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Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis a
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Mana Iizuka , Yuhya Wakasa , Hiroto Tsuboi , Hiromitsu Asashima , Tomoya Hirota , Yuya a
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Kondo , Isao Matsumoto , Takayuki Sumida & Fumio Takaiwa
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Faculty of Medicine, Department of Internal Medicine, University of Tsukuba, Tsukuba, Japan b
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan Published online: 10 Jul 2014.
To cite this article: Mana Iizuka, Yuhya Wakasa, Hiroto Tsuboi, Hiromitsu Asashima, Tomoya Hirota, Yuya Kondo, Isao Matsumoto, Takayuki Sumida & Fumio Takaiwa (2014) Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis, Bioscience, Biotechnology, and Biochemistry, 78:10, 1662-1668, DOI: 10.1080/09168451.2014.936349 To link to this article: http://dx.doi.org/10.1080/09168451.2014.936349
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 10, 1662–1668
Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis Mana Iizuka1, Yuhya Wakasa2, Hiroto Tsuboi1, Hiromitsu Asashima1, Tomoya Hirota1, Yuya Kondo1, Isao Matsumoto1, Takayuki Sumida1 and Fumio Takaiwa2,* 1
Faculty of Medicine, Department of Internal Medicine, University of Tsukuba, Tsukuba, Japan; 2Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan
Received February 20, 2014; accepted May 5, 2014
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http://dx.doi.org/10.1080/09168451.2014.936349
Rheumatoid arthritis is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We generated transgenic rice seeds expressing three types of altered peptide ligands (APL) and the T cell epitope of type II collagen (CII256–271). When these transgenic rice and non-transgenic rice seeds were orally administrated to DBA/1 J mice once a day for 14 days, followed by immunization with CII, the clinical score of collagen-induced arthritis (CIA) was reduced and inflammation and erosion in the joints were prevented in mice fed APL7 transgenic rice only. IL-10 production against the CII antigen significantly increased in the splenocytes and iLN of CIA mice immunized with the CII antigen, whereas IFN-γ, IL-17, and IL2 levels were not altered. These results suggest that IL-10-mediated immune suppression is involved in the prophylactic effects caused by transgenic rice expressing APL7. Key words:
altered peptide ligands; autoreactive T cells; rheumatoid arthritis; transgenic rice; type II collagen
Rheumatoid arthritis (RA) is a common autoimmune disease primarily manifesting as chronic synovitis that leads to cartilage destruction, bone erosion, periarticular decalcification, and ultimately impaired joint function. Although the pathogenesis of RA remains unclear, there is sufficient evidence to suggest the involvement of T cells in the inflammatory process, such as the infiltration of T cells, especially CD4+ T cells, in RA joints.1–3) Type II collagen (CII) is an abundant extracellular matrix protein present in joint cartilage, and plays a critical role in the smooth movement of joints. CII is one of the target autoantigens in RA and its reactive T
cell clones have been established in vitro from the synovial T cells of RA.4,5) Altered peptide ligands (APL) are peptides that have substitutions in amino acid residues at T cell receptor contact sites.6,7) We reported previously that peripheral blood mononuclear cells from HLA-DRB1*0101 Japanese patients with RA were highly reactive to the 256–271 peptide of CII, and also that designed APLs suppressed the T cell response to its immunodominant epitope (CII256–271, GEPGIAGFKGEQGPKG).8) APL6 (G262A) and APL7 (K264A), in particular, were shown to have a potential inhibitory effect on the T cell response.9) Antagonistic APLs can inhibit the function of limited T cell populations; therefore, they may be useful as an antigen-specific therapy for autoimmune diseases. It has been reported in animal model of RA that repeated oral administration of CII peptide antigen induces immune tolerance, resulting in the suppression of collagen-induced arthritis (CIA).10–12) Plants are an ideal production platform for high value products such as biopharmaceuticals.13,14) In addition to the scalability, safety, and cost-effectiveness commonly associated with plant production, seeds are especially advantageous in terms of high productivity and stability. Rice seeds represent a potential candidate for the development of plant-derived edible drugs because even artificial products can be easily accumulated, and products bioencapsulated in specific organelles called protein bodies (PBs) can be protected from harsh conditions and proteolysis in the gastrointestinal tract.15,16) In the present study, we developed a transgenic rice plant expressing the APLs of CII256–271 in its rice seeds, which were orally administered to DBA/1 J mice to examine their prophylactic effects against a CII challenge. Furthermore, arthritis-suppressive immune mechanisms were investigated using mice in which prophylactic effects were observed.
*Corresponding author. Email: [email protected] Abbreviations: CII, collagen type II; CIA, collagen-induced arthritis; CSP, callus specific promoter; FBS, fetal bovine serum; iLN, inguinal lymph nodes; IL, interleukin; INF, interferon; mALS, mutated acetolactate synthase; mLN, mesenteric lymph nodes; PBS, phosphate buffered saline; RA, Rheumatoid arthritis. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Transgenic rice for prophylaxis of rheumatoid arthritis
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Materials and methods Generation of transgenic rice expressing APLs and the T cell epitope of CII in seeds. DNA fragments encoding the three-tandem repeat of the T cell epitope of collagen type II (CII256–271) or its analog peptides (APL4 [G262D; as a control of APLs], APL6 and APL7) were inserted into the C-termini of the acidic subunit of modified glutelins (GluA-2, GluB-1, and GluC). Three expression cassettes, each composed of modified glutelins containing individual peptides directed by the endosperm-specific glutelin 2.3 kb GluB-1, and 0.8 kb 10 kDa prolamin and 0.9 kb 16 kDa prolamin promoters,17) were transferred into the binary vector CSP::mALS 43 GWII18) using the Multisite Gateway system (Invitrogen), and subsequently introduced into the rice genome of low glutelin mutant a123 (cv. Koshihikari variety) via Agrobacterum-mediated transformation.19) Transgenic rice plants were selected based on their resistance to the herbicide Bispyribac and grown in a closed greenhouse. Homozygous transgenic lines were used for this study. At least four dry seeds for each independent transgenic rice line were ground to a fine powder using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan). Total seed protein was extracted using a buffer containing 50 mM Tris–HCl (pH6.8), 4% SDS, 8 M urea, 5% 2mercaptoethanol (2-ME), 20% glycerol, and 0.01% bromophenol blue. The expression of the APLs or CII peptides in mature seeds was detected immunologically using a rabbit antibody raised against the CII256-271 peptide (GEQGPKGEPGI) derived from the consensus region of the three-tandem repeated CII256-271, APL4, APL6, and APL7.
Mice with CIA. DBA/1 J mice were purchased from the Charles River Laboratory (Yokohama, Japan). They were maintained in specific pathogen-free conditions in the laboratory animal resource center. The Ethics Review Committee of Tsukuba University approved the study and all experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals of the same university. Mice were immunized intradermally with 100 μg bovine type II collagen (CII: Collagen Research Center) in Complete Freund’s adjuvant (CFA; Difco). Each mouse received a booster dose of 100 μg CII intraperitoneally on day 21. Mice were observed at 3-day intervals and evaluated for the severity of arthritis by scoring each paw. The score 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 in the entire paw and joints). The score used for each animal was the sum of the scores in all four paws. Treatment protocol for CIA. Transgenic rice seed powder (80 to 135 mg) containing CII256–271, APL4, APL6, or APL7 was adjusted to administer almost same amount of recombinant protein as glutelin fusion protein (700–800 μg) per mouse, which include 100–120 μg as
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APL peptide. On the other hand, 100 mg of non-transgenic rice seed powder was administered to each mouse as a control. Rice seed powder suspended in 500–750 μL of phosphate buffered saline (PBS) was orally administrated to DBA/1J mice once a day for 2 weeks, which were then immunized with the CII antigen to induce the arthritis. Measurement of the ex-vivo CII-specific T cell response. DBA/1 J mice were immunized intradermally with 100 μg bovine CII emulsified in CFA containing 250 μg of inactivated Mycobactrium tuberculosