A Marine Carotenoid, Fucoxanthin, Induces Regulatory T Cells and Inhibits Th17 Cell Differentiation in Vitro

110459 (355)

Biosci. Biotechnol. Biochem., 75 (10), 110459-1–4, 2011 Note A Marine Carotenoid, Fucoxanthin, Induces Regulatory T Cells and Inhibits Th17 Cell Differentiation in Vitro

Tadaomi KAWASHIMA

Research and Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278-0037, Japan

Received June 14, 2011; Accepted July 16, 2011; Online Publication, October 7, 2011 [doi:10.1271/bbb.110459]

Fucoxanthin is a non-provitamin A carotenoid con- subset of integrin chain CD103-expressing dendritic tained in brown seaweeds. We found that it suppressed cells (DCs) was identified in murine mesenteric lymph interleukin-17 secretion from CD4þ T cells under IL-17- nodes.15) CD103þ DCs promote the development of producing T (Th17) cell development conditions. By Foxp3þ Treg cell responses depending on TGF- and evaluating T cell differentiation in vitro, fucoxanthin the dietary metabolite RA.16) There are many reports on and its metabolite fucoxanthinol inhibited T cell differ- the immunological effects of vitamin A and its metab- entiation into Th17 cells. This suggests that fucoxanthin olite, RA, as described above, while no report on the can improve inflammatory diseases due to Th17 cells. inhibitory effect of non-provitamin A carotenoids against Th17 cell development has appeared. Hence Key words: fucoxanthin; interleukin-17; interleukin-17- we evaluated the impact of these carotenoids on Th17 Advanceproducing T (Th17) cell; regulatory View T cell in vitro cell differentiation . To assess the effect of non-provitamin A carotenoids Fucoxanthin is a non-provitamin A carotenoid in as to the suppression of IL-17 production, CD4þ T cells brown seaweeds, Undaria pinnatifida. There are many were collected from spleens of C57BL/6 mice (Japan reports on the beneficial effects of fucoxanthin on Crea, Tokyo) using CD4 microbeads (Miltenyi Biotec, human health including anti-tumor and anti-obesity Bergisch Gladbach, Germany) and MACS (Miltenyi effects.1–3) Recently, a protective effect of topical Biotec). Anti-CD3 and anti-CD28 monoclonal antibod- fucoxanthin treatment against skin photodamage was ies for T cell activation and proliferation were purchased reported.4) As for immunological functions, it has been from eBioscience (San Diego, CA). After culturing found that intravenous injection of fucoxanthin protects CD4þ T cells with non-provitamin A carotenoids the eyes from lipopolysaccharide-induced inflammation (fucoxanthin, astaxanthin,Proofs lycopene, and lutein, from in rats.5) We hypothesized that non-provitamin A Wako, Osaka, Japan) in the presence of IL-6 (20 ng/mL, carotenoids, including fucoxanthin, can affect T cell R&D Systems, Minneapolis, MN) and TGF- (2 ng/mL, differentiation due to a chemical structure similar to R&D Systems) for 3 d, IL-17 production was measured vitamin A, which is metabolized into retinoic acid (RA) by a Mouse IL-17 ELISA Set (eBioscience). All-trans and induces forkhead box P3þ (Foxp3þ) regulatory RA (ATRA, Sigma, St. Louis, MO) was added as T (Treg) cells, resulting in inhibition of interleukin positive control of suppression of Th17 cell differ- 17-producing T (Th17) cell development in vitro and entiation. ATRA and carotenoid solutions were prepared in vivo.6–8) at 5 and 10 mM in dimethyl sulfoxide (DMSO). Then Foxp3þ Treg cells are induced in the presence of these solutions were diluted and added to a culture transforming growth factor (TGF)- and RA in vitro, medium at 2 or 4 mM (final concentration). As control, while Th17 cell development is induced in the presence the same concentration of DMSO (0.04% v/v) was of IL-6 and TGF-.6,7) Elevated levels of IL-17 are added to the culture medium. This concentration of associated with the pathogenesis of many inflammatory DMSO did not affect the experimental model as to diseases, including autoimmune diseases, inflammatory cytotoxicity or cytokine production. IL-17 secretion was bowel diseases, allergic asthma, and atopic dermati- suppressed only by the addition of fucoxanthin in a tis.9–12) Therefore, Th17 cell differentiation or IL-17 dose-dependent manner, while the other carotenoids secretion is a target of therapeutic drugs and foods to be did not suppress IL-17 production by CD4þ T cells used against these inflammatory diseases. Clinical (Fig. 1). To analyze the gene expression of retinoic effects of synthetic retinoid Am80, an agonist of RA acid receptor-related orphan nuclear receptor (ROR)t, receptors, on autoimmune diseases has been found in a transcription factor expressed in Th17 cells,17) mice,13,14) and a commensal bacterium in the intestine of and Foxp3, quantitative RT-PCR was performed infants, Bifidobacterium infantis, has been reported to using PrimeScript RT reagent (Takara, Ohtsu, Japan), suppress IL-17 production ex vivo using a dextran SYBR Premix Ex Taq (Takara), and specific primers sodium sulfate-treated colon as a model of inflammatory as follows: 50-TGTCCTGGGCTACCCTACTG-30 and bowel diseases.15) Recently, a functionally distinct 50-GTGCAGGAGTAGGCCACATT-30 for RORgt, 50-

