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DOI 10.1002/mnfr.201400909
Mol. Nutr. Food Res. 2015, 59, 1406–1410
FOOD & FUNCTION
Oral administration of apple condensed tannins delays rheumatoid arthritis development in mice via downregulation of T helper 17 (Th17) cell responses Kosuke Nakamura1∗ , Hideki Matsuoka1∗ , Syohei Nakashima2 , Tomomasa Kanda2 , Tomoko Nishimaki-Mogami1 and Hiroshi Akiyama1 1 2
National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan Asahi Group Holdings, Ltd., Moriya, Ibaraki, Japan
Apples are known to contain high concentrations of phenolic compounds such as condensed tannins. Consumption of condensed tannins has been reported to reduce the risk of many types of chronic diseases including allergies. However, their therapeutic effectiveness and potential in treating autoimmune disease remain controversial. Here, the effect of oral administration of apple condensed tannins (ACT) prepared from apples (Malus pumila cv. Fuji) on bovine type II collagen (CII)-induced arthritis in DBA1/J mice, a well-established murine model of human rheumatoid arthritis (RA), was evaluated. As compared to the control (without ACT administration) group, RA development was delayed and a significant reduction in the RA clinical score was observed in the ACT-administered group. Using cultured splenocytes isolated from CII-immunized mice, ACT-administration was shown to decrease the CII-induced increases in IL-17 expression and production in vitro. We propose that downregulation of T helper (Th) 17 cells is responsible for the ACT-induced RA suppression. Keywords: Apple condensed arthritis / T helper
tannins / Oral
Received: December 15, 2014 Revised: March 12, 2015 Accepted: April 7, 2015
administration / Phytochemical / Rheumatoid
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Several lines of evidence indicate that daily incorporation of fruit into the diet may reduce the risk of many types of chronic diseases including allergy [1]. Apples are known to contain high concentrations of phytochemicals, phenolic compounds such as chlorogenic acid, (+)-catechin, (-)-epicatechin, phlorodzin, and rutin [2–4], and condensed tannins such as procyanidins B1, B2, and C1 [5, 6]. The antiallergy activities of condensed tannins purified from apples (apple condensed tannins; ACT) have previously been investigated. The studies demonstrated that ACT inhibits histamine release from cultured rat basophilic leukemia (RBL-2H3) and peritoneal mast cells [7, 8] as well as food-allergen induced Correspondence: Dr. Kosuke Nakamura E-mail: [email protected] Abbreviations: ACT, apple condensed tannins; CIA, collageninduced arthritis; CII, bovine type II collagen; RA, rheumatoid arthritis; Treg, regulatory T; TGF1, transforming growth factor 1; Th, T helper C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
oral sensitization in mice [9,10], and improves the symptoms of atopic dermatitis in humans [11]. Although the source of condensed tannins differs, Miyake et al. also reported that condensed tannins purified from a South American herb, Jatoba, exerted rheumatoid arthritis (RA) inhibition, whereas green tea polyphenols facilitated disease development [12]. However, little is known about the inhibitory mechanism of these condensed tannins in RA, and further investigation is required. To study the effect of ACT on RA severity and incidence, we employed a well-known animal model of human RA (collagen-induced arthritis; CIA) [13], in which RA is induced by injecting bovine type II collagen (CII) into DBA1/J mice (for details, see Supporting Information). ACT was prepared from apples (Malus pumila cv. Fuji) following the previously described method [14], with slight modifications (for details, see Supporting Information Fig. 1). Previous studies ∗ These
authors contributed equally to this study. www.mnf-journal.com
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Figure 1. Effect of ACT-administration on RA severity and incidence in mice. ACT was administered from 2 weeks prior to CII immunization and throughout the experimental period. After RA induction, both ACT-administered and control groups continued to have free access to Mouse Flat diet with the ACT solution and water, respectively. Mice were immunized with CII and received the secondary immunization (day 0) after 3 weeks. The severity (A) and incidence (B) of RA were evaluated by clinical scoring of paw swelling in the wateradministered control group (circle, n = 4) and the ACT-administered group (square, n = 4). ***p < 0.01 as determined by analysis of variance on day 20. (C) Representative clinical signs of RA on day 20. The control group (total arthritis score, 3.8), the ACT-administered group (total arthritis score, 1.1).
