Piceatannol inhibits effector T cell functions by suppressing TcR signaling

International Immunopharmacology 25 (2015) 285–292

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Piceatannol inhibits effector T cell functions by suppressing TcR signaling Do-Hyun Kim a,b,1, Yong-Gab Lee c,d,1, Hong-Jai Park a,b, Jung-Ah Lee a,b, Hyun Jung Kim e, Jae-Kwan Hwang f,⁎, Je-Min Choi a,b,⁎⁎ a

Department of Life Science, Hanyang University, Seoul 133-791, South Korea Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, South Korea Department of Biomaterials Science and Technology, Yonsei University, Seoul 120-749, South Korea d OTTOGI Research Institute, OTTOGI Corporation, Gyeonggi-do 431-070, South Korea e Food Safety Research Group, Korea Food Research Institute, 1201-62, Anyangpangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-746, South Korea f Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, South Korea b c

a r t i c l e

i n f o

Article history: Received 22 October 2014 Received in revised form 30 December 2014 Accepted 30 January 2015 Available online 9 February 2015 Keywords: Piceatannol T cells TcR signaling T cell proliferation T cell differentiation

a b s t r a c t Piceatannol, a metabolite of resveratrol found in red wine and grapes, displays a wide spectrum of biological activity. Although the anti-oxidant, anti-inflammatory, and anti-tumorigenesis activity of piceatannol has been extensively studied, its role in the adaptive immune response has received less attention. Here we investigated the role of piceatannol, a well-known Syk inhibitor, in T cell activation, proliferation, and differentiation using isolated murine splenic T cells from C57BL/6 mice. Piceatannol treatment inhibited surface expression of CD4 and CD8 T cell activation markers CD25 and CD69, reduced production of cytokines IFNγ, IL-2, and IL-17, and suppressed proliferation of activated T cells. Moreover, piceatannol treatment significantly inhibited differentiation of CD4+CD25−CD62L+ naïve CD4 T cells into Th1, Th2, and Th17 cells, presumably due to inhibition of TcR signaling through p-Erk, p-Akt, and p-p38. Piceatannol appears to be a useful nutritional or pharmacological biomolecule that regulates effector T cell functions such as cytokine production, differentiation, and proliferation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Piceatannol, a natural analogue of resveratrol with a stilbene-based backbone with four hydroxyl groups (Supplementary Fig. 1), was originally found in the roots of Japanese knotweed and is prevalent in grapes and red wine [1–4]. Many studies have reported that piceatannol displays a wide spectrum of biological activity. The anti-oxidant effects of piceatannol mainly originate from the easily oxidized catechol group [5] and have been reported in hydrogen peroxide-treated human lung fibroblasts [6]. In addition, piceatannol inhibited CHP-induced cellular radical generation without significant cytotoxicity in C6 astroglioma cells [7]. A mechanism for the anti-oxidative effects of piceatannol through suppression of mushroom tyrosinase activity was recently described in B16 melanoma cells [8]. In addition, piceatannol increased apoptosis through increased expression of Bid, Bok, and microRNA-129 [9, 10] and plays regulatory roles in tumorigenesis and inflammation. ⁎ Correspondence to: J.-K. Hwang, B510, Engineering Building #2, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 134 Sinchon-dong, Seodaemun-gu, Seoul 120-749, South Korea. Tel.: +82 2 2123 5881. ⁎⁎ Correspondence to: J.-M. Choi, #519, Natural Science Building, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 133-791, South Korea. Tel.: +82 2 2220 4765; fax: +82 2 2200 3495. E-mail addresses: [email protected] (J.-K. Hwang), [email protected] (J.-M. Choi). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.intimp.2015.01.030 1567-5769/© 2015 Elsevier B.V. All rights reserved.