Correspondence: Tel/Fax: +81-4-7123-5529; E-mail: [email protected] Abbreviations: IL, interleukin; Th17 cell, IL-17-producing T cell; Treg cell, regulatory T cell; RA, retinoic acid; Foxp3, forkhead box P3; ROR, retinoic acid receptor-related orphan nuclear receptor; TGF, transforming growth factor; RT-PCR, reverse transcriptase-polymerase chain reaction 110459-2 T. KAWASHIMA

ATRA ** 250 200 150 100

IL-17 (pg/mL) 50 0 Sample (µM) 0 0 1 IL-6/TGF-β - + + αCD3/αCD28 + + +

Fucoxanthin Astaxanthin ** 250 * 250 200 200 150 150 100 100 IL-17 (pg/mL) 50 IL-17 (pg/mL) 50 0 0 Sample (µM) 0 0 2 4 Sample (µM) 0 0 2 4 IL-6/TGF-β - + + + IL-6/TGF-β - + + + αCD3/αCD28 + + + + αCD3/αCD28 + + + +

Lycopene Lutein Advance250 View250 200 200 150 150 100 100 IL-17 (pg/mL) 50 IL-17 (pg/mL) 50 0 0 Sample (µM) 0 0 2 4 Sample (µM) 0 0 2 4 IL-6/TGF-β - + + + IL-6/TGF-β - + + + αCD3/αCD28 + + + + αCD3/αCD28 + + + +

Fig. 1. Fucoxanthin Suppressed IL-17 Production by CD4þ T Cells. CD4þ T cells (5 105) from the spleens of C57BL/6 mice were cultured with all-trans retinoicProofs acid (ATRA) or carotenoids in the presence of anti-CD3 monoclonal antibody (CD3, plate-bound, 5 mg/mL), anti-CD28 monoclonal antibody (CD28, soluble, 5 mg/mL), IL-6 (20 ng/mL), and TGF- (2 ng/mL) for 3 d. The IL-17 concentration in culture supernatants was determined by ELISA. The data are shown as mean SD of triplicate cell cultures. They are representative of three independent experiments. p < 0:05, p < 0:01 (Student’s t-test).

CCCAGGAAAGACAGCAACCTT-30 and 50-TTCTC- the cells were fixed and permeabilized with a Foxp3 ACAACCAGGCCACTTG-30 for Foxp3, and 50-GCTA- Staining Set (eBioscience) and stained with FITC CAGCTTCACCACCACAG-30 and 50-GGTCTTTAC- conjugated IL-17 antibody (eBioscience) and phycoery- GGATGTCAACGTC-30 for -actin. thrin (PE) conjugated Foxp3 antibody (eBioscience). In Under Th17 cell development conditions, RORt an analysis of Th17 (IL-17þ) and Treg (Foxp3þ) cell expression was suppressed by fucoxanthin (Fig. 2A). populations by FACS (BD Bioscience), fucoxanthin Moreover, in the presence of TGF- without IL-6, gene suppressed Th17 cell development and increased the expression encoding Foxp3 was upregulated by fucox- number of Foxp3þ Treg cells in a dose-dependent anthin supplementation (Fig. 2B). These results indicate manner (Fig. 3). Other carotenoids (astaxanthin, lyco- that fucoxanthin suppresses Th17 cell development and pene, and lutein) did not affect T cell differentiation like induces Foxp3þ Treg cell differentiation as RA. fucoxanthin (data not shown). This result is in accord- Next we evaluated the T cell differentiation of ance with the results for the suppressive effect on IL-17 naı¨ve T cells (CD4þ CD62Lþ T cells) into Th17 cells. production (Fig. 1). In an analysis of T cell differ- Naı¨ve T cells were prepared from C57BL/6 mice entiation, fucoxanthinol (Wako), a metabolite of fucox- by negatively isolating CD4þ T cells using CD4þ T anthin by gut microflora,18) was also tested. It was found cell Isolation Kit II (Miltenyi Biotec), followed by that fucoxanthinol inhibited Th17 cell development as the collection of CD62Lþ cells using fluorescein well as fucoxanthin did (Fig. 3). This indicates that oral isothiocyanate (FITC) conjugated CD62L antibody administration of fucoxanthin should exert the same (eBioscience) and FITC-labeled microbeads (Miltenyi effect as to T cell differentiation after absorption in the Biotec). Naı¨ve T cells were cultured with fucoxanthin in intestine. the presence of IL-6 and TGF- for 3 d, and then the We found that fucoxanthin and its metabolite fucox- cells were stimulated with phorbol-12-myristate-13- anthinol in the gut inhibited Th17 cell differentiation acetate (PMA)/ionomycin (100 ng/mL and 500 ng/mL and induced Treg cell development, resulting in the respectively, Sigma) and GolgiStop (BD Bioscience, suppression of inflammatory IL-17 production. Many San Jose, CA) for intracellular cytokine analysis. Then studies have found that RA receptor agonists such as Fucoxanthin Inhibits Th17 Cell Differentiation 110459-3 A ATRA Fucoxanthin 80 80