on atopic dermatitis patients indicated that itching and sleep disturbance scores after ACT supplement even for 2 weeks were significantly decreased [11]. Also, 1% w/v ACT intake for 2 weeks would inhibit the development of oral sensitization and prevent the development of food allergies in mice [9]. Thus, 1% w/v ACT was ad libitum administered from 2 weeks prior to CII immunization and throughout the experimental period. Each mouse drank 5–7 mL 1% w/v ACT per day as in the case of water, and no significant difference in body weight and the amount of feed intake was observed between the ACT-administered and water-administered (control) group. Figure 1 shows the severity and incidence of RA in the ACT-administered and control groups (four mice in each group) after secondary immunization with CII (day 0). As shown in Fig. 1A, the control group showed rapid RA development and the severity decreased after 3 weeks. Definite acute and chronic phases (days 2–20 and 20–55, respectively) were observed. In the control group, RA developed with joint inflammation of the paws to a maximum mean score (3.5 ± 0.3) by day 20, and a constant decrease in the score to 2.4 ± 0.2 was observed by day 37 (Fig. 1A). The onset of RA in the ACT-administered group was delayed as compared with the control group, and the severity score of RA was reduced to a maximum score of 1.6 ± 0.6 at day 37 (Fig. 1A). Attenuation of RA symptoms continued until the end of the observation period (day 55). In the control group, one of the mice developed RA at day 2 after the secondary CII injection, and the incidence rate increased up to day 12 when all mice showed RA symptoms (Fig. 1B). In the ACT-administered group, one of the mice C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
developed RA at day 7 after the secondary CII immunization. All mice in the ACT-administered group acquired RA by day 49 (Fig. 1B). The incidence of RA was lower as compared with the control group. Representative clinical symptoms of RA on both the forepaws and hindpaws on day 20 are shown in Fig. 1C. Control group showed more severe swelling in digit, wrist, and ankle as compared with the ACT-administered group. In a separate experiment, we confirmed that mice without CII injection did not develop RA, and that ACT administration had no effect (data not shown). Taken together, we found that the oral administration of ACT solution to DBA1/J mice delayed CII-elicited RA development. To evaluate whether the alleviation of RA in the ACTadministered group was associated with altered T helper (Th) responses, splenocytes were isolated at day 14 after primary CII immunization from the ACT-administered and control groups (three mice in each group), and were cultured and restimulated with CII (at a final concentration of 25 g/mL) in vitro. After 72 h culture, cytokine (transforming growth factor 1 [TGF1], IL-17, IL-4, IL-10, and IFN-␥) expression was quantified at the mRNA and protein levels (Fig. 2A; for details, see Supporting Information). Real-time PCR (Fig. 2B and C) showed that, in the control group, CII restimulation significantly induced the expression of IL-17A by approximately tenfold (relative expression from 0.13 ± 0.04 to 1.00 ± 0.25; p < 0.01, n = 6), while TGF1 expression was unchanged. In the ACT-administered group, IL-17A expression was significantly reduced by approximately
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Figure 2. Cytokine expression analysis in cultured splenocytes. (A) Scheme of experimental procedures. (B, C) Cytokine expression measured using real-time PCR. Fourteen days after primary CII immunization in the ACT-administered group and control group, splenocytes were isolated and restimulated for 72 h with CII. Total RNAs were isolated from the cells, and cytokine (B, TGF1; C, IL-17A) mRNA levels were determined in duplicate. Data represent means ± SD (n = 3). Significant differences between groups are indicated.
