JOURNAL OF MEDICINAL FOOD J Med Food 16 (11) 2013, 1039–1045 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.2911

Water-Soluble Undenatured Type II Collagen Ameliorates Collagen-Induced Arthritis in Mice Orie Yoshinari,1,2 Yoshiaki Shiojima,1 Hiroyoshi Moriyama,1,2 Junichi Shinozaki,2 Takahisa Nakane,2 Kazuo Masuda,2 and Manashi Bagchi1 1

Development Division, Ryusendo Co. Ltd., Toshimaku, Tokyo, Japan. Faculty of Pharmaceutical Sciences, Showa Pharmaceutical University, Machida, Tokyo, Japan.

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ABSTRACT Earlier studies have reported the efficacy of type II collagen (C II) in treating rheumatoid arthritis (RA). However, a few studies have investigated the ability of the antigenic collagen to induce oral tolerance, which is defined as active nonresponse to an orally administered antigen. We hypothesized that water-soluble undenatured C II had a similar effect as C II in RA. The present study was designed to examine the oral administration of a novel, water-soluble, undenatured C II (commercially known as NEXT-II) on collagen-induced arthritis (CIA) in mice. In addition, the underlying mechanism of NEXT-II was also identified. After a booster dose (collagen-Freund’s complete adjuvant), mice were assigned to control CIA group, or NEXT-II treatment group, to which saline and NEXT-II were administered, respectively. The arthritis index in the NEXT-II group was significantly lower compared with the CIA group. Serum IL-6 levels in the NEXT-II group were significantly lower compared with the CIA group, while serum IL-2 level was higher. Furthermore, oral administration of NEXT-II enhanced the proportion of CD4 + CD25 + T (Treg) cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cells such as forkhead box p3 (Foxp3), transforming growth factor (TGF)-b1, and CD25. These results demonstrated that orally administered water-soluble undenatured C II (NEXT-II) is highly efficacious in the suppression of CIA by inducing CD4 + CD25 + Treg cells. KEY WORDS:  CD4 + CD25 + Treg  collagen-induced arthritis  Foxp3  oral tolerance  rheumatoid arthritis  TGF-b  undenatured type II collagen

forkhead box p3 (Foxp3), respectively. IL-17-producing Th17 is thought to be a major effector of autoimmune disease.3 Th17 cells are present in the T-cell-rich area of RA synovium.4 Patients with RA have higher levels of IL-17 in their serum and synovial fluid than normal controls.5,6 CD4 + CD25 + Treg cells constitute 5–10% of the peripheral CD4 + T cells,7 play a major role in the maintenance of immune tolerance, and also control autoimmunity.8,9 CD4 + CD25 + Treg cells have been reported to suppress development of methylated bovine serum albumin or CIA.10,11 CD4 + T cells activated with IL-2 and transforming growth factor (TGF)-b1 become Treg cells,12,13 whereas activation with IL-6 and TGF-b results in Th17 cells.14,15 IL-6 inhibits the function of Treg cells,16 and Th17 cells reinforce immune reactions. Alternatively, IL-2 acts through STAT5, resulting in the inhibition of the key transcription factor Th17 and the up-regulation of RORct, while activating Treg cell production.17 The cytokine milieu influences the balance of differentiating inducible Treg and Th17 cells.17 Induction of oral tolerance as an active nonresponse to an antigen ingested orally has long been considered a promising approach to the treatment of chronic autoimmune diseases, including RA. The antigen characteristics,

INTRODUCTION

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heumatoid arthritis (RA) is a severe inflammatory autoimmune disorder with a prevalence of 0.5–1% in the global population.1 It is characterized by persistent inflammation of the synovium and progressive erosion of cartilage and bone, while infiltrated with activated immune cells such as CD4 + T cells, B cells, dendritic cells (DCs), and macrophages. A commonly employed model to study RA involves the use of small animals such as rats and mice in collagen-induced arthritis (CIA) that elicits both antibody and T-cell responses to type II collagen (C II),2 thereby offering a powerful tool for evaluating the efficacy of potential therapeutic agents for treating RA. CD4 + T cells comprise different subsets with distinct differentiation profiles and functional characteristics. The characterized subsets are Th1, Th2, Th17, and CD4 + CD25 + regulatory T (Treg), whose development is specified by the transcription factors Tbets, GATA-3, RORct, and Manuscript received 8 April 2013. Revision accepted 25 June 2013. Address correspondence to: Orie Yoshinari, PhD, Development Division, Ryusendo Co. Ltd., 1-5-3 Nishiikebukuro, Toshimaku, Tokyo 171-0021, Japan, E-mail: [email protected] ryusendo.co.jp

