. J. Nutr. 2015, 114, 673−683.

433

(5) Huang, Y. Q.; Lu, X.; Min, H.; Wu, Q. Q.; Shi, X. T.; Bian, K. Q.; Zou, X. P.

434

Green tea and liver cancer risk: A meta-analysis of prospective cohort studies in Asian

435

populations. Nutrition 2016, 32, 3−8.

436

(6) Li, Q.; Zhang, X. Epigallocatechin-3-gallate attenuates bone cancer pain

437

involving decreasing spinal Tumor Necrosis Factor-α expression in a mouse model.

438

Int. Immunopharmacol. 2015, 29, 818−823.

439

(7) Chakrabarty, S.; Ganguli, A.; Das, A.; Nag, D.; Chakrabarti, G.

440

Epigallocatechin-3-gallate shows anti-proliferative activity in HeLa cells targeting

441

tubulin-microtubule equilibrium. Chem.-Biol. Interact. 2015, 242, 380−389.



442

(8) Tsukamoto, S.; Huang, Y.; Umeda, D.; Yamada, S.; Yamashita, S.; Kumazoe,

443

M.; Kim, Y.; Murata, M.; Yamada, K.; Tachibana, H. 67-kDa laminin

444

receptor-dependent protein phosphatase 2A (PP2A) activation elicits

445

melanoma-specific antitumor activity overcoming drug resistance. J. Biol. Chem.

446

2014, 289, 32671−32681.

447

(9) Kumazoe, M.; Fujimura, Y.; Hidaka, S.; Kim, Y.; Murayama, K.; Takai, M.;

448

Huang, Y.; Yamashita, S.; Murata, M.; Miura, D.; Wariishi, H.; Maeda-Yamamoto,

449

M.; Tachibana, H. Metabolic profiling-based data-mining for an effective chemical

450

combination to induce apoptosis of cancer cells. Sci. Rep. 2015, 5, 9474.

451

(10) Chen, L.; Lee, M. J.; Li, H.; Yang, C. S. Absorption, distribution, and

452

elimination of tea polyphenols in rats. Drug Metab. Dispos. 1997, 25, 1045−1050.

453

(11) Takagaki, A.; Nanjo, F. Metabolism of (−)-epigallocatechin gallate by rat

454 455

intestinal flora. J. Agric. Food Chem. 2010, 58, 1313−1321. (12) Takagaki, A.; Nanjo, F. Effects of metabolites produced from

456

(−)-epigallocatechin gallate by rat intestinal bacteria on angiotensin I-converting

457

enzyme activity and blood pressure in spontaneously hypertensive rats. J. Agric. Food

458

Chem. 2015, 63, 8262−8266.

459

(13) Buttgereit, F.; Burmester, G. R.; Brand, M. D. Bioenergetics of immune

460

functions: fundamental and therapeutic aspects. Immunol. Today 2000, 21, 192−199.

461

(14) Kowalski, R. J. In vitro CMITM: rapid assay for measuring cell-mediated

462

immunity. Luminescence Biotechnology Instruments and Applications; Van Dyke, K.,

463

Van Dyke, C., Woodfork, K.; CRC Press, 2002; 331−344.


Page 20 of 33

Page 21 of 33


464

(15) Sottong, P. R.; Rosebrock, J. A.; Britz, J. A.; Kramer, T. R. Measurement of

465

T-lymphocyte responses in whole blood cultures using newly synthesized DNA and

466

ATP. Clin. Diag. Lab. Immunol. 2000, 7, 307−311.

467 468 469

(16) Halsey, J. F. New methods, clinical applications of T cell functional assays. Adv. Lab. 2001, 10, 67−72. (17) Hooper, E.; Hawkins, D. M.; Kowalski, R. J.; Post, D. R.; Britz, J. A.; Brooks,

470

K. C.; Turman, M. A. Establishing pediatric immune response zones using the Cylex

471

ImmuKnow assay. Clin Transplant. 2005, 19, 834−839.

472

(18) Shino, M. Y.; Weigt, S. S.; Saggar, R.; Elashoff, D.; Derhovanessian, A.;

473

Gregson, A. L.; Saggar, R.; Reed, E. F.; Kubak, B. M.; Lynch, J. P. 3rd; Belperio, J. A.;

474

Ardehali, A.; Ross, D. J. Usefulness of immune monitoring in lung transplantation

475

using adenosine triphosphate production in activated lymphocytes. J Heart Lung

476

Transplant. 2012, 31, 996−1002.

477

(19) Vivier, E.; Raulet, D. H.; Moretta, A.; Caligiuri, M. A.; Zitvogel, L.; Lanier, L.

