Plant Biotechnology Journal (2014), pp. 1–10

doi: 10.1111/pbi.12223

Suppression of collagen-induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of type II collagen Mana Iizuka1, Yuhya Wakasa2, Hiroto Tsuboi1, Hiromitsu Asashima1, Tomoya Hirota1, Yuya Kondo1, Isao Matsumoto1, Fumio Takaiwa2,* and Takayuki Sumida1,* 1

Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

2

Functional Transgenic Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan

Received 11 January 2014; revised 25 May 2014; accepted 1 June 2014. *Correspondence (Tel +81 29 838 8373; fax +83 29 838 8397; email [email protected] (FT) and Tel/fax +81 29 853 7388; email [email protected] (TS))

Keywords: altered peptide ligands, glutelin, rheumatoid arthritis, transgenic rice seed, type II collagen.

Summary Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We previously reported that altered peptide ligands (APLs) of type II collagen (CII256-271) suppress the development of collagen-induced arthritis (CIA). In this study, we generated transgenic rice expressing CII256-271 and APL6 contained in fusion proteins with the rice storage protein glutelin in the seed endosperm. These transgene products successfully and stably accumulated at high levels (7–24 mg/g seeds) in protein storage vacuoles (PB-II) of mature seeds. We examined the efficacy of these transgenic rice seeds by performing oral administration of the seeds to CIA model mice that had been immunized with CII. Treatment with APL6 transgenic rice for 14 days significantly inhibited the development of arthritis (based on clinical score) and delayed disease onset during the early phase of arthritis. These effects were mediated by the induction of IL-10 from CD4+ CD25 T cells against CII antigen in splenocytes and inguinal lymph nodes (iLNs), and treatment of APL had no effect on the production of IFN-c, IL-17, IL-2 or Foxp3+ Treg cells. These findings suggest that abnormal immune suppressive mechanisms are involved in the therapeutic effect of rice-based oral vaccine expressing high levels of APLs of type II collagen on the autoimmune disease CIA, suggesting that the seed-based mucosal vaccine against CIA functions via a unique mechanism.

Introduction Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent inflammatory synovitis, varying degrees of cartilage destruction, bone erosion, joint deformity and loss of joint function. Although the pathogenesis of RA is not clear, there is sufficient evidence to suggest the involvement of T cells in the inflammatory process, for example, infiltration of T cells, especially CD4+ T cells, in RA joints (Hovdenes et al., 1989; Struyk et al., 1995). Furthermore, susceptibility to RA is associated with the expression of specific HLA class II alleles, especially HLA-DR4 (De Rosa et al., 2010; Roudier, 2000; Taneja et al., 2007, 2008). Type II collagen (CII), a molecule abundant in the articular cartilage, is considered to be one of the target autoantigens in RA. Autoantibodies to CII are commonly detected in patients with RA. In addition, CII-reactive T-cell clones have been established in vitro from synovial T cells of RA (Londei et al., 1989). Sekine et al. (1999) suggested that the expansion of oligoclonal T cells in RA joints is promoted by stimulation of CII. Furthermore, the pathology of collagen-induced arthritis (CIA) in mice is similar to that in human RA synovium. The susceptibility to CIA is determined by I-Aq, a major histocompatibility complex

(MHC) class II molecule, and the immunodominant CII256-271 region of CII (256–271 amino acid region of collagen type II) can bind to I-Aq molecules (Luross and Williams, 2001; Wooley et al., 1981). Altered peptide ligands (APLs) are peptides with substitutions in amino acid residues at T-cell receptor (TCR) contact sites; APLs can either be agonistic or antagonistic with partial activation (Chen et al., 1996; Sloan and Allen, 1996). Antagonistic APLs can inhibit the function of limited T-cell populations, which indicates that they may be potentially useful for antigen-specific therapy for autoimmune diseases in which T cells play a pathogenic role. We previously reported that peripheral blood mononuclear cells from HLA-DRB1*0101 Japanese patients with RA were highly reactive to the 256–271 peptide of CII. The designed APLs suppressed T-cell responses to the immunodominant epitope (CII256-271) of CII (Ohnishi et al., 2006). Moreover, we showed that APL6 downregulates the progression of arthritis following intraperitoneal or intradermal administration (Wakamatsu et al., 2009). Plants have recently been employed as bioreactors for the production of recombinant proteins such as pharmaceuticals (Tiwari et al., 2009; Yusibov and Rabindran, 2008). Rice seed is