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administration protocol, and primary contact with the immune system regulate the induction of tolerance,18 and agents that enhance anti-inflammatory cytokine profiles from immune cells can activate the induction of oral tolerance. Oral administration of C II has been proved to improve signs and symptoms in CIA animals and human clinical studies for RA.19–21 The present study was designed to examine the effects of the water-soluble, undenatured C II (NEXT-II) on CIA mice. The mechanism of immune suppression mediated by orally administered NEXT-II was also clarified by measuring IgG response to C II, and IL-2 and IL-6 levels. The proportion of CD4 + IL-10 + , CD4 + CD25 + T cells, and gene expressions of TGF-b1, Foxp3, and CD25 were also analyzed using a flow cytometer and real-time PCR. MATERIALS AND METHODS Preparation of NEXT-II The water-soluble, undenatured C II (NEXT-II) was provided by Ryusendo Co., Ltd. (Tokyo, Japan). Briefly, sternum cartilages of commercially available chickens (age: 4–6 months) were sterilized in 5% sodium hypochlorite solution and pulverized using a food grinder. The ground cartilage was extracted with 0.05 M acetic acid, and the reaction was initiated by adding 1.0% pepsin (Amano Enzyme Co., Ltd., Tokyo, Japan) at 4C for 60 h while stirring. The mixture was filtered to remove nondissolved particles, evaporated under a vacuum, and further diluted with deionized water. The presence of epitope in undenatured C II was measured by using a commercial kit (Native Type II Collagen Capture Kit; Astarte Biologics, LLC., Redmond, WA, USA).

On day 39, the collagen-induced group mice were divided into three groups (CIA [n = 8], glucosamine hydrochloride [Glu] ([n = 8], and NEXT-II [n = 9] group), and administered with glucosamine hydrochloride (300 mg/kg of body weight), NEXT-II (1 mg/kg, as undenatured C II level), or saline only (for CIA group) until day 48. The oral dose of NEXT-II was decided by measuring undenatured C II. Glucosamine hydrochloride, as an index of the improvement of CIA, was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Normal group mice were treated with saline only as the CIA group from day 39 to 48. The development of arthritis was assessed every 3 days. The clinical severity of arthritis in each paw was quantified according to a graded scale from 0 to 4, as follows: 0, no swelling; 1, swelling in one digit or mild edema; 2, moderate swelling affecting several digits; 3, severe swelling affecting most digits; and 4, the most severe swelling and/or ankylosis.22 Total arthritis index per mouse was determined by summarizing the indexes of all four extremities. On day 48, abdominal surgeries on mice were performed after anesthesia, and blood was collected by cardiac puncture. Serum was prepared by centrifugation of the collected blood at 1000 g for 15 min. The spleen and the hind limbs were removed from each mouse. Serum anti-C II IgG, IL-2, and IL-6 levels Serum anti-C II IgG levels were measured using an IgG Anti-Mouse Type II Collagen Antibody ELISA kit (Chondrex, Inc., Redmond, WA, USA). Serum IL-2 and IL-6 levels were measured using commercial kits, Mouse IL-2 and Mouse IL-6 ELISA Kits (Thermo Fisher Scientific K.K., Kanagawa, Japan).

Animals

Histopathology

All animal experiments were performed according to the Guidelines of Animal Experiment Protocol of Showa Pharmaceutical University. Male 7-week-old DBA1/J mice were purchased from Sankyo Labo Service Co., Inc. (Tokyo, Japan) and used after an acclimatization period of 1 week. Mice were maintained under controlled conditions (12:12 h light-dark cycle, 22C – 2C, and 40–60% humidity). Animals were housed in polystyrene cages and were fed commercial rodent chow, and tap water was given ad libitum.