Increased caspase activity is found in piceatannol-treated tumor cells [10,11], and piceatannol specifically inhibits the Wnt/β-catenin signaling critical for melanoma cell growth [12]. Piceatannol also reduced iNOS induction in LPS-sensitized macrophages [13] and suppressed TNF-α induced NF-κB activation through inhibition of IκB kinase and phosphorylation of p65, as well as the cytokine-mediated JAK/STAT signaling pathway [14,15]. Importantly, piceatannol is a well-known inhibitor of Syk [16,17], a critical kinase involved in immune recognition receptor signaling [18,19]. Despite extensive study of the biological functions of piceatannol, its role in T cell immunity has not yet been fully described. Previous studies of the Syk inhibitory function of piceatannol prompted us to investigate the regulatory role of piceatannol in CD4 or CD8 T cell activation, proliferation, and differentiation with isolated murine splenocytes or MACS-purified naïve CD4 T cells (CD4+CD25− CD62L+). The SH2 domain of Syk, along with ZAP-70, interacts with ITAM (Immuno-receptor Tyrosine-based Activation Motif) in T cell receptors or costimulatory molecules to transmit signals downstream. Here, we used both TcR dependent (anti-CD3/28 antibodies) and independent (PMA/ionomycin) stimulation to determine that piceatannol inhibited activation marker CD25 and CD69 expression on both CD4 and CD8 T cells upon TcR dependent and independent stimuli. The presence of piceatannol down-regulated the production of cytokines IFNγ, IL-17, and IL-2 and significantly reduced PMA/ionomycin activated T cell proliferation. Moreover, piceatannol inhibited differentiation of

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naïve CD4 T cells into Th1, Th2, and Th17 cells with a reduction of p-Erk, p-Akt, and p-p38 in activated T cells, suggesting that piceatannol inhibits effector T cell functions via regulation of the TcR signaling pathway. 2. Materials and methods 2.1. Animals Female C57BL/6 mice, 6 to 8 weeks old, were obtained from Orient Bio (Daejeon, Korea). The mice were housed and maintained in a specific pathogen-free facility at Hanyang University in controlled conditions with temperature (21 ± 1 °C), humidity (50 ± 5%), and 12 h light/dark cycle with regular chow (PicoLab Rodent Diet) and autoclaved water. All animal protocols used in this study were approved by the Hanyang Animal Care and Use Committee. 2.2. Reagents Endotoxin-free piceatannol was purchased from Sigma Aldrich (St. Louis, MO), dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution, and stored at 4 °C. PMA and ionomycin (Sigma Aldrich) were stored at −20 °C. RPMI 1640, penicillin/streptomycin and FBS were purchased from HyClone (South Logan, UT). Mouse anti-CD3 (145-2C11), anti-CD28 (37.51) antibodies, mouse recombinant IL-6, and anti-IFNγneutralizing and anti-IL-4-neutralizing antibodies were obtained from BD Biosciences (Franklin Lakes, NJ) and recombinant mouse IL-12, IL4, IL-6, and IL-23 cytokines were acquired from eBioscience (San Diego, CA). Recombinant IL-2 was obtained from Peprotech (Rocky Hill, NJ), and TGFβ1 and IL-1β cytokines were acquired from R&D systems (Minneapolis, MN). Annexin-V staining and intracellular cytokine fixation kits were purchased from BD Biosciences. Fluorescently labeled anti-mouse antibodies against CD69 (FITC), IFNγ (FITC), CD4 (PE and PerCP-Cy5.5), IL-4 (PE), CD8 (PerCP-Cy5.5), CD25 (APC), and IL-17A (APC), as well as Cell Stimulation Cocktail with protein transport inhibitors were obtained from eBioscience. CCK-8 was purchased from Dojindo (Kumamoto, Japan) and Dynabeads were obtained from Invitrogen (Life Technologies, Carlsbad, CA). For Western blot, anti-pErk, anti-p-Akt, anti-p-p38 and anti-rabbit-HRP antibodies were purchased from Cell Signaling Technology (Danvers, MA), and anti-βactin-HRP was obtained from Santa Cruz (Dallas, TX). For naïve CD4 T cell sorting, a Naïve T cell Isolation Kit II and LS column were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). 2.3. T cell activation Spleens were isolated from 6 to 8 week-old female C57BL/6 mice and RBCs were lysed with ACK buffer for 1 min at room temperature. The cells were washed with PBS, re-suspended in RPMI 1640 media with 10% FBS and 1% penicillin/streptomycin (complete media), and pre-incubated with the indicated concentration of piceatannol for 1 h at 37 °C. The cells were then washed with complete media and stimulated with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies or PMA (10 ng/ml) and ionomycin (400 ng/ml) in 96-well plates for 24 h at 37 °C. The cells were stained with antimouse CD4-PE, CD8-PerCP-Cy5.5, CD69-FITC, and CD25-APC antibodies for 15 min and analyzed by flow cytometry. IL-2, IFNγ, and IL-17 expression in culture supernatants was measured by ELISA. 2.4. CFSE assay To measure cell proliferation, prepared splenocytes were stained with CFSE (1.5 μM) for 10 min at 37 °C and then washed with complete media. The cells were pre-incubated with piceatannol for 1 h at 37 °C, and then stimulated with plate bound anti-CD3 and soluble anti-CD28 antibodies or PMA and ionomycin for 3 or 5 days. The dividing cells

were analyzed by flow cytometry following staining with anti-mouse CD4-PerCP-Cy5.5 and anti-mouse CD8-APC antibodies. 2.5. Cytotoxicity assay Splenocytes were incubated with complete media containing CCK-8 for 3 h, and the accumulated live cells were determined at an optical density of 450 nm. To measure apoptosis, cells were stained with Annexin-V-PE and 7-AAD-PerCP-Cy5.5 for 15 min, and then analyzed by flow cytometry. 2.6. T cell differentiation Naïve CD4 T cells were isolated using the mouse CD4+CD25− CD62L+ T Cell Isolation Kit II according to the manufacturer's protocols. The purity was around 95%. Purified naïve CD4 T cells were preincubated with piceatannol for 1 h. The cells were differentiated under exposure to the following cytokines for 5 days: IL-12 (2 ng/ml), IL-2 (50 U/ml), and anti-IL-4 (5 μg/ml) for Th1; IL-4 (30 ng/ml), IL-2 (50 U/ml), and anti-IFNγ (5 μg/ml) for Th2; and TGF-β (0.5 ng/ml), IL6 (30 ng/ml), IL-23 (20 ng/ml), IL-1β (20 ng/ml), anti-IFNγ (5 μg/ml), and anti-IL-4 (5 μg/ml) for Th17 differentiation. To determine intracellular cytokine levels, the cells were re-stimulated with eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors) for 5 h, and then stained with anti-mouse CD4-PerCP-Cy5.5 for 15 min. The cells were further fixed, permeabilized, and stained with anti-mouse IFNγ-FITC, IL-4-PE, and IL-17-APC antibodies and then analyzed by flow cytometry. IFNγ, IL-13, and IL-17 were measured in culture supernatants by ELISA. 2.7. Western blot MACS-purified naïve CD4 T cells were pre-incubated with 20 μM of piceatannol for 1 h, and then activated with anti-CD3 and anti-CD28 antibody-coated Dynabeads at 37 °C for 0, 5, 10 and 20 min. The cells were lysed with RIPA buffer (Cell Signaling Technology) on ice for 30 min and the amount of protein in the lysate was determined by Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). After SDS-PAGE, proteins were transferred onto a PVDF membrane (Millipore, Temecula, CA). The membrane was blocked with skim milk in Tris buffered saline containing 0.1% Tween-20 and 5% BSA. After blocking, the membrane was incubated with anti-p-Erk, p-Akt, and p-p38 antibodies overnight at 4 °C and anti-β-actin antibody for 1 h at room temperature. Next, the membrane was incubated with HRPconjugated anti-rabbit secondary antibody for 1 h at room temperature. Band intensity was measured by Fusion-Solo and gated band intensity from the acquired image was analyzed by Fusion-capt advance (Vilber Lourmat). 2.8. Quantitative real-time PCR mRNA from differentiated helper T cells was isolated with an RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. cDNA was synthesized by ReverTra Ace qPCR RT master mix (Toyobo, Osaka, Japan). Real-time PCR was performed on a Bio-Rad CFX Connect real-time PCR detection system using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The primer sequences used were: T-bet (forward): 5′-AGCAAGGACGGCGAATGTT-3′; T-bet (reverse): 5′GGGTGG ACATATAAGCGGTTC-3′; GATA3 (forward): 5′-CTCGGCCATT CGTACATGGAA-3′; G ATA3 (reverse): 5′-GGATACCTCTGCACCGTAGC3′; RORγt (forward): 5′-GACCCACA CCTCACAAATTGA-3′; RORγt (reverse): 5′-AGTAGGCCACATTACACTGCT-3′; β-actin (forward): 5′-TGTC CCTGTATGCCTCTGGT-3′; β-actin (reverse): 5′-CACGCACGATTT CCCT CTC-3′.

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Fig. 1. Piceatannol inhibits expression of CD25 and CD69 in activated splenic CD4 T cells. Mouse splenocytes were pre-treated with piceatannol for 1 h and then stimulated with anti-CD3/ 28 antibodies or PMA/ionomycin for 24 h. The expression level of (A, B) CD25 and (C, D) CD69 gated from CD4 positive cells was analyzed by flow cytometry. Data are represented as mean ± SD of three independent experiments. *p b 0.05, **p b 0.01 versus DMSO control.

All data were analyzed by two-tailed Student's t-test using Prism5 (GraphPad). p-Values of less than 0.05 were considered statistically significant.

independent stimuli (Fig. 2A, B). CD69 expression on CD8 T cells was also down-regulated by piceatannol in a concentration dependent manner (Fig. 2C–D). Collectively, piceatannol inhibited the expression of CD25 and CD69 on activated CD4 and CD8 T cells, suggesting that piceatannol could be a negative regulator of T cell activation.

3. Results

3.2. Piceatannol negatively regulates cytokine production and proliferation in activated CD4 and CD8 T cells

2.9. Statistical analysis

3.1. Piceatannol inhibits activation marker expression on activated CD4 and CD8 T cells To investigate the role of piceatannol in T cells, cell surface expression levels of activation markers CD25 and CD69 were measured by flow cytometry in splenocytes pre-treated with piceatannol and stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies (TcR dependent) or PMA/ionomycin (TcR independent) for 24 h. Expression of CD25, the IL-2 receptor alpha chain, was significantly induced in activated CD4 T cells, but down-regulated by pre-treatment with 20 or 40 μM of piceatannol (Fig. 1A, B). In addition, expression of the early T cell activation marker CD69 was also reduced by piceatannol (Fig. 1C, D), suggesting that piceatannol regulates early activation of CD4 T cells. Similarly, piceatannol pre-treatment of splenocytes led to reduced expression of CD25 on activated CD8 T cells upon TcR dependent and

To determine whether piceatannol inhibits cytokine production from activated T cells, we analyzed culture supernatants from activated splenocytes. As shown in Fig. 3A, pre-treatment with both 20 and 40 μM of piceatannol significantly reduced IL-2 production upon stimulation with both anti-CD3/CD28 and PMA/ionomycin. Production of the inflammatory cytokines IFNγ and IL-17 by activated T cells was also significantly down-regulated by piceatannol pre-treatment (Fig. 3B, C), suggesting that piceatannol negatively regulates cytokine production in activated T cells in both a TcR dependent and independent manner. Next, to investigate the effect of piceatannol on proliferation of activated T cells, splenocytes were pre-treated with 5–40 μM of piceatannol for 1 h then stimulated with anti-CD3/28 or PMA/ ionomycin for 96 h. Based on the CCK-8 assay, there was an increase of accumulated live cells in activated splenocytes, but pre-treatment

Fig. 2. Piceatannol inhibits expression of CD25 and CD69 in activated splenic CD8 T cells. Mouse splenocytes were pre-treated with piceatannol for 1 h and then stimulated with anti-CD3/ 28 antibodies or PMA/ionomycin for 24 h. The expression level of (A, B) CD25 and (C, D) CD69 gated from CD8 positive cells was analyzed by flow cytometry. Data are represented as mean ± SD of three independent experiments. *p b 0.05, **p b 0.01 versus DMSO control.