Please cite this article as: Iizuka, M., Wakasa, Y., Tsuboi, H., Asashima, H., Hirota, T., Kondo, Y., Matsumoto, I., Takaiwa, F. and Sumida, T. (2014) Suppression of collagen-induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of type II collagen. Plant Biotechnol. J., doi: 10.1111/pbi. 12223

ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd

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2 Mana Iizuka et al. considered to be one of the most attractive plant-based production platforms. The potential benefits of rice seed include costeffectiveness, high stability at room temperature, scalability and safety, as rice seed is not contaminated with mammalian pathogens (Stoger et al., 2002; Wakasa and Takaiwa, 2013). Thus, rice seed represents a potential candidate for the production of plantderived edible drugs due to its very low level of toxins and food allergens (Khan et al., 2012; Takaiwa, 2013). For example, oral administration of transgenic rice seeds containing T-cell epitope peptides derived from the major Japanese cedar pollen allergens Cry j 1 and Cry j 2 alleviates pollen-induced clinical symptoms such as nasal sneezing in mice (Takagi et al., 2005). Given these findings, oral administration of transgenic rice seeds containing APLs specific for autoantigens could theoretically prevent autoimmune diseases. In this study, we generated transgenic rice seeds accumulating CII256-271 and APL6 and subsequently examined the therapeutic effects of APL transgenic rice on arthritis in CIA mice. We also investigated the mechanisms underlying the arthritis-suppressive actions of APL transgenic rice.

Results Development of transgenic rice plants accumulating a T-cell epitope of collagen type II and its analogue peptide in seeds We previously designed APL6, the analogue peptides of CII256271, which suppress CII256-271 autoantigen-reactive T cells. In this study, we generated transgenic rice plants expressing

tandemly repeated CII256-271 and APL6 in their endosperm to create a rice seed-based oral vaccine against the autoimmune disease RA. Approximately 30 independent transgenic rice lines were generated by Agrobacterium-mediated transformation for individual CII256-271 and APL6 expression constructs (Figure 1b). We examined the accumulation levels of transgenic products in mature seeds by immunoblot analysis using anti-CII peptide antibody. The anti-CII antibody reacted with the tandem repeat (trimer) of CII256-271 as well as that of APL6 (Figure 1a). New products were clearly detected as visible bands on CBBstained SDS-PAGE gels (Figure 1c, arrowheads). Immunoblot analysis using anti-CII antibody revealed a band representing a single major acidic subunit, as well as minor precursor bands, in transgenic rice expressing the CII256-271/glutelin fusion product. On the other hand, transgenic rice seeds expressing APL6 glutelin fusion products exhibited two or three acidic subunits in addition to a single precursor signal (Figure 1d). Although the same strategy was used for the production of the CII256-271 and APL6 peptides, the accumulation patterns of these peptides differed between them. We compared the accumulation levels of individual fusion products of individual homozygous lines by immunoblot analysis with four types of antibodies (anti-CII, anti-GluA, anti-GluB and anti-GluC antibodies), as shown in Figure 1d. When anti-CII or GluA antibodies were used, the detected signals were only derived from the transgene products, as no signal was detected with anti-CII or GluA antibodies in nontransgenic rice. The glutelin GluA was not present in nontransgenic rice due to the use of the a123 variety lacking functional GluA1, GluA2 and

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Figure 1 Expression of CII256-571 and APL6 in transgenic rice. (a) Amino acid sequences of CII256-271 and APL6. Identical amino acid residues are shown as ‘-’. Recognition sites of anti-CII antibodies are marked with asterisks. (b) Schematic representation of the plasmid used for transformation. DNA fragment encoding the major rice seed storage proteins, glutelins, under the control of the GluB1, GluA2 and GluC promoters. CSP, callus-specific promoter; mALS, mutated acetolactate synthase coding region; 10 kD T, 10 kDa prolamin terminator; 16 kD pro, 16 kDa prolamin promoter; GluB1, modified Glutelin B1 coding region; 16 kD T, 16 kDa prolamin terminator; GluB1 pro, Glutelin B1 promoter; GluA2, modified Glutelin A2 coding region, GluB1T, Glutelin B1 terminator; 10 kD pro, 10 kDa prolamin promoter; GluC, modified Glutelin C coding region; LB, left border; RB, right border. (c) SDSPAGE and (d) immunoblot analyses of transgenic rice seed proteins. The presence of glutelin-fused CII256-271 and APL6 was determined using specific antibodies. Arrowheads indicate the positions of transgene products derived from the introduced gene cassettes. ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–10