The hind limbs were dissected and fixed in 4% paraformaldehyde. After decalcification with saturated ethylene diamine tetraacetic acid, the tissues were processed and embedded in paraffin. Five-micrometer tissue sections were prepared and stained with hematoxylin and eosin using standard methods.23 The microscopic images demonstrated damage in the joint tissues.

Induction, treatment, and evaluation of CIA mouse After the acclimatization period, mice (8-week-old) were divided into two groups: normal group (n = 7) and collageninduced group (n = 25). Bovine C II (Collagen Research Center, Tokyo, Japan) was dissolved in 0.05 M acetic acid at 4 mg/mL and emulsified in an equal volume of Freund’s complete adjuvant (Difco Laboratories, Detroit, MI, USA). One hundred microliters of this emulsion was injected intradermally into the dorsal root of the tail of the collageninduced group mice. This day of treatment was defined as day 0. On day 21, the same booster injection was also administered to the collagen-induced group.

Immunofluorescence flow cytometry Spleen tissues were minced, and the cells were filtered through a 100 lm nylon mesh and centrifuged at 1000 g for 10 min at room temperature. The cell pellet was resuspended in wash buffer (PBS with 5% BSA) and incubated with fluorescein isothiocyanate (FITC)-anti-CD4 (eBioscience, San Diego, CA, USA) and phycoerythrin (PE)-anti-CD25 (BioLegend, Inc., San Diego, CA, USA) monoclonal antibody (10 lg/mL) for 45 min at room temperature in the dark. After washing, the labeled cells were detected immediately. For IL-10 detection, splenocytes pellets were resuspended in RPMI-1640 medium to a concentration of 1 · 106 cells/ well in 12-well plates and incubated with 40 lg/mL C II in a humidified 5% CO2 atmosphere at 37C for 48 h. Then, the

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cells were incubated with FITC-anti-CD4, washed, fixed, permeabilized with 0.1% saponin, and incubated with PEanti-IL-10 (BioLegend, Inc.) for 45 min at room temperature. Cells were analyzed using an FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). FITC and PE-IgG1 antibody (Beckman Coulter, Inc., Brea, CA, USA) were used as isotype controls. Real-time PCR The expression levels of Foxp3, TGF-b1, CD25, and b-actin mRNA in the spleen were estimated by quantitative real-time PCR using a StepOne Real-Time PCR System (Applied Biosystems, Inc., Carlsbad, CA, USA). Total cellular RNA was extracted using RNeasy Mini kit (Qiagen, Hilden, Germany). The first strand of cDNA was synthesized using SuperScript Z Reverse Transcriptase (Invitrogen Co., Carlsbad, CA, USA). cDNA was added to SYBR Green Real-time PCR Master Mix-Plus (Toyobo Co., Ltd., Osaka, Japan) to generate sensitive quantitative gene expression data. The primers (Takara Bio, Inc., Tokyo, Japan) used in the present study were as follows: Foxp3: Forward, 50 -TGC CTT CAG ACG AGA CTT GGA-30 ; Reverse, 50 -GGC ATT GGG TTC TTG TCA GAG-30 TGF-b1: Forward, 50 -GTG TGG AGC AAC ATG TGG AAC TCT A-30 ; Reverse, 50 -CGC TGA ATC GAA AGC CCT GTA-30 CD25: Forward, 50 -TGA TGC TTT GTC ACG GTG ATG-30 ; Reverse, 50 -TGC CTG AGT CTG GCT GAG ATG-30 b-actin: Forward, 50 -CAT CCG TAA AGA CCT CTA TGC CAA C-30 ; Reverse, 50 -ATG GAG CCA CCG ATC CAC A-30 The program profile was 95C for 30 sec, followed by 40 cycles of denaturation at 95C for 5 sec, annealing for 55C for 15 sec, and extension at 72C for 15 sec. The data are expressed as relative values to b-actin. Statistical analysis Data were expressed as mean – standard error. The nonparametric Kruskal-Wallis test was used to determine statistical significance of the differences in the arthritis index between groups. Other data were statistically analyzed by one-way analysis of variance. A post-hoc analysis of significance was made by using Tukey’s test. P < .05 was considered significant for all data comparisons. RESULTS Inhibition of arthritis development in CIA mice The time course of the arthritis index of each group is shown in Figure 1. The severity of arthritis remained low in the mice until 21 days after primary immunization. After the booster injection, the arthritis index in the collagen-induced group mice began to increase. On day 39, mice in the collagen-induced group were assigned to Glu, NEXT-II, and