half as compared with the control group (relative expression from 1.00 ± 0.25 to 0.54 ± 0.35; p < 0.05, n = 6) (Fig. 2C). The TGF1 expression level remained unchanged with or without ACT administration (Fig. 2B). To evaluate cytokine release into the medium, ELISA was used to determine cytokine levels. The concentration of IL17A/F (heterodimer) in the ACT-administered and control groups was 1992 pg/mL and 3054 pg/mL, respectively (Fig. 3B). The concentration of IFN-␥ in the ACT-administered and control groups was 3015 pg/mL and 12520 pg/mL, respectively (Fig. 3D). The levels of IL-17A/F and IFN-␥ in the ACT-administered group were significantly (p < 0.05 and p < 0.01 [n = 6], respectively) lower than those of the control group (Fig. 3B and D). The levels of IL-4 and IL-10 were < 100 pg/mL and 5000 pg/mL, respectively, and no significant differences in those cytokine levels were observed between the ACT-administered and control groups (Fig. 3A and C). Taken together, ACT-administration significantly downregulated the production of both IL-17A and IFN-␥ in splenocytes as compared with the control group. In human RA patients, elevated levels of the proinflammatory cytokine IL17 were reported [15]. In addition, defects in IFN-␥ signaling have been shown to cause uncontrolled IL-17 production [16]. IL-17 has been reported to play an important role in the development of various autoimmune diseases [17]. The IL-17 produced by Th17 cells contributes to both the induction and progression of RA [18, 19]. The development of CIA is suppressed in IL-17-deficient mice [18]. Treatment with IL-17 neutralizing antibodies or IL-17 receptor antagonists reduces the symptoms of RA [19]. Th17 cells simultaneously produce IL-17A and IL-17F, which bind IL-17 receptor complexes consisting of IL-17RA and IL-17RC in the form of heterodimeric or homodimeric complexes of IL-17 A/F [20]. IL-17A plays C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. The effect of ACT-administration on cytokine expression measured using ELISA. Splenocytes were isolated and restimulated with CII as described in Fig. 2. (A) IL-4, (B) IL-17A/F, (C) IL-10, and (D) IFN-␥ released to the culture medium were measured in duplicate. Data represent means ± SD (n = 3). Significant differences between groups are indicated.
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a major role in T cell-dependent autoimmune and allergic responses, whereas IL-17F alone contributes marginally to these responses [21]. In this study, the IL-17A mRNA levels in splenocytes were elevated tenfold after RA induction in vitro. Our results showed that ACT administration inhibited IL-17 expression. IL-17 is known to initiate positive feedback loop mechanisms, leading to the expansion of Th17 cells and thereby increasing the production of IL-17 [22]. Therefore, the oral-administration of ACT may suppress the CII-induced overproduction of Th17 by reducing IL-17 expression. Increases in Th1 cytokines are observed in human RA patients, and the IFN␥/IL-4 ratio is closely related to the ratio of Th1-/Th2-type cytokine production [23]. In this study, ACT-administration significantly decreased IFN-␥ expression, while no significant differences in IL-4 expression were observed, although the levels of secreted IL-4 were very low, suggesting that ACT-administration repressed Th1-type cytokine production. Th2-type cytokines (IL-4 and IL-13) were also detected at mRNA level, but no significant difference was observed upon ACT administration (data not shown). Previous studies have revealed the critical role that IFN-␥ plays in RA development [15, 16, 24, 25]. The development of RA was inhibited by neutralization of IFN-␥ during the induction of CIA (early treatment), while the neutralization of IFN-␥ after the induction of CIA (late treatment) exacerbated the severity of CIA [24]. Thus, our findings suggested that ACT administration prior to RA induction may be effective in attenuating the Th1 response, leading in RA attenuation. Ke et al. demonstrated that anti-CD3 antibody treatment in an