Immune suppression of arthritis by transgenic rice 3 GluB4 genes (Iida et al., 1997). On the other hand, endogenous GluB and GluC were detected in nontransgenic rice via immunoblotting using anti-GluB and anti-GluC antibodies. Therefore, it was difficult to precisely detect the transgene products derived from GluB and GluC due to their overlap with endogenous proteins in transgenic rice. We selected the transgenic rice line with the highest accumulation level of transgene product from the T1 generation for each construct (CII256-271 and APL6). Homozygous lines were selected from the subsequent generations through self-crossing (T2 generation). We then determined the T-DNA copy numbers of these transgenic rice lines by Southern blot analysis using the mALS region as a probe. Two and one copy of T-DNA were introduced into the genomes of transgenic rice plants expressing CII256-271 and APL6, respectively (Figure S1). We then analysed the subcellular localization of the transgene products in endosperm cells of the rice lines by immunocytochemical confocal microscopy using the anti-CII antibody (Figure 2). Rice endosperm cells have two distinct types of protein bodies (PBs), that is, the ER-derived PB-I and the protein storage vacuole PB-II. Seed storage protein prolamins are deposited into the spherical PB-I, whereas glutelins and globulin are stored in the irregularly shaped PB-II (Krishnan et al., 1986). The specificity of this intracellular localization of transgene products in rice seeds was confirmed by immunoblot analysis as well as rhodamine staining. Rhodamine-stained PB-I appears as an intense spherical signal, while rhodamine-stained PB-II appears as a weak, indeterminate signal in endosperm cells (Onda et al., 2009) (Figure 2). In transgenic rice seeds expressing glutelin-fused CII256-271 or APL6, signals of transgenen products (green) were merged with rhodamine-stained PB-II (pale red) (Figure 2), suggesting that transgene products were exclusively transported to PB-II, while no signal was detected in PB-I or other organelles (Figure 2). Furthermore, these recombinant proteins were efficiently processed into acidic and basic subunits like endogenous glutelins (Figure 1c,d). It is well

known that glutelin precursor is cleaved by cysteine protease after sorting into PB-II (Wang et al., 2009). These observations suggest that modified glutelins containing CII or APL6 were trafficked into PB-II in a manner similar to that of native glutelins (Figure 2). We then quantified the accumulation levels of the transgene products by immunoblot analysis. First, it was essential to determine whether the reactivity to the anti-CII antibody differed among the GluA2-fusion, GluB1-fusion and GluC-fusion proteins. Therefore, we produced the acidic subunit regions of GluA2-fused CII256-271, GluB1-fused CII256-271 and GluCfused CII256-271 individually in an E. coli expression system and subjected these fusion proteins to immunoblot analysis. As shown in Figure S2a, reactivity to the anti-CII antibody was very similar among the three types of glutelin fusion proteins. Then, to examine the accumulation levels of transgene products in individual transgenic rice seeds, we carried out immunoblot analysis using anti-CII antibody and purified GluA2-fused CII256271or GluA2-fused APL6 acidic subunit. The accumulation levels were subsequently quantified using NIH Image J software (Figure S2). The accumulation levels of CII256-271 and APL6 as fusion proteins with glutelin acidic subunit in the transgenic lines with the highest expression levels were estimated to be approximately 7.2 mg/g seeds (CII256-271) and 24.6 mg/g seeds (APL6), on average, in T2 homozygous seeds (Figure S2). It is important to note that the majority of transgene products accumulated as GluA-2 fusion products, as shown in Figure 1. This may be related to the fact that there was no endogenous GluA product in the host used in this study, and thus, there was no competition for deposition into PB-II between the endogenous GluA glutelins and GluA fusion product, as there was ample space in PB-II due to lack of some glutelins in a123. Although a high level of accumulation was achieved in these transgenic rice seeds, it is notable that the seed phenotypes were almost same as that of nontransgenic rice grains (Figure S3). We therefore successfully obtained transgenic rice seeds with high levels of the epitope of collagen type II (CII256-271) and its analogue peptide (APL6).