FIG. 1. Effects of glucosamine hydrochloride and NEXT-II on CIA mice. Mice were immunized with 200 lg bovine C II on day 0 and boosted in the same way on day 21. Glucosamine hydrochloride and NEXT-II were administered intragastrically to the Glu, NEXT-II group mice from day 39 to 48. Arthritis index was detected every 3 days. Each value is mean – SE. abcGroups without a common letter at the same time point differ significantly (P < .05). C II, type II collagen; CIA, collagen-induced arthritis; Glu, glucosamine hydrochloride; SE, standard error.

CIA groups, which had about the same average arthritis index. Each group was administered glucosamine hydrochloride, NEXT-II or saline only from day 39 to 48. On day 42, the arthritis index in the NEXT-II group was significantly decreased in comparison with that of the CIA group (P = .0091). On day 45, the arthritis index of the Glu group also significantly decreased as compared with the CIA group (P = .0458). However, the decrease in the NEXT-II group was more than that of the Glu group from day 45 to 48 (day 45, P = .0234; day 48, P = .0030). This result demonstrates that NEXT-II is more effective than glucosamine hydrochloride on the CIA mice in the suppression of arthritis development. Changes in the level of serum anti-C II IgG, IL-2 and IL-6 To examine systemic changes caused by NEXT-II, we measured anti-C II IgG, IL-2, and IL-6 levels in the serum of normal, CIA, and NEXT-II group mice. Anti-C II IgG production in the NEXT-II group was significantly lower compared with that of the CIA group (P = .03; Fig. 2A). IL-2 levels in the NEXT-II group were significantly higher than the CIA group (P = .04; Fig. 2B). On the contrary, IL-6 levels of the NEXT-II group were remarkably lower compared with the increased level in the CIA group of mice (P = .04; Fig. 2C). Histopathology In order to ascertain the inhibitory effects of NEXT-II on arthritis, we performed a histopathological analysis. Mice in the CIA group exhibited typical pathogenesis of RA such as inflammatory cell infiltration with extensive pannus formation, cartilage destruction, and irregular joint space compared with the normal control group (Fig. 3). The NEXT-II–treated group demonstrated limited synovial

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FIG. 2. Serum anti-C II IgG (A), IL-2 (B), and IL-6 (C) levels in normal mice and in control and NEXT-II–treated CIA mice. On day 48, serum was prepared by centrifugation of the collected blood at 1000 g for 10 min. Anti-C II IgG, IL-2, and IL-6 levels were measured by a commercial kit. Values are means – SE in each group. abGroups without a common letter differ significantly (P < .05).

infiltration, well-defined articular cartilage, and improved degenerative joints. In addition to the evaluation of clinical index, histological findings demonstrated the anti-arthritic effect of NEXT-II. Flow cytometric analysis of T cells Lymphocytes were isolated from the spleen, stained for the presence of CD4 + IL-10 + T and CD4 + CD25 + T cells, and analyzed by flow cytometry. As shown in Figure 4A, the proportion of CD4 + IL-10 + T cells in the CIA group tended to be lower than the normal control group (P = .09), but not significantly. However, these parameters were higher in the NEXT-II group than in the CIA group (P = .01). Furthermore, flow cytometric analysis tended to decrease CD4 + CD25 + T cells in CD4 + T cells in the CIA mice as compared with the normal control mice (P = .09; Fig. 4B). However, administration of NEXT-II significantly increased the proportion of CD4 + CD25 + T cells in CD4 + T cells (P = .02). TGF-b1, Foxp3, and CD25 expression levels The expression levels of TGF-b1, Foxp3, and CD25 in splenocytes were measured using primers specific to each gene and real-time quantitative PCR. As shown in Figure 5, the expression levels of TGF-b1, Foxp3, and CD25 mRNA were not different in the CIA or normal control mice.