experimental model of autoimmune uveitis in mice inhibited disease development by inducing regulatory T (Treg) cells via both IL-10 and TGF1 induction [26]. Also, IL-10 and TGF1 were previously found to protect against experimental autoimmune encephalomyelitis disease severity by suppressing encephalitogenic T effector cells and expanding or sustaining functional Treg cells [27]. Both IL-10 and TGF1 expression levels were not significantly affected by ACT administration in this study. Therefore, RA amelioration by ACT was not mediated by the induction of Treg in splenocytes. Together, oral administration of the 1% w/v ACT solution delayed RA development in DBA1/J mice. IL-17 expression was significantly reduced in cultured splenocytes from ACT-administered mice during collagen-driven T-cell development. Oral administration of ACT likely affected RA responses via downregulation of Th17 cell development. Further studies are required to investigate the ACT’s effective administration amount and method for RA amelioration. This study was supported by a grant from the Japanese Ministry of Health, Labor and Welfare, a grant from the Japan Health Sciences Foundation, and a grant from the Food Safety Commission. The authors have declared no conflict of interest.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
References [1] Boyer, J., Liu R. H., Apple phytochemicals and their health benefits. Nutr. J. 2004, 3, 1–15. [2] Delage, E., Bohuon, G., Baron, A., Drilleau, J. F., Highperformance liquid chromatography of the phenolic compounds in the juice of some French cider apple varieties. J. Chromatogr. A 1991, 555, 125–136. [3] Valles, B. S., Victorero, J. S., Alonso, J. J. M., Gomis, D. B., High-performance liquid chromatography of the neutral phenolic compounds of low molecular weight in apple juice. J. Agric. Food Chem. 1994, 42, 2732–2736. [4] Wilson, E. L., High-pressure liquid chromatography of apple juice phenolic compounds. J. Sci. Food Agric. 1981, 32, 257– 264. [5] Perez-Ilzarbe, F. J., Martinez, V., Hernandez, T., Estrella, I., Liquid chromatographic determination of apple pulp procyanidins. J. Liq. Chromatogr. 1992, 15, 637–646. [6] Schmidt, H. W. H., Neukom, H., Identification of the main oligomeric proanthocyanidin occurring in apples. J. Agric. Food Chem. 1969, 17, 344–346. [7] Akiyama, H., Sakushima, J., Taniuchi, S., Kanda, T. et al., Antiallergic effect of apple polyphenols on the allergic model mouse. Biol. Pharm. Bull. 2000, 23, 1370–1373. [8] Kanda, T., Akiyama, H., Yanagida, A., Tanabe, M. et al., Inhibitory effects of apple polyphenol on induced histamine release from RBL-2H3 cells and rat mast cells. Biosci. Biotechnol. Biochem. 1998, 62, 1284–1289. [9] Akiyama, H., Sato, Y., Watanabe, T., Nagaoka, M. H. et al., Dietary unripe apple polyphenol inhibits the development of food allergies in murine models. FEBS Lett. 2005, 579, 4485–4491. [10] Katayama, S., Kukita, T., Ishikawa, E., Nakashima, S. et al., Apple polyphenols suppress antigen presentation of ovalbumin by THP-1-derived dendritic cells. Food Chem. 2013, 138, 757–761. [11] Kojima, T., Akiyama, H., Sasai, M., Taniuchi, S. et al., Antiallergic effect of apple polyphenol on patients with atopic dermatitis: a pilot study. Allergol. Int. 2000, 49, 69–73. [12] Miyake, M., Ide, K., Sasaki, K., Matsukura, Y. et al., Oral administration of highly oligomeric procyanidins of Jatoba reduces the severity of collagen-induced arthritis. Biosci. Biotechnol. Biochem. 2008, 72, 1781–1788. [13] Bevaart, L., Vervoordeldonk, M. J., Tak, P. P., Evaluation of therapeutic targets in animal models of arthritis. Arthritis Rheum. 2010, 62, 2192–2205. [14] Shoji, T., Masumoto, S., Moriichi, N., Kanda, T., Ohtake, Y., Apple (Malus pumila) procyanidins fractionated according to the degree of polymerization using normal-phase chromatography and characterized by HPLC-ESI/MS and MALDITOF/MS. J. Chromatogr. A 2006, 1102, 206–213. [15] Afzali, B., Lombardi, G., Lechler, R. I., Lord, G. M., The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin. Exp. Immunol. 2007, 148, 32–46.
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[16] Irmler, I. M., Gajda, M., Brauer, R., Exacerbation of antigeninduced arthritis in IFN-gamma-deficient mice as a result of unrestricted IL-17 response. J. Immunol. 2007, 179, 6228– 6236. [17] Fouser, L. A., Wright, J. F., Dunussi-Joannopoulos, K., Collins, M., Th17 cytokines and their emerging roles in inflammation and autoimmunity. Immunol. Rev. 2008, 226, 87– 102. [18] Nakae, S., Nambu, A., Sudo, K., Iwakura, Y., Suppression of immune induction of collagen-induced arthritis in IL-17deficient mice. J. Immunol. 2003, 171, 6173–6177. [19] Lubberts, E., Koenders, M. I., vanden Berg, W. B., The role of T-cell interleukin-17 in conducting destructive arthritis: lessons from animal models. Arthritis Res. Ther. 2005, 7, 29– 37. [20] Toy, D., Kugler, D., Wolfson, M., Vanden Bos, T. et al., Cutting edge: interleukin 17 signals through a heteromeric receptor complex. J. Immunol. 2006, 177, 36–39. [21] Ishigame, H., Kakuta, S., Nagai, T., Kadoki, M. et al., Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity 2009, 30, 108–119.
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[22] Benedetti, G., Miossec, P., Interleukin 17 contributes to the chronicity of inflammatory diseases such as rheumatoid arthritis. Eur. J. Immunol. 2014, 44, 339–347. [23] Miossec, P., vanden Berg, W., Th1/Th2 cytokine balance in arthritis. Arthritis Rheum. 1997, 40, 2105–2115. [24] Boissier, M. C., Chiocchia, G., Bessis, N., Hajnal, J. et al., Biphasic effect of interferon-gamma in murine collageninduced arthritis. Eur. J. Immunol. 1995, 25, 1184–1190. ˜ ´ J., Sanmart´ı, R. et al., [25] Canete, J.D., Mart´ınez, S.E., Farres, Differential Th1/Th2 cytokine patterns in chronic arthritis: interferon gamma is highly expressed in synovium of rheumatoid arthritis compared with seronegative spondyloarthropathies. Ann. Rheum. Dis. 2000, 59, 263–268. [26] Ke, Y., Jiang, G., Sun, D., Kaplan, H. J., Shao, H., Anti-CD3 antibody ameliorates experimental autoimmune uveitis by inducing both IL-10 and TGF- dependent regulatory T cells. Clin. Immunol. 2011, 138, 311–320. ´ [27] Jun, S., Ochoa-Reparaz, J., Zlotkowska, D., Hoyt, T., Pascual, D. W., Bystander-mediated stimulation of proteolipid protein-specific regulatory T (Treg) cells confers protection against experimental autoimmune encephalomyelitis (EAE) via TGF-. J. Neuroimmunol. 2012, 245, 39–47.