Therapeutic and prophylactic application of APL transgenic rice in CIA mice

Figure 2 Intracellular localization of transgene products. Red (rhodamine) signals of panels of nontransgenic and transgenic rice seeds (Glu-CII256-271and Glu-APL6) reveal PB-I and PB-II. White arrowheads and arrows indicate PB-I and PB-II, respectively. Green (Alexa 488) signals show the localization of transgene products (glutelin-fused CII256-271, and APL6). Green signals were completely merged with PB-II.

To investigate the therapeutic effects of APL transgenic rice in CIA mice, CII256-271, APL6 transgenic rice or nontransgenic rice were orally administered for 1 week starting on day 24 after the first immunization of CII antigen. As shown in Figure S4, the highest improvement in clinical score and incidence of arthritis was observed in mice that were fed APL6 transgenic rice. To confirm the efficacy of this APL6 transgenic rice, CIA mice were first immunized with CII and then orally fed CII256-271 and APL6 transgenic rice or nontransgenic rice once per day for 2 weeks (14 days), according to the treatment schedule shown in Figure 3a. The clinical scores were significantly reduced by treatment with APL6 transgenic rice (Figure 3b), especially during the early phase of the disease, which resulted in delayed onset. The incidence of arthritis in mice treated with APL6 transgenic rice was significantly decreased compared with those in control groups at early phase of the disease (Figure 3c). Furthermore, histologic analyses of the joints obtained from mice 33 days after immunization revealed that cellular infiltration in joint was suppressed in mice treated with APL6 transgenic rice compared with those treated with CII267-271 transgenic or nontransgenic rice (Figure 3d). These results indicate that oral administration of

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Figure 3 Oral administration of APL transgenic rice inhibits the development of CIA. (a) Schematic illustration showing the schedule of oral administration/ treatment of CIA mice. APL6 transgenic rice (APL) (n = 10), CII256-271-transgenic rice (CII256-471) (n = 6) or nontransgenic rice (non-TG) (n = 6) was orally administered for 2 weeks beginning on day 7 after the first immunization of CII antigen. (b) Clinical score and (c) incidence of arthritis. Data are mean  SE. *P < 0.05 vs. non-TG, **P < 0.05 vs. CII256-271 (clinical score was evaluated with Student’s t-test and the incidence score by chi-square test). Representative data of two independent experiments. (d) Hematoxylin- and eosin-stained sections of the hind paws of mice obtained 33 days after the first immunization. Arrows points indicated cellular infiltrations. Original magnification 409. Bar = 70 lm.

APL6 transgenic rice downregulates the development of CIA. Thus, APL transgenic rice may have potential therapeutic effects against CIA.

Increased production of IL-10 in APL transgenic rice-treated CIA mice To determine the mechanisms underlying the anti-CIA actions of APL transgenic rice, we analysed the effects of APL6 transgenic rice treatment on T cell-derived pro- and antiinflammatory cytokines such as IFN-c, IL-17, IL-2 and IL-10 in the spleen, inguinal lymph nodes (iLNs) and mesenteric lymph nodes (mLNs; Figures 4). In CIA mice treated with APL6 transgenic rice, the production of regulatory cytokine IL-10 against CII antigen significantly increased in the spleen, compared with mice treated with nontransgenic rice (Figure 4a). However, transgenic rice had little effect on the levels of IFN-c, IL-17 and IL-2 in iLN, as revealed by examination of supernatants from cultured spleens (Figure 4a,b), although the production of IFN-c and IL-2 appeared to decrease, but not significantly, in mLN (Figure 4c).

Effects of treatment with APL transgenic rice on regulatory T-cell differentiation The production of IL-10 increased in splenic and iLN cells of APL6treated mice cultured in vitro with antigen CII (Figure 4a,b). Therefore, we examined the effects of APL6 transgenic rice treatment on the differentiation of regulatory T cells. However, unexpectedly, treatment with APL6 transgenic rice did not affect the population of CD4+ CD25+ Foxp3+ regulatory T cells (Figure 5a,b). We also examined the effects of APL transgenic rice on the production of anti-CII antibodies in serum. A subtype of IgG1 is associated with anti-inflammatory responses, whereas IgG2a is a mediator of inflammation in CIA (Butler et al., 1999; Mukherjee et al., 2003). As shown in Figure 6, the titre of total anti-CII antibodies was similar in the APL transgenic rice-treated group and the control. However, specific IgG1 and IgG2a levels slightly (but not significantly) increased and decreased, respectively, in mice fed APL6 transgenic rice (Figure 6). We also measured the levels of the activation marker CD44 in T cells in CIA mice treated