However, administration of NEXT-II markedly elevated the expression levels of these mRNAs in spleen lymphocytes compared with the CIA group (TGF-b1, P = .02; Foxp3, P = .05; CD25, P = .03). DISCUSSION Earlier studies have reported that oral administration of low doses of C II significantly ameliorated the arthritic index in the CIA mice.20 In the present study, a low dose of NEXT-II also caused a significant improvement in CIA mice. Previous studies have also examined the effectiveness of orally administered C II in the amelioration of arthritis. NEXT-II was administrated in the present study after the development of arthritis. In addition, the dose of NEXT-II administered to CIA mice was determined by measuring undenatured C II using a commercial ELISA kit. NEXT-II group mice were treated with 1 mg/kg of body weight/day, as undenatured C II levels. These results are different from earlier reports.24,25 Some groups have reported the presence of major T-cell epitopes in C II,24,25 even if the C II is denatured immunological tolerance may occur by retaining the structure of the epitopes. However, the integrity of denatured structures is easily lost by resolution of protease. Therefore, in this study, undenatured C II level was measured in animal experiments with low dose. The oral administration of glucosamine hydrochloride or NEXT-II suppressed the arthritic indices in CIA mice.

FIG. 3. Histopathologic features of hind limb joints in normal mice (A) and in control (B) and NEXT-II–treated (C) CIA mice. All were stained hematoxylin and eosin ( · 50). The microscopic images were indicative of damage in the joint tissues.

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FIG. 4. Effects of NEXT-II on spleens of CIA mice. Flow cytometry was used to determine the proportion of CD4 + IL-10 + T cells (A) and CD4 + CD25 + T cells in CD4 + T cells (B) in spleens of normal mice and control and NEXT-II–treated CIA mice. Values are means – SE in each group. abGroups without a common letter differ significantly (P < .05).

Glucosamine has been reported to be effective in animal arthritis.26–28 In our preliminary study, several doses of NEXT-II (1, 10, or 100 mg/kg, as undenatured C II) were administrated in CIA mice. The arthritic index was not different among these three treatment groups, which indicates that NEXT-II has no dose-dependent effect (data not shown). Thus, NEXT-II may not show the dose-dependent effect in these ranges. Under these circumstances, the present study indicates that administration of NEXT-II is more efficacious than glucosamine in the amelioration of arthritis (Fig. 1). In the histopathological study, the NEXT-II group showed limited synovial infiltration, well-kept articular cartilage, and improved degenerative joints. Furthermore, orally administered NEXT-II reduced the antibody response to C II. These results clearly demonstrate that orally administered NEXT-II is efficacious in CIA mice. Moreover, the present study demonstrated that the proportion of CD4 + CD25 + T cells in CD4 + T cells, CD4 + IL-10 + T cells in the NEXT-II group increased compared with the CIA group. It has been reported that in RA patients, a reduction in the frequency of CD4 + IL-10 + T cells in inflamed synovium and peripheral blood is a contributing factor to the loss of tolerance.29 There is a greater production of IL-10 in the CD4 + CD25 + T cells in the spleens of mice fed with C II before CIA induction.30 In this study, it is possible that the production of IL-10 occurs from CD4 + CD25 + T cells. Phenotypically, these CD4 + CD25 + T cells resemble Treg cells, based on the expression of their intracellular and surface markers (Foxp3 and CD25). Foxp3 has been identified as a specific molecular marker for Treg cells, and its expression is essential for the programming of