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with 20–40 μM piceatannol significantly reduced the accumulation of live cells following 96 h of stimulation (Supplementary Fig. 2). This finding suggests that piceatannol might suppress activated T cell proliferation rather than induce cell death. To confirm, CFSE-labeled splenocytes were stimulated with PMA/ionomycin for 3 and 5 days following pre-treatment with 5–40 μM of piceatannol. Flow cytometric analysis demonstrated that division of both CD4 (Fig. 4A, B) and CD8 (Fig. 4C, D) T cells was significantly reduced by piceatannol, providing further evidence that it negatively regulates activated T cell proliferation. 3.3. Piceatannol inhibits CD4 T cell differentiation into Th1, Th2, and Th17 Based on previous reports that high dose piceatannol induces apoptosis in leukemia cells [20], we examined apoptosis in splenocytes. We found that 20 μM piceatannol did not induce apoptosis, while 40 μM led to about a 2 fold increase of Annexin-V/7-AAD double positive cells (Supplementary Fig. 3). Accordingly, we chose 20 μM of piceatannol to test the next hypothesis. Because TcR signaling is critical for differentiation of naïve CD4 T cells into Th1, Th2, and Th17 effector cells, we hypothesized that piceatannol would strongly inhibit effector T cell differentiation. CD4+CD25−CD62L+ naïve T cells were isolated by MACS, then pre-treated with 20 μM piceatannol for 1 h. Those cells were then skewed to Th1, Th2, and Th17 cells by exposure to specific

media, and the cytokine producing cells were analyzed by flow cytometry. As shown in Fig. 5A and B, IFNγ, IL-4 and IL-17 production by each skewed T cell subset was significantly down-regulated by piceatannol pre-treatment. Interestingly, piceatannol did not regulate the lineage specific transcription factor of T-bet or GATA3 mRNA expression in Th1 and Th2 cells, respectively, but did significantly reduce RORγt mRNA in Th17 cells, suggesting that there might be specific regulation by piceatannol in Th17 cells (Fig. 5C). To address the question of whether the effect of piceatannol on T cell differentiation was due to inhibition of early T cell activation, we examined accumulated cytokines in cultured supernatants from Th1, Th2, and Th17 cells from days 2 to 5 following stimulation (Fig. 5D–F). Piceatannol significantly inhibited effector T cell differentiation on day 2 of activation, with reduction in accumulated cytokines in the supernatant by day 5. These results suggest that piceatannol can critically impact effector CD4 T cell differentiation through regulation of early T cell activation with possible specificity on Th17 differentiation. 3.4. Piceatannol inhibits phosphorylation of Erk, Akt, and p38 following TcR stimulation Since piceatannol is a well-known Syk inhibitor and Syk is an important kinase in the TcR signaling pathway for cytokine production,

Fig. 3. Piceatannol negatively regulates cytokine production in splenocytes upon TcR dependent or independent stimuli. Production of (A) IL-2, (B) IFNγ, and (C) IL-17 in the supernatants of anti-CD3/28 or PMA/ionomycin stimulated splenocytes for 96 h was evaluated by ELISA. Data are represented as mean ± SD of three independent experiments. *p b 0.05, **p b 0.01 versus DMSO control.

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Fig. 4. Piceatannol negatively regulates proliferation of activated T cells. CFSE-labeled splenocytes were stimulated with PMA/ionomycin for 3 or 5 days and cell division of (A, B) CD4 and (C, D) CD8 positive cells was evaluated by flow cytometry. Data are represented as mean ± SD of three independent experiments. *p b 0.05, **p b 0.01 versus DMSO control.

proliferation, and differentiation [18,19], we hypothesized that TcR downstream signaling, including Erk, Akt, and p38, would be regulated by piceatannol treatment. We stimulated MACS-purified naïve CD4 T cells with anti-CD3/28-coated beads following 20 μM piceatannol pretreatment. As shown in Fig. 6A, piceatannol pre-treatment significantly reduced band intensity for phosphorylated Erk, Akt, and p38 compared to DMSO pre-treatment. Densitometric quantitative analysis normalized to β-actin suggested that piceatannol inhibited those signaling molecules with different kinetics. p-Erk was down-regulated after 10– 20 min, p-Akt was significantly reduced after 20 min, and p-p38 was reduced after 5 min (Fig. 6B–D). The raw densitometer band intensity data without normalization are summarized in Supplementary Table 1. Collectively, these results suggest that piceatannol negatively regulates components of the common TcR signaling pathway such as Erk, Akt, and p38 phosphorylation, which could result in a reduction in cytokine production, proliferation, and differentiation in effector T cells.