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Immune suppression of arthritis by transgenic rice 5 (a)

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Figure 4 Production of cytokines from CII-reactive T cells of CIA mice treated with APL6 transgenic rice. Mice were immunized with CII and treated with APL6 or control rice. On day 24 after the first immunization, the mice were euthanized and the spleens (a), iLNs (b) and mLNs (c) were cultured with CII for 72 h. IFN-c, IL-2, IL-10 and IL-17 concentrations in the culture supernatants were measured by ELISA. Data are mean  SD of three mice per group. *P < 0.05 (Student’s t-test).

with APL6 transgenic rice (Figure S5a,b) and found that for CD4+ T cells, APL treatment had no effect on the expression of CD44 in the spleen, iLN or mLN.

IL-10 production by CD4+ CD25 T cells in CIA mice treated with APL transgenic rice To determine the types of cells that produce IL-10, we isolated CD4+ CD25+ T and CD4+ CD25 T cells from mice fed APL6 transgenic rice and cultured the cells with CII antigen. IL-10 was detected in the supernatants of cultured CD4+ CD25 T cells but not in that of CD4+ CD25+ T cells (Figure 7a). These results suggest that oral administration of APL6 transgenic rice seeds

induces IL-10-producing CII-specific CD4+ CD25 T cells in CIA mice. Foxp3 was not expressed in CD4+ CD25 cells (data not shown). Therefore, we measured the expression levels of Egr2 and LAG3 in CD4+ CD25 cells to determine whether the IL-10producing cells were Tr1 or LAG3 Treg cells that did not express Foxp3 or CD25. The expression of Egr2 in CD4+ CD25 T cells was similar between freshly isolated splenocytes from CIA mice treated with APL6 vs. nontransgenic rice, but the expression of LAG3 was slightly higher in the APL6 group (Figure 7b,c). These results suggest that treatment with APL6 transgenic rice induces the production of IL-10 from CD4+ CD25 T cells but not from Foxp3+ Treg, Tr1 or LAG3 Treg cells.

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6 Mana Iizuka et al. (a)

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Figure 5 Treatment of CIA mice with APL transgenic rice did not induce Treg cell. (a) Mice were immunized with CII and treated with APL6 or control rice, and spleens, iLNs and mLNs were harvested on day 24 and stained with CD25 and Foxp3. Flow cytometric analysis was gated for CD4+ T cells. Representative data of two independent experiments. (b) Proportion of CD4+ CD25+ Foxp3+ cells in spleen, iLN and mLN. Data are mean  SD of four mice per group. N.S.; not significant.

Discussion In this study, we generated two types of transgenic rice plants accumulating high levels of T-cell epitope of CII (CII256-271) and its analogue peptide (APL6) as fusion proteins with the major rice storage protein glutelin in the edible parts (endosperm) of seeds. Modified glutelins derived from the transgene products accumulated to high levels (7–24 mg/g seed) in PB-II in the mature, processed form. Recombinant proteins deposited into PB-I or PB-II in rice seeds can withstand digestive enzymes (such as pepsin and pancreatin) in the gastrointestinal tract compared with naked protein. Therefore, rice seed-based recombinant peptides are effectively delivered to gut-associated lymphoid tissue (GALT) in the intestinal tract via oral administration (Takagi et al., 2010; Takaiwa, 2013). Notably, the fusion of these peptides to glutelin did not perturb the protein trafficking from the endoplasmic reticulum (ER) to PB-II or the final refolding and assembly within

PB-II in the endosperm cells, as the glutelin fusion products were transported to PB-II at the same deposition site as the native glutelins and the seed phenotypes were almost identical to those of nontransgenic rice seeds. These results are in marked contrast to the results of experiments involving some other recombinant proteins (e.g. b-amyloid) expressed in rice seed, which induce the ER stress response, resulting in severe abnormal phenotypes such as shrunken seeds (Figure S2) (Oono et al., 2010; Wakasa et al., 2012). In the current study, the ER stress marker proteins OsBiP4 and OsBiP5 (Wakasa et al., 2012) were not detected by immunoblot analysis (Figure S3). In this study, we demonstrated that oral administration of APL6 transgenic rice significantly inhibited the clinical score of arthritis. This finding suggests that transgenic rice seeds expressing APL6 peptides represent a promising therapy for the treatment of CIA. Therefore, oral vaccination via transgenic rice seed, which can accumulate high amounts of analogue peptides, may provide a

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Immune suppression of arthritis by transgenic rice 7

Figure 6 Production of anti-CII antibodies in CIA mice treated with APL transgenic rice. Serum samples were obtained on day 27 after the first immunization from mice treated with APL6 transgenic rice or control rice. Each symbol represents a single mouse. Data are mean optimal density  SD.