Treg-cell development and function.31 In the last decade, active suppression of autoreactive T lymphocytes by Treg cells has also emerged as an important factor in the maintenance of self-tolerance. Frey et al. reported a significant role of Treg cells in the control of chronic arthritis.10 Wang et al. reported significantly higher frequencies of CD4 + CD25 + T cells in spleens of normal controls compared with CIA rats.32 With regard to Foxp3, TGF-b1 has reported that Foxp3 expression is required for the maintenance and suppression of Treg cells.33,34 IL-2 is also required for the maintenance of Foxp3 expression.35 In the present study, high levels of serum IL-2 in the NEXT-II group regulate the up-regulation of CD25 (IL-2 receptor a-chain) expression. Conversely, IL-6 inhibits Treg cell function,20 expansion,36 and TGF-b activity.37 These results demonstrate that administration of NEXT-II may induce increased production of Treg cells compared with the CIA group. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in Treg cells has also been reported to suppress CD80/ 86 expression in DCs.38,39 It is reported that DCs downregulating the expression of CD80/86 cannot activate T cells.38,39 As a result of the induction of Treg cells by NEXT-II, it may be possible that the immune response is controlled and down-regulated. Alternatively, Zheng et al. have reported that CD40-, CD80/86-silenced DCs generated Treg cells.40 The results cited earlier indicate that NEXT-II may potentially act on DCs, resulting in the induction of Treg cells. In the present study, immune responses may have been adjusted through an interaction of Treg cells with CD40, CD80/86 of the DCs. Additional mechanistic studies are warranted to unveil the mechanism of NEXT-II.

FIG. 5. Expression of TGF-b1 (A), Foxp3 (B), and CD25 (C) in spleens of normal mice and control and NEXT-II–treated CIA mice. Expression levels were normalized to b-actin level and expressed as means – SE in each group. abGroups without a common letter differ significantly (P < .05). Foxp3, forkhead box p3; TGF, transforming growth factor.

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In conclusion, oral administration of NEXT-II resulted in decreased arthritis in CIA mice in conjunction with suppressed production of anti-C II IgG and IL-6, and enhanced secretion of IL-2. The proportion of CD4 + CD25 + T cells in the NEXT-II group also increased compared with that of the CIA group. Furthermore, gene expressions of Foxp3, TGF-b1, and CD25 in the NEXT-II group were significantly increased. From these results, it may be concluded that oral administration of NEXT-II may induce CD4 + CD25 + T cells in the amelioration of arthiritis. The efficacy of NEXT-II in animals is highly encouraging; however, wellmonitored, placebo-controlled human clinical studies are needed to demonstrate the efficacy of NEXT-II in humans.

ACKNOWLEDGMENTS This study was supported by a research grant from Ryusendo Co. Ltd., Japan. O.Y. analyzed the data, designed the research, and wrote the article; Y.S., H.M., J.S., T.N., K.M., and M.B. conducted and directed the research. All authors read and approved the final manuscript. AUTHOR DISCLOSURE STATEMENT O.Y., Y.S., H.M., and M.B., are associated with Ryusendo Co. Ltd., Japan, which funded the study. REFERENCES 1. Feldmann M: Pathogenesis of arthritis: recent research progress. Nat Immunol 2001;2:771–773. 2. Wooley PH, Luthra HS, Stuart JM, David CS: Type II collageninduced arthritis in mice. I. Major histocompatibility complex (I region) linkage and antibody correlates. J Exp Med 1981;154:688–700. 3. Romagnani S: Human Th17 cells. Arthritis Res Ther 2008;10: 206–213. 4. Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, Pin JJ, Garrone P, Garcia E, Saeland S, Blanchard D, Gaillard C, Das Mahapatra B, Rouvier E, Golstein P, Banchereau J, Lebecque S: T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med 1996;183:2593–2603. 5. Kotake S, Udagawa N, Takahashi N, Matsuzaki K, Itoh K, Ishiyama S, Saito S, Inoue K, Kamatani N, Gillespie MT, Martin TJ, Suda T: IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 1999;103:1345–1352. 6. Ziolkowska M, Koc A, Luszczykiewicz G, Ksiezopolska-Pietrzak K, Klimczak E, Chwalinska-Sadowska H, Maslinski W: High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism. J Immunol 2000;164:2832–2838. 7. Baecher-Allan C, Wolf E, Hafler DA: Functional analysis of highly defined, FACS-isolated populations of human regulatory CD4 + CD25 + T cells. Clin Immunol 2005;115:10–18. 8. Kohm AP, Carpentier PA, Anger HA, Miller SD: Cutting edge: CD4 + CD25 + regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol 2002;169:4712–4716.