60 60

40 40 t expression t expression

Relative γ Relative 20 γ 20 ROR 0 ROR 0 Sample (µM) 0 0 10-3 10-1 10 Sample (µM) 0 0 1 2 4 IL-6/TGF-β - + + + + IL-6/TGF-β - + + + + αCD3/αCD28 + + + + + αCD3/αCD28 + + + + +

B ATRA Fucoxanthin 8 6

6 4 4 Relative Relative 2 2 Foxp3 expression Foxp3 expression 0 0 Sample (µM) 0 0 10-3 10-1 10 Sample (µM) 0 0 1 2 4 TGF-β - + + + + TGF-β - + + + + αCD3/αCD28 + + + + + αCD3/αCD28 + + + + +

Fig. 2. Fucoxanthin Suppressed RORt mRNA Expression and Increased Foxp3 mRNA Expression in CD4þ T Cells. A, CD4þ T cells (5 105) from the spleens of C57BL/6 mice were cultured with all-trans retinoic acid (ATRA) or fucoxanthin in the Advancepresence of anti-CD3 monoclonal antibody (CD3, View plate-bound, 5 mg/mL), anti-CD28 monoclonal antibody (CD28, soluble, 5 mg/mL), IL-6 (20 ng/mL), and TGF- (2 ng/mL) for 3 d. RORt mRNA expression was determined by quantitative RT-PCR. The data are representative of three independent experiments. B, CD4þ T cells (5 105) were cultured with ATRA or fucoxanthin in the presence of CD3, CD28, and TGF- for 3 d. Foxp3 mRNA expression was measured by quantitative RT-PCR. The data are representative of three independent experiments. A and B, The data are shown as expression relative to cells stimulated with CD3/CD28 only.