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Figure 7 Treatment with APL increases IL-10 production from CD4+ CD25 cells. Mice were immunized with CII and treated with APL6 or control rice. On day 24 after the first immunization, (a) CD4+ CD25+ T or CD4+ CD25 T cells were isolated from spleens and then cultured with CII for 72 h. IL-10 concentrations in the culture supernatant were measured by ELISA. Data are mean  SD of two mice per group. (b) Flow cytometry analysis was gated in splenic CD4+ CD25 T cells. (c) Bar graph shows the mean fluorescent intensity (MFI) in CD4+ CD25 T cells. Three mice per group were analysed.

potential therapeutic treatment against autoimmune diseases including chronic diseases. What are the modes of action of transgenic rice seeds containing ALP6 against CIA? After an orally administered antigen is taken up by mucosal dendritic cells (DCs) or macrophages via M cells or lamina propria (LP) in the intestine, it migrates to Peyer’s patches (PP) or the MLN to activate na€ıve T cells through antigen presentation. Furthermore, the antigen is systemically transferred into the spleen and peripheral lymph nodes (Takakura et al., 2011). Previous studies indicate that immunization with rice-based oral vaccine expressing cholera toxin B subunit (CT-B) successfully induces protective immunity in

both systemic and intestinal (mucosal) tissues in mice. Rice-based oral vaccine is taken up by M cells lining the PP of the intestine, and it induces the formation of toxin-specific serum IgG and mucosal IgA antibodies with neutralizing activity (Nochi et al., 2007). We previously demonstrated that intraperitoneally or intradermally administered APLs inhibit arthritis in autoimmune models of RA, such as CIA and glucose-6-phosphate isomerase (GPI)-induced arthritis (Iwanami et al., 2009; Wakamatsu et al., 2009). These APLs function at different levels, including suppressing IL-17 production and reducing antigen-specific autoantibody production (total IgG) in serum. On the other

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8 Mana Iizuka et al. hand, in this study, we found that oral administration of APL6 transgenic rice delayed the onset of arthritis via enhancement of IL-10 production. Thus, although the effects of different forms of APL treatment in the RA model are identical with respect to the suppression of arthritis, these treatments may utilize different mechanism regarding immune prevention. In the light of the observation that APL transgenic rice can ameliorate CIA, what molecular mechanisms are involved in preventing the development of arthritis in CIA mice through oral administration? First, APLs in transgenic rice may induce antigen-specific T-cell anergy (Ohnishi et al., 2006). However, there was no evidence for this effect in the present study, as the transgenic rice had little effect on inflammatory cytokines (IFN-c and IL-17) produced by CII antigen-specific CD4+ T cells or CD44 expression in CD4+ T cells. Second, APL6 can induce IL10-producing regulatory T cells. Indeed, Jinxia and colleagues (Zhao et al., 2008) reported that APLs inhibit CIA in rats by inducing IL-10-producing regulatory T cells. Our study also observed a significant increase in APLs-induced IL-10 production in CIA mice; however, the mechanisms responsible for the production of CII antigen-activated IL-10 remain elusive. In the present study, administration of APL6 transgenic rice had no effect on the differentiation of CD4+ CD25+ Foxp3+ regulatory T cells (Treg). Surprisingly, the predominant IL-10-producing cells were CD4+ CD25 cells. Previous studies have demonstrated that certain IL-10-producing regulatory T cells express CD4, but not CD25, such as type 1 regulatory T (Tr1) cells (Gagliani et al., 2013; Groux et al., 1997; Vieira et al., 2004) and lymphocyte activation gene-3 (LAG3) regulatory T cells (LAG3+ Treg) (Okamura et al., 2009, 2012), which suppress autoimmunity based on IL-10 production. Tr1 cells prevented inflammation in an animal model of colitis, in which the pathogenic CD45RBhighCD4+ T cells were transferred into SCID mice (Groux et al., 1997). Similarly, in a mouse model of experimental autoimmune encephalomyelitis (EAE), transfer of antigen-specific Tr1 cells prevented the development of neurological symptoms (Barrat et al., 2002). In another study, IL-10-secreting LAG3+ Treg cells expressed LAG3 and early growth response gene-2 (EGR2), which also suppressed the function of na€ıve CD4+ T cells (Iwasaki et al., 2013). However, in the current study, treatment with APL6 transgenic rice in CIA did not significantly increase the expression of Egr2 and LAG3 in CD4+ CD25 T cells, although LAG3 expression was slightly upregulated. Thus, these results suggest that IL-10 producing CD4+ CD25 T cells induced by orally administered APLs in transgenic rice may represent novel regulatory T cells rather than the standard Tr1 cells and LAG3+ Treg cells. The high levels of APLs that were administered orally may be related to this novel suppression. The precise mechanisms of IL-10 production by CII-specific CD4+ CD25 T cells induced by APL transgenic rice should be examined in future studies. In conclusion, treatment with APL transgenic rice suppressed inflammation of CIA through the induction of a novel IL-10 producing CD4+ CD25 regulatory T cells. This study demonstrates the potential utility of APLs for antigen-specific immunotherapy of autoimmune diseases.