9. Morgan ME, Flierman R, van Duivenvoorde LM, Witteveen HJ, van Ewijk W, van Laar JM, de Vries RR, Toes RE: Effective treatment of collagen-induced arthritis by adoptive transfer of CD25 + regulatory T cells. Arthritis Rheum 2005;52:2212– 2221. 10. Frey O, Petrow PK, Gajda M, Siegmund K, Huehn J, Scheffold A, Hamann A, Radbruch A, Bra¨uer R: The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4 + CD25 + T cells. Arthritis Res Ther 2005;7:R291–R301. 11. Morgan ME, Sutmuller RP, Witteveen HJ, van Duivenvoorde LM, Zanelli E, Melief CJ, Snijders A, Offringa R, de Vries RR, Toes RE: CD25 + cell depletion hastens the onset of severe disease in collagen-induced arthritis. Arthritis Rheum 2003;48:1452–1460. 12. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA: IL-2 is essential for TGF-beta to convert naive CD4 + CD25- cells to CD25 + Foxp3 + regulatory T cells and for expansion of these cells. J Immunol 2007;178:2018–2027. 13. Davidson TS, DiPaolo RJ, Andersson J, Shevach EM: Cutting Edge: IL-2 is essential for TGF-beta-mediated induction of Foxp3 + T regulatory cells. J Immunol 2007;178:4022–4026. 14. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, Wang Y, Hood L, Zhu Z, Tian Q, Dong C: A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 2005;6:1133–1141. 15. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B: TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006;24:179–189. 16. Pasare C, Medzhitov R: Toll pathway-dependent blockade of CD4 + CD25 + T cell-mediated suppression by dendritic cells. Science 2003;299:1033–1036. 17. Ivanov II, Zhou L, Littman DR: Transcriptional regulation of Th17 cell differentiation. Semin Immunol 2007;19:409–417. 18. Weiner HL: Oral tolerance, an active immunologic process mediated by multiple mechanisms. J Clin Invest 2000;106:935–937. 19. Nagler-Anderson C, Bober LA, Robinson ME, Siskind GW, Thorbecke GJ: Suppression of type II collagen-induced arthritis by intragastric administration of soluble type II collagen. Proc Natl Acad Sci USA 1986;83:7443–7446. 20. Thorbecke GJ, Schwarcz R, Leu J, Huang C, Simmons WJ: Modulation by cytokines of induction of oral tolerance to type II collagen. Arthritis Rheum 1999;42:110–118. 21. Park KS, Park MJ, Cho ML, Kwok SK, Ju JH, Ko HJ, Park SH, Kim HY: Type II collagen oral tolerance; mechanism and role in collagen-induced arthritis and rheumatoid arthritis. Mod Rheumatol 2009;19:581–589. 22. Sagawa K, Nagatani K, Komagata Y, Yamamoto K: Angiotensin receptor blockers suppress antigen-specific T cell responses and ameliorate collagen-induced arthritis in mice. Arthritis Rheum 2005;52:1920–1928. 23. Bancroft JD, Steven A: Theory and Practice of Histological Techniques, 3rd edition. Churchill Livingstone, New York, 1990. 24. Kobayashi S, Terato K, Harada Y, Moriya H, Taniguchi M: Suppression of type II collagen-induced arthritis by monoclonal antibodies. Arthritis Rheum 1991;34:48–54. 25. Ohnishi Y, Tsutsumi A, Sakamaki T, Sumida T: T cell epitopes of type II collagen in HLA-DRB1*0101 or DRB1*0405-positive Japanese patients with rheumatoid arthritis. Int J Mol Med 2003; 11:331–335.

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Water-soluble undenatured type II collagen ameliorates collagen-induced arthritis in mice.

Earlier studies have reported the efficacy of type II collagen (C II) in treating rheumatoid arthritis (RA). However, a few studies have investigated ...
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