αCD3 + αCD28

IL-6 + TGF-β Control Control ATRA (1 µM) Proofs 2.1 10.1 1.2

2.4 10.6 38.3 Fucoxanthin Fucoxanthin (2 µM) (4 µM) 7.8 3.8

15.8 23.0

FITC-IL-17 Fucoxanthinol Fucoxanthinol (2 µM) (4 µM) PE-Foxp3 7.1 5.2

18.1 23.3

Fig. 3. Fucoxanthin and Fucoxanthinol Inhibited Th17 Cell Development and Induced Foxp3þ Regulatory T Cell Differentiation. Naı¨ve T cells (5 105) from the spleens of C57BL/6 mice were cultured with all-trans retinoic acid (ATRA), fucoxanthin, and fucoxanthinol in the presence of anti-CD3 monoclonal antibody (CD3, plate-bound, 5 mg/mL), anti-CD28 monoclonal antibody (CD28, soluble, 5 mg/mL), IL-6 (20 ng/mL), and TGF- (2 ng/mL) for 3 d. The cells were collected, incubated with PMA/ionomycin and GolgiStop, and fixed and permeabilized. They were analyzed by FACS staining with FITC conjugated IL-17 antibody and PE conjugated Foxp3 antibody. The data are representative of three independent experiments. Values represent ratios of cell populations. 110459-4 T. KAWASHIMA ATRA and synthetic retinoids suppress Th17 cell References development,6–8,13,14) and hence we assume that fucox- anthin and fucoxanthinol exert activity similar to ATRA 1) Das SK, Hashimoto T, Shimizu K, Yoshida T, Sakai T, Sowa Y, via RA receptors. Although fucoxanthin and fucoxan- Komoto A, and Kanazawa K, Biochim. Biophys. Acta, 1726, 328–335 (2005). thinol were effective at higher concentrations than 2) Maeda H, Hosokawa M, Sashima T, and Miyashita K, J. Agric. ATRA, a difference in affinity for RA receptors might Food Chem., 55, 7701–7706 (2007). be involved in the difference in effective concentrations. 3) Matsumoto M, Hosokawa M, Matsukawa N, Hagio M, Shinoki According to a previous report, the structure, especially A, Nishimukai M, Miyashita K, Yajima T, and Hara H, Eur. J. the H6–H7 loop, is involved in the interaction between Nutr., 49, 243–249 (2010). several synthetic retinoids and RA receptors.19) Thus 4) Urikura I, Sugawara T, and Hirata T, Biosci. Biotechnol. Biochem., 75, 757–760 (2011). structural differences among the tested carotenoids 5) Shiratori K, Ohgami K, Ilieva I, Jin XH, Koyama Y, Miyashita and their different affinities for RA receptors might K, Yoshida K, Kase S, and Ohno S, Exp. Eye Res., 81, 422–428 contribute to their suppressive effects on Th17 cell (2005). development. 6) Elias KM, Laurence A, Davidson TS, Stephens G, Kanno Y, In conclusion, our results suggest that oral admin- Shevach EM, and O’Shea JJ, Blood, 111, 1013–1020 (2008). istration of fucoxanthin inhibits Th17 cell development, 7) Xiao S, Jin H, Korn T, Liu SM, Oukka M, Lim B, and Kuchroo probably through RA receptors, and works against VK, J. Immunol., 181, 2277–2284 (2008). 8) Bai A, Lu N, Guo Y, Liu Z, Chen J, and Peng Z, J. Leukoc. Th17-dependent inflammatory diseases such as auto- Biol., 86, 959–969 (2009). immune diseases, inflammatory bowel diseases, and 9) Zepp J, Wu L, and Li X, Trends Immunol., 32, 232–239 (2011). asthma. In regard to the safety of oral administration of 10) Hemdan NY, Birkenmeier G, Wichmann G, Abu El-Saad AM, fucoxanthin, it has been reported that no abnormal Krieger T, Conrad K, and Sack U, Autoimmun. Rev., 9, 785–792 changes were observed in the liver, kidney, spleen, (2010). or gonadal tissues under oral administration of 1,000 11) Geremia A, Arancibia-Ca´rcamo CV, Fleming MP, Rust N, J. Exp. Med. mg/kg of body weight over 30 d.20) Since it is expected Singh B, Mortensen NJ, Travis SP, and Powrie F, , Advance View208, 1127–1133 (2011). that the RA-like activity of fucoxanthin might affect the 12) Souwer Y, Szegedi K, Kapsenberg ML, and de Jong EC, Curr. homeostasis of vitamin A metabolism, we intend to Opin. Immunol., 22, 821–826 (2010). investigate the in vivo effects of oral administration of 13) Klemann C, Raveney BJ, Klemann AK, Ozawa T, von Ho¨rsten fucoxanthin on inflammatory diseases without side S, Shudo K, Oki S, and Yamamura T, Am. J. Pathol., 174, effects on vitamin A metabolism using mouse model 2234–2245 (2009). such as experimental autoimmune encephalomyelitis or 14) Keino H, Watanabe T, Sato Y, and Okada AA, Invest. Ophthalmol. Vis. Sci., 52, 1548–1556 (2011). experimental colitis. We also intend to investigate the 15) Tanabe S, Kinuta Y, and Saito Y, Int. J. Mol. Med., 22, 181–185 mechanisms of these different activities among fucox- (2008). anthin, fucoxanthionol, and other non-provitamin A 16) Coombes JL, Siddiqui KR, Arancibia-Ca´rcamo CV, Hall J, Sun carotenoids, focusing on structural features and the CM, Belkaid Y, and Powrie F, J. Exp. Med., 204, 1757–1764 affinity for RA receptors. (2007). Proofs 17) Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Acknowledgments Lafaille JJ, Cua DJ, and Littman DR, Cell, 126, 1121–1133 (2006). 18) Asai A, Sugawara T, Ono H, and Nagao A, Drug Metab. We thank Dr. A. Obata, Dr. N. Kajiyama, Dr. I. Dispos., 32, 205–211 (2004). Nishimura, and Mr. K. Matsushima for scientific 19) Mailfait S, Thoreau E, Belaiche D, Formstecher P, and discussion. We also thank Mrs. K. Nagamura for Sablonnie`re B, J. Mol. Endocrinol., 24, 353–364 (2000). technical assistance. 20) Beppu F, Niwano Y, Tsukui T, Hosokawa M, and Miyashita K, J. Toxicol. Sci., 34, 501–510 (2009).