GluA2 and GluB4) (Iida et al., 1997), was used as the host plant for transformation, because low glutelin mutants, such as a123, can accrue high levels of transgenic products (Tada et al., 2003).

Experimental procedures

The MH2-GEQGPKGEPGI-OH peptide derived from the conserved region of three-tandem repeated-CII256-271 and APL6 (Figure 1a) was synthesized and used to raise anti-CII polyclonal antibody in a rabbit (Scrum Inc., Tokyo, Japan). Anti-CII antibody can detect signals for glutelin-fused CII and APL6 (Figure 1a).

Plant materials The rice (Oryza sativa L.) seed storage protein mutant a123 (cv. ‘Koshihikari’ background), which lacks three glutelins (GluA1,

Production of transgenic rice Two types of gene expression cassettes were prepared harbouring seed-specific promoters, modified seed storage protein glutelin genes and terminators: (i) GluB1 promoter::modified GluA2 coding region::GluB1 terminator; (ii) 16 kDa prolamin promoter::modified GluB1 coding region::16 kDa prolamin terminator and (iii) 10 kDa prolamin promoter::modified GluC coding region::10 kDa prolamin terminator. DNA fragments encoding the three-tandem repeat of T-cell epitope of collagen type II (CII256-271) or its analogue peptides (APL6; Figure 1a) were inserted into the C-termini of the acidic subunit of modified glutelins. These gene cassettes were subcloned into MultiSite Gateway (Invitrogen, Carlsbad, CA) entry clones (pKS4-1 harbouring 16 kDa prolamin promoter::GluB1-fused CII, APL6::16 kDa prolamin terminator, pKS221 harbuoring GluB1 promoter::GluA2-fused CII, APL6::GluB1 terminator and pKS2-3 harbouring 10 kDa prolamin promoter::GluC-fused CII and APL6::10 kDa prolamin terminator). Subsequently, these gene cassettes were transferred into binary vector CSP::mALS 43 GWII (Wakasa et al., 2006) via the MultiSite Gateway LR Clonase Reaction (Invitrogen). CSP::mALS is a selectable marker gene cassette derived from the rice genome consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP; CSP::mALS). The mALS marker has significantly reduced affinity for sulphonylurea, imidazolinone and pyrimidinyl carboxy herbicides and has been used to develop transformation systems (Chaleff and Mauvais, 1984; Shimizu et al., 2002). The completed binary vector constructs are shown in Figure 1b. These binary vector plasmids were introduced into the a123 genome via Agrobacterium-mediated transformation (Goto et al., 1999).

Protein extraction and immunoblot analysis Mature seeds from the transgenic rice lines were harvested. Each seed was ground into a fine powder. For total protein extraction, 500 lL of extraction buffer [50 mM Tris-HCl pH 6.8, 8 M urea, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue (BPB)] was added to the seed powder and vortexed for more than 1 h at room temperature. The mixture was centrifuged at 12 000 g for 10 min at room temperature, and the total protein sample was transferred to a fresh tube. Protein samples (2 lL) were subjected to immunoblot analysis after electrophoresis on 12% SDS-PAGE gels. After electrophoresis, the proteins were transferred onto an Immobilon-P PVDF Transfer membrane (Millipore, Billerica, MA). The membrane was reacted with the primary antibody at 4 °C for 16 h after blocking with 5% skim milk for 1 h, followed by incubation with secondary anti-rabbit IgG-conjugated HRP antibody for 3 h. The signals were detected using ECL-Western Blotting Detection Reagent (GE Healthcare UK, Little Chalfont, England) and X-ray film (Fujifilm, Tokyo, Japan).