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DOI 10.1002/mnfr.201400909
Mol. Nutr. Food Res. 2015, 59, 1406–1410
FOOD & FUNCTION
Oral administration of apple condensed tannins delays rheumatoid arthritis development in mice via downregulation of T helper 17 (Th17) cell responses Kosuke Nakamura1∗ , Hideki Matsuoka1∗ , Syohei Nakashima2 , Tomomasa Kanda2 , Tomoko Nishimaki-Mogami1 and Hiroshi Akiyama1 1 2
National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan Asahi Group Holdings, Ltd., Moriya, Ibaraki, Japan
Apples are known to contain high concentrations of phenolic compounds such as condensed tannins. Consumption of condensed tannins has been reported to reduce the risk of many types of chronic diseases including allergies. However, their therapeutic effectiveness and potential in treating autoimmune disease remain controversial. Here, the effect of oral administration of apple condensed tannins (ACT) prepared from apples (Malus pumila cv. Fuji) on bovine type II collagen (CII)-induced arthritis in DBA1/J mice, a well-established murine model of human rheumatoid arthritis (RA), was evaluated. As compared to the control (without ACT administration) group, RA development was delayed and a significant reduction in the RA clinical score was observed in the ACT-administered group. Using cultured splenocytes isolated from CII-immunized mice, ACT-administration was shown to decrease the CII-induced increases in IL-17 expression and production in vitro. We propose that downregulation of T helper (Th) 17 cells is responsible for the ACT-induced RA suppression. Keywords: Apple condensed arthritis / T helper
tannins / Oral
Received: December 15, 2014 Revised: March 12, 2015 Accepted: April 7, 2015
administration / Phytochemical / Rheumatoid
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Several lines of evidence indicate that daily incorporation of fruit into the diet may reduce the risk of many types of chronic diseases including allergy [1]. Apples are known to contain high concentrations of phytochemicals, phenolic compounds such as chlorogenic acid, (+)-catechin, (-)-epicatechin, phlorodzin, and rutin [2–4], and condensed tannins such as procyanidins B1, B2, and C1 [5, 6]. The antiallergy activities of condensed tannins purified from apples (apple condensed tannins; ACT) have previously been investigated. The studies demonstrated that ACT inhibits histamine release from cultured rat basophilic leukemia (RBL-2H3) and peritoneal mast cells [7, 8] as well as food-allergen induced Correspondence: Dr. Kosuke Nakamura E-mail: [email protected] Abbreviations: ACT, apple condensed tannins; CIA, collageninduced arthritis; CII, bovine type II collagen; RA, rheumatoid arthritis; Treg, regulatory T; TGF1, transforming growth factor 1; Th, T helper C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
oral sensitization in mice [9,10], and improves the symptoms of atopic dermatitis in humans [11]. Although the source of condensed tannins differs, Miyake et al. also reported that condensed tannins purified from a South American herb, Jatoba, exerted rheumatoid arthritis (RA) inhibition, whereas green tea polyphenols facilitated disease development [12]. However, little is known about the inhibitory mechanism of these condensed tannins in RA, and further investigation is required. To study the effect of ACT on RA severity and incidence, we employed a well-known animal model of human RA (collagen-induced arthritis; CIA) [13], in which RA is induced by injecting bovine type II collagen (CII) into DBA1/J mice (for details, see Supporting Information). ACT was prepared from apples (Malus pumila cv. Fuji) following the previously described method [14], with slight modifications (for details, see Supporting Information Fig. 1). Previous studies ∗ These
authors contributed equally to this study. www.mnf-journal.com
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Figure 1. Effect of ACT-administration on RA severity and incidence in mice. ACT was administered from 2 weeks prior to CII immunization and throughout the experimental period. After RA induction, both ACT-administered and control groups continued to have free access to Mouse Flat diet with the ACT solution and water, respectively. Mice were immunized with CII and received the secondary immunization (day 0) after 3 weeks. The severity (A) and incidence (B) of RA were evaluated by clinical scoring of paw swelling in the wateradministered control group (circle, n = 4) and the ACT-administered group (square, n = 4). ***p < 0.01 as determined by analysis of variance on day 20. (C) Representative clinical signs of RA on day 20. The control group (total arthritis score, 3.8), the ACT-administered group (total arthritis score, 1.1).