4. Discussion In the present study, we investigated the regulatory effect of piceatannol on activated T cells using murine splenocytes and naïve CD4 T cells. Piceatannol pre-treatment inhibited expression of activation markers CD25 and CD69 on both CD4 and CD8 T cells with a reduction in cytokine expression, including IL-2, IFNγ, and IL-17, upon both TcR dependent and independent stimuli. In addition, piceatannol pretreatment negatively regulated proliferation of activated T cells and effector CD4 T cell differentiation into Th1, Th2, and Th17 cells. We propose that piceatannol suppresses phosphorylation of Erk, Akt, and p38 to regulate effector T cell functions.

Piceatannol, abundant in grapes and red wine, is an analogue and metabolite of resveratrol, which has a wide spectrum of biological activities and one less hydroxyl group than piceatannol. Piceatannol consists of a stilbene structure with hydroxyl groups, and the function of piceatannol and other polyhydroxylated stilbenes is associated with the hydrogen bonding capability and stable resonance structure based on the location and number of phenolic hydroxyl groups [21]. The similarity of the chemical structures of piceatannol and resveratrol correlates to similar biological effects, such as antioxidative activities [5,6,8,22–26]. Resveratrol inhibited IL-2, IL-4, and IFNγ cytokine production in anti-CD3/28 stimulated human PBMCs [27]. In addition, resveratrol and curcumin treatment reduced T cell proliferation and cytokine production through suppression of CD28 expression [28]. More recently, one report suggested that the inhibitory function of resveratrol in activated T cells could be related to up-regulated Sirt1 activity [29]. Piceatannol also induced expression of Sirt1 in THP-1 cells [30], suggesting that, like resveratrol, piceatannol may regulate T cell activation via regulation of Sirt1 expression or activity. The Syk family tyrosine kinase is a critical initiator of the TcR proximal signaling pathway [18,19]. Upon TcR stimulation, Fyn and lck phosphorylates ITAMs in the TcR which recruit Syk/ZAP-70 to phosphorylate SLP-76 [31] and LAT [32]. This proximal TcR signaling is critical to recruiting PLCγ, which propagates further signaling and eventually activates transcription factors such as NFAT, NF-κB, and AP-1, resulting in cytokine production, proliferation and differentiation into effector T cells [33–36]. Piceatannol is a well-known Syk inhibitor [37,38] that reportedly regulates LFA-1 dependent migration of TAM2D2 T cell hybridoma cells by blocking ZAP-70 activity [39]. In addition, piceatannol showed inhibitory function on FcεRl-mediated activation of mast cells in a Syk-dependent manner [17]. In the present study, we observed

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Fig. 5. Piceatannol inhibits effector CD4 T cell differentiation. MACS-purified CD4+CD25-CD62L+ naïve T cells were cultured under Th1, 2, and 17 conditions with DMSO or 20 μM piceatannol for 5 days. (A) Intracellular cytokine levels were analyzed by flow cytometry. (B) Quantitative proportion of cytokine producing cells. (C) Relative mRNA expression of lineage specific transcription factors was analyzed by real-time PCR. Production of (D) IFNγ in Th1, (E) IL-13 in Th2, and (F) IL-17 in Th17 conditions was measured by ELISA. Data are represented as mean ± SD of three independent experiments. *p b 0.05, **p b 0.01 versus DMSO control.