Antibody preparation

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Immune suppression of arthritis by transgenic rice 9 Anti-GluA, GluB and GluC antibodies were produced as described previously (Wakasa and Takaiwa, 2013). Anti-GluA antibody reacts with endogenous GluA1, GluA2 and recombinant GluA2fused CII256-271 and APL6 Anti-GluB antibody reacts with endogenous GluB1, GluB2, GluB4 and recombinant GluB1-fused CII256-271 and APL6. Anti-GluC antibody reacts with endogenous GluC and GluC-fused CII256-271 and APL6.

Confocal immune-histochemical analysis Immature seeds were collected at 15 days after flowering (DAF) and used for confocal microscopy. The samples were crosssectioned with a DTK-1000 Microslicer (DOSAKA EM Co., Ltd., Kyoto, Japan) to approximately 200 lm in thickness, and the sections were used for immunocytochemical confocal microscopic analysis using the anti-CII antibody as described by Yasuda et al., 2006. After immuno-staining the sections, rhodamine B was used to stain PB-I and PB-II. The samples were observed under a confocal laser scanning microscope (FLUOVEIW; OLYMPUS, Tokyo, Japan).

Mice with CIA DBA/1 J mice were purchased from The Charles River Laboratory (Yokohama, Japan). The mice were maintained under specific pathogen-free conditions at the laboratory animal resource center. The Ethics Review Committee of Tsukuba University approved the study, and all experiments were performed according to the Guide for the Care and Use of Laboratory Animals from the same university. The mice were immunized intradermally with 100 lg bovine type II collagen (CII: Collagen Research Center, Tokyo, Japan) in Complete Freund’s Adjuvant (CFA; Difco, Detroit, MI). Each mouse received a booster dose of 100 lg CII intraperitoneally on day 21. The mice were observed at 3-day intervals and evaluated for the severity of arthritis by scoring each paw. The scores ranged from 0 to 3 (0, no swelling or redness; 1, swelling or redness in one joint; 2, involvement of two or more joints; 3, severe arthritis of the entire paw and joints). The score for each animal is the sum of scores for all four paws.

Therapeutic and prophylactic treatment with APL transgenic rice Altered peptide ligands transgenic rice was dissolved in phosphate-buffered solution (PBS) and administered to mice orally at 400 lg for 2 weeks after the first immunization.

Measurement of ex vivo CII-specific T-cell response DBA/1 mice were immunized intradermally with 100 lg bovine CII emulsified in CFA containing 250 lg of inactivated Mycobacterium tuberculosis H37Ra. After CII/CFA immunization, splenocytes, inguinal lymph node cells and mesenteric lymph node cells were restimulated with 100 lg/mL bovine CII for 72 h at 37 °C under a 5% CO2–95% air environment. CD4+ CD25+ T cells or CD4+ CD25 T cells were isolated from the spleen by magneticactivated cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were restimulated with 100 lg/mL bovine CII for 72 h in the presence of irradiated splenocytes as antigenpresenting cells (APC). Cell suspensions were prepared in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich Co., St. Louis, MO) containing 10% foetal bovine serum (FBS), penicillin–streptomycin (100 U/mL) and 5.5 mM 2-mercaptoethanol (2-ME). The concentrations of IFN-c, IL-2, IL-10 and IL-17 in the culture supernatant were measured by ELISA using a Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Acknowledgements We thank Dr. F. G. Issa for the critical reading of the manuscript and Ms. M. Utsuno, Ms. K. Miyashita, Ms. Y. Ikemoto and Ms.H.Yajima for technical assistance. This work was supported by Agri-Health Translational Research Project from the Ministry of Agriculture, Forestry and Fisheries of Japan and the Research Program for Intractable Diseases, Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare, Japan, and the Ministry of Education, Culture, Sports, Science and Technology.

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Supporting information Additional Supporting information may be found in the online version of this article: Figure Figure Figure Figure Figure

S1 S2 S3 S4 S5

Southern blot analysis. Quantitative analysis of transgene products. Seed phenotypes. Oral administration of APL transgenic rice in CIA. Expression of activation markers in CD4+ T cells.

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