on atopic dermatitis patients indicated that itching and sleep disturbance scores after ACT supplement even for 2 weeks were significantly decreased [11]. Also, 1% w/v ACT intake for 2 weeks would inhibit the development of oral sensitization and prevent the development of food allergies in mice [9]. Thus, 1% w/v ACT was ad libitum administered from 2 weeks prior to CII immunization and throughout the experimental period. Each mouse drank 5–7 mL 1% w/v ACT per day as in the case of water, and no significant difference in body weight and the amount of feed intake was observed between the ACT-administered and water-administered (control) group. Figure 1 shows the severity and incidence of RA in the ACT-administered and control groups (four mice in each group) after secondary immunization with CII (day 0). As shown in Fig. 1A, the control group showed rapid RA development and the severity decreased after 3 weeks. Definite acute and chronic phases (days 2–20 and 20–55, respectively) were observed. In the control group, RA developed with joint inflammation of the paws to a maximum mean score (3.5 ± 0.3) by day 20, and a constant decrease in the score to 2.4 ± 0.2 was observed by day 37 (Fig. 1A). The onset of RA in the ACT-administered group was delayed as compared with the control group, and the severity score of RA was reduced to a maximum score of 1.6 ± 0.6 at day 37 (Fig. 1A). Attenuation of RA symptoms continued until the end of the observation period (day 55). In the control group, one of the mice developed RA at day 2 after the secondary CII injection, and the incidence rate increased up to day 12 when all mice showed RA symptoms (Fig. 1B). In the ACT-administered group, one of the mice C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
developed RA at day 7 after the secondary CII immunization. All mice in the ACT-administered group acquired RA by day 49 (Fig. 1B). The incidence of RA was lower as compared with the control group. Representative clinical symptoms of RA on both the forepaws and hindpaws on day 20 are shown in Fig. 1C. Control group showed more severe swelling in digit, wrist, and ankle as compared with the ACT-administered group. In a separate experiment, we confirmed that mice without CII injection did not develop RA, and that ACT administration had no effect (data not shown). Taken together, we found that the oral administration of ACT solution to DBA1/J mice delayed CII-elicited RA development. To evaluate whether the alleviation of RA in the ACTadministered group was associated with altered T helper (Th) responses, splenocytes were isolated at day 14 after primary CII immunization from the ACT-administered and control groups (three mice in each group), and were cultured and restimulated with CII (at a final concentration of 25 g/mL) in vitro. After 72 h culture, cytokine (transforming growth factor 1 [TGF1], IL-17, IL-4, IL-10, and IFN-␥) expression was quantified at the mRNA and protein levels (Fig. 2A; for details, see Supporting Information). Real-time PCR (Fig. 2B and C) showed that, in the control group, CII restimulation significantly induced the expression of IL-17A by approximately tenfold (relative expression from 0.13 ± 0.04 to 1.00 ± 0.25; p < 0.01, n = 6), while TGF1 expression was unchanged. In the ACT-administered group, IL-17A expression was significantly reduced by approximately
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Figure 2. Cytokine expression analysis in cultured splenocytes. (A) Scheme of experimental procedures. (B, C) Cytokine expression measured using real-time PCR. Fourteen days after primary CII immunization in the ACT-administered group and control group, splenocytes were isolated and restimulated for 72 h with CII. Total RNAs were isolated from the cells, and cytokine (B, TGF1; C, IL-17A) mRNA levels were determined in duplicate. Data represent means ± SD (n = 3). Significant differences between groups are indicated.