that piceatannol pre-treatment significantly reduced effector T cell functions, including cytokine production, proliferation and even differentiation, suggesting that inhibition of proximal TcR signaling by piceatannol may strongly suppress early activation of T cells. At the same time, piceatannol also inhibited T cells stimulated by PMA and ionomycin, which bypass TcR proximal signaling by increasing calcium influx, implying there could be alternative TcR downstream target molecules regulated by piceatannol. Interestingly, piceatannol also reported that it inhibited calcium signaling in a Syk-independent manner [40] and, in one other report, it regulated the function of PI3K in human aortic smooth muscle cells upon PDGF-BB induction [41]. The regulatory effect of piceatannol on PI3K in breast cancer cells has been investigated [42], as well as the inhibitory effect on PKC in PMA-stimulated human neutrophils [43] and on p56lck in the LSTRA leukemic cell line [44]. Accordingly, we expect that piceatannol might regulate multiple kinases in activated T cells to influence the TcR signaling pathway. Although piceatannol strongly suppressed Th1, Th2, and Th17 differentiation in our tests, only RORγt mRNA was significantly reduced by piceatannol treatment, suggesting the possible specific regulation of Th17 differentiation. Piceatannol showed inhibitory effect on tyrosine phosphorylation of STAT3 and STAT5 in the RAMOS cell line [15], and may regulate IL-6-mediated STAT3 phosphorylation in DU145 prostate cancer cells [45]. STAT3 is an important transcription factor under the IL-6 receptor that is critical for Th17 differentiation [46], implying that piceatannol might regulate STAT3 in Th17 cells to inhibit RORγt

expression. We plan to further investigate its role in Th17 cells and autoimmune diseases. Piceatannol has been widely demonstrated to show pro-apoptotic effects in various types of tumor cell lines [5,43,47], with proposed mechanisms of up-regulation of caspase-8, 9 in prostate cancer cells [10] and induction of Fas and FasL expression in U937 leukemia cells [20]. In the present study, we observed that 40 μM of piceatannol induced apoptosis in activated T cells while 20 μM of piceatannol did not. The effect of 40 μM piceatannol on cytokine production and proliferation was extremely strong, possibly due to the apoptosis of activated T cells. However, the effect of 20 μM or less piceatannol appears to be apoptosis-independent regulation of effector T cells via suppression of TcR signaling. One report has suggested a biphasic effect of resveratrol, with enhanced activation of T cells at low doses [27]. Proliferation and cytokine expression of anti-CD3/28-stimulated human PBMCs are lower with 10 μg/ml of resveratrol, while 2.5 μg/ml of resveratrol actually induces more cytokine production. In this study, however, piceatannol did not alter CD25 and CD69 expression and cytokine production at low doses (Supplementary Fig. 4), giving piceatannol a potential advantage over resveratrol as a drug supplement. There have been attempts to apply piceatannol in the disease model of colitis. Oral administration of piceatannol ameliorates tissue inflammation of DSS-induced colitis by inhibition of NF-κB and Erk [48,49]. In addition, subcutaneously injected piceatannol negatively regulates endotoxin-induced ocular inflammation in rats, with reduced cytokine

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Fig. 6. Piceatannol regulates phosphorylation of Erk, Akt and p38 upon TcR stimulation. MACS-purified naïve CD4 T cells were pre-incubated with DMSO or 20 μM piceatannol for 1 h and then activated with anti-CD3/28 coated Dynabeads for 0 to 20 min. (A) p-Erk, p-Akt, and p-p38 protein expression was determined by Western blot analysis. (B–D) Relative densitometric analysis of Western blot results normalized to β-actin. Data are represented as mean ± SD of three independent experiments. *p b 0.05, **p b 0.01 versus DMSO control.

production and NF-κB activation [50]. This reduction in the symptoms of inflammation might also be related to inhibition of effector T cells in vivo by piceatannol. We plan to further investigate the in vivo effect of piceatannol in T cell-mediated allergic or autoimmune diseases. Long-term consumption of a polyphenol-rich diet has been strongly encouraged for benefits to host immunity [51]. Based on our current findings, piceatannol represents a valuable nutritional and pharmacological supplement for regulating abnormal T cell function and related diseases.

Acknowledgments This study is supported by the Basic Science Research Program through the grants from the National Research Foundation of Korea (NRF-2013R1A1A2A10060048) and Hanyang University (HY-201100000001004).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2015.01.030.

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