478

L.; Yokoyama, W. M.; Ugolini, S. Innate or adaptive immunity? The example of

479

natural killer cells. Science 2011, 331, 44−49.

480

(20) Hu, J. Y.; Zhang, J.; Cui, J. L.; Liang, X. Y.; Lu, R.; Du, G. F.; Xu, X. Y.; Zhou,

481

G. Increasing CCL5/CCR5 on CD4+ T cells in peripheral blood of oral lichen planus.

482

Cytokine 2013, 62, 141−145.

483

(21) Imai, K.; Matsuyama, S.; Miyake, S.; Suga, K.; Nakachi, K. Natural cytotoxic

484

activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up

485

study of a general population. Lancet 2000, 356, 1795−1799.



486

(22) Smyth, M. J.; Hayakawa, Y.; Takeda, K.; Yagita, H. New aspects of

487

natural-killer-cell surveillance and therapy of cancer. Nat. Rev. Cancer 2002, 2,

488

850−861.

489

(23) Coca, S.; Perez-Piqueras, J.; Martinez, D.; Colmenarejo, A.; Saez, M. A.;

490

Vallejo, C.; Martos, J. A.; Moreno, M. The prognostic significance of intratumoral

491

natural killer cells in patients with colorectal carcinoma. Cancer 1997, 79,

492

2320−2328.

493

(24) Ishigami, S.; Natsugoe, S.; Tokuda, K.; Nakajo, A.; Che, X.; Iwashige, H.;

494

Aridome, K.; Hokita, S.; Aikou, T. Prognostic value of intratumoral natural killer cells

495

in gastric carcinoma. Cancer 2000, 88, 577−583.

496

(25) Villegas, F. R.; Coca, S.; Villarrubia, V. G.; Jiménez, R.; Chillón, M. J.;

497

Jareño, J.; Zuil, M.; Callol, L. Prognostic significance of tumor infiltrating natural

498

killer cells subset CD57 in patients with squamous cell lung cancer. Lung Cancer

499

2002, 35, 23−28.

500

(26) Takagaki, A.; Kato, Y.; Nanjo, F. Isolation and characterization of rat

501

intestinal bacteria involved in biotransformation of (-)-epigallocatechin. Arch

502

Microbiol. 2014, 196, 681−695.

503

(27) Yang, C. S.; Lambert, J. D.; Hou, Z.; Ju, J.; Lu, G.; Hao, X. Molecular targets

504

for the cancer preventive activity of tea polyphenols. Mol. Carcinog. 2006, 45,

505

431−435.

506 507

(28) Chen, L.; Lee, M.-J.; Li, H.; Yang, C. S. Absorption, distribution, and elimination of tea polyphenols in rats. Drug Metab. Dispos. 1997, 25, 1045−1050.


Page 22 of 33

Page 23 of 33


508

(29) Kohri, T.; Matsumoto, N.; Yamakawa, M.; Suzuki, M.; Nanjo, F.; Hara, Y.;

509

Oku, N. Metabolic fate of (−)-[4-3H]epigallocatechin gallate in rats after oral

510

administration. J. Agric. Food Chem. 2001, 49, 4102− 4112.

511

(30) Kohri, T.; Suzuki, M.; Nanjo, F. Identification of metabolites of

512

(−)-epicatechin gallate and their metabolic fate in the rat. J. Agric. Food Chem. 2003,

513

51, 5561−5566.

514

(31) Takagaki, A.; Otani, S.; Nanjo, F. Antioxidative activity of microbial

515

metabolites of (−)-epigallocatechin gallate produced in rat intestines. Biosci.

516

Biotechnol. Biochem. 2011, 75, 582−585.

517

(32) Lambert, J. D.; Rice, J. E.; Hong, J.; Hou, Z.; Yang, C. S. Synthesis and

518

biological activity of the tea catechin metabolites, M4 and M6 and their

519

methoxy-derivatives. Bioorg. Med. Chem. Lett. 2005, 15, 873−876.

520

(33) Unno, T.; Tamemoto, K.; Yayabe, F.; Kakuda, T. Urinary excretion of

521

5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, a ring-fission metabolite of

522

(−)-epicatechin, in rats and its in vitro antioxidant activity. J. Agric. Food Chem.

523

2003, 51, 6893−6898.

524

(34) Uhlenhut, K.; Högger, P. Facilitated cellular uptake and suppression of

525

inducible nitric oxide synthase by a metabolite of maritime pine bark extract

526

(pycnogenol). Free Radical Biol. Med. 2012, 53, 305−313.

527

(35) Sang, S.; Lee, M.-J.; Yang, I.; Buckley, B.; Yang, C. S. Human urinary

528

metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray

529

ionization tandem mass spectrometry with data-dependent acquisition. Rapid

530

Commun. Mass Spectrom. 2008, 22, 1567−1578.



531

(36) Del Rio, D.; Calani, L.; Cordero, C.; Salvantore, S.; Pellegrini, N.; Brighenti,

532

F. Bioavailability and catabolism of green tea flavan-3-ols in humans. Nutrition 2010,

533

26, 1110−1116.

534

(37) Kohri, T.; Matsumoto, N.; Yamakawa, M.; Suzuki, M.; Nanjo, F.; Hara, Y.;

535

Oku, N. Metabolic fate of (−)-[4-3H]epigallocatechin gallate in rats after oral

536

administration. J. Agric. Food Chem. 2001, 49, 4102−4112.

537

(38) Li, C.; Lee, M.-J.; Sheng, S.; Meng, X.; Prabhu, S.; Winnik, B.; Huang, B.;

538

Chung, J. Y.; Yan, S.; Ho, C.-T.; Yang, C. S. Structural identification of two

539

metabolites of catechins and their kinetics in human urine and blood after tea

540

ingestion. Chem. Res. Toxicol. 2000, 13, 177−184.

541

(39) Suzuki, Y.; Orellana, M. A.; Schreiber, R. D.; Remington, J. S.

542

Interferon-gamma: the major mediator of resistance against Toxoplasma gondii.

543

Science 1988, 240, 516−518.

544 545 546 547

(40) Zhu J, Paul WE. CD4 T cells: fates, functions, and faults. Blood 2008, 112, 1557−1569. (41) Pahl, J.; Cerwenka, A. Tricking the balance: NK cells in anti-cancer immunity. Immunobiology 2015 pii: S0171-2985, 30031−0.

548

(42) Zhou, Q.; Li, H.; Zhou, J. G.; Ma, Y.; Wu, T.; Ma, H. Green tea, black tea

549

consumption and risk of endometrial cancer: a systematic review and meta-analysis.

550

Arch Gynecol Obstet. 2016, 293, 143−155.

551

(43) Henning, S. M.; Wang, P.; Said, J. W.; Huang, M.; Grogan, T.; Elashoff, D.;

552

Carpenter, C. L.; Heber, D.; Aronson, W. J. Randomized clinical trial of brewed green


Page 24 of 33

Page 25 of 33


553

and black tea in men with prostate cancer prior to prostatectomy. Prostate 2015, 75,

554

550−559.

555

(44) Yin, X.; Yang, J.; Li, T.; Song, L.; Han, T.; Yang, M.; Liao, H.; He, J.; Zhong,

556

X. The effect of green tea intake on risk of liver disease: a meta analysis. Int J Clin

557

Exp Med 2015, 8, 8339−8346.

558

(45) Yamada, S.; Tsukamoto, S.; Huang, Y.; Makio, A.; Kumazoe, M.; Yamashita,

559

S.; Tachibana, H. Epigallocatechin-3-O-gallate up-regulates microRNA-let-7b

560

expression by activating 67-kDa laminin receptor signaling in melanoma cells. Sci

561

Rep. 2016, 6, 19225.

562

(46) Huang, Y.; Kumazoe, M.; Bae, J.; Yamada, S.; Takai, M.; Hidaka, S.;

563

Yamashita, S.; Kim, Y.; Won, Y.; Murata, M.; Tsukamoto, S.; Tachibana, H. Green tea

564

polyphenol epigallocatechin-O-gallate induces cell death by acid sphingomyelinase

565

activation in chronic myeloid leukemia cells. Oncol. Rep. 2015, 34, 1162−1168.

566 567 568 569 570

(47) Robertson, M. J. Role of chemokines in the biology of natural killer cells. J Leukoc Biol. 2002, 71, 173−183. (48) Sinija, V. R.; Mishra, H. N. Green tea: Health benefits. J Nutr Environ Med 2008, 17, 232−242. (49) He, X. S.; Draghi, M.; Mahmood, K.; Holmes, T. H.; Kemble, G. W.; Dekker,

571

C. L.; Arvin, A. M.; Parham, P.; Greenberg, H. B. T cell-dependent production of

572

IFN-gamma by NK cells in response to influenza A virus. J Clin Invest. 2004, 114,

573

1812−1819.



574

(50) Kuo, C. L.; Chen, T. S.; Liou, S. Y.; Hsieh, C. C. Immunomodulatory effects of

575

EGCG fraction of green tea extract in innate and adaptive immunity via T regulatory

576

cells in murine model. Immunopharmacol Immunotoxicol 2014, 36, 364−370.


Page 26 of 33

Page 27 of 33


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

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