half as compared with the control group (relative expression from 1.00 ± 0.25 to 0.54 ± 0.35; p < 0.05, n = 6) (Fig. 2C). The TGF1 expression level remained unchanged with or without ACT administration (Fig. 2B). To evaluate cytokine release into the medium, ELISA was used to determine cytokine levels. The concentration of IL17A/F (heterodimer) in the ACT-administered and control groups was 1992 pg/mL and 3054 pg/mL, respectively (Fig. 3B). The concentration of IFN-␥ in the ACT-administered and control groups was 3015 pg/mL and 12520 pg/mL, respectively (Fig. 3D). The levels of IL-17A/F and IFN-␥ in the ACT-administered group were significantly (p < 0.05 and p < 0.01 [n = 6], respectively) lower than those of the control group (Fig. 3B and D). The levels of IL-4 and IL-10 were < 100 pg/mL and 5000 pg/mL, respectively, and no significant differences in those cytokine levels were observed between the ACT-administered and control groups (Fig. 3A and C). Taken together, ACT-administration significantly downregulated the production of both IL-17A and IFN-␥ in splenocytes as compared with the control group. In human RA patients, elevated levels of the proinflammatory cytokine IL17 were reported [15]. In addition, defects in IFN-␥ signaling have been shown to cause uncontrolled IL-17 production [16]. IL-17 has been reported to play an important role in the development of various autoimmune diseases [17]. The IL-17 produced by Th17 cells contributes to both the induction and progression of RA [18, 19]. The development of CIA is suppressed in IL-17-deficient mice [18]. Treatment with IL-17 neutralizing antibodies or IL-17 receptor antagonists reduces the symptoms of RA [19]. Th17 cells simultaneously produce IL-17A and IL-17F, which bind IL-17 receptor complexes consisting of IL-17RA and IL-17RC in the form of heterodimeric or homodimeric complexes of IL-17 A/F [20]. IL-17A plays C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. The effect of ACT-administration on cytokine expression measured using ELISA. Splenocytes were isolated and restimulated with CII as described in Fig. 2. (A) IL-4, (B) IL-17A/F, (C) IL-10, and (D) IFN-␥ released to the culture medium were measured in duplicate. Data represent means ± SD (n = 3). Significant differences between groups are indicated.
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a major role in T cell-dependent autoimmune and allergic responses, whereas IL-17F alone contributes marginally to these responses [21]. In this study, the IL-17A mRNA levels in splenocytes were elevated tenfold after RA induction in vitro. Our results showed that ACT administration inhibited IL-17 expression. IL-17 is known to initiate positive feedback loop mechanisms, leading to the expansion of Th17 cells and thereby increasing the production of IL-17 [22]. Therefore, the oral-administration of ACT may suppress the CII-induced overproduction of Th17 by reducing IL-17 expression. Increases in Th1 cytokines are observed in human RA patients, and the IFN␥/IL-4 ratio is closely related to the ratio of Th1-/Th2-type cytokine production [23]. In this study, ACT-administration significantly decreased IFN-␥ expression, while no significant differences in IL-4 expression were observed, although the levels of secreted IL-4 were very low, suggesting that ACT-administration repressed Th1-type cytokine production. Th2-type cytokines (IL-4 and IL-13) were also detected at mRNA level, but no significant difference was observed upon ACT administration (data not shown). Previous studies have revealed the critical role that IFN-␥ plays in RA development [15, 16, 24, 25]. The development of RA was inhibited by neutralization of IFN-␥ during the induction of CIA (early treatment), while the neutralization of IFN-␥ after the induction of CIA (late treatment) exacerbated the severity of CIA [24]. Thus, our findings suggested that ACT administration prior to RA induction may be effective in attenuating the Th1 response, leading in RA attenuation. Ke et al. demonstrated that anti-CD3 antibody treatment in an experimental model of autoimmune uveitis in mice inhibited disease development by inducing regulatory T (Treg) cells via both IL-10 and TGF1 induction [26]. Also, IL-10 and TGF1 were previously found to protect against experimental autoimmune encephalomyelitis disease severity by suppressing encephalitogenic T effector cells and expanding or sustaining functional Treg cells [27]. Both IL-10 and TGF1 expression levels were not significantly affected by ACT administration in this study. Therefore, RA amelioration by ACT was not mediated by the induction of Treg in splenocytes. Together, oral administration of the 1% w/v ACT solution delayed RA development in DBA1/J mice. IL-17 expression was significantly reduced in cultured splenocytes from ACT-administered mice during collagen-driven T-cell development. Oral administration of ACT likely affected RA responses via downregulation of Th17 cell development. Further studies are required to investigate the ACT’s effective administration amount and method for RA amelioration. This study was supported by a grant from the Japanese Ministry of Health, Labor and Welfare, a grant from the Japan Health Sciences Foundation, and a grant from the Food Safety Commission. The authors have declared no conflict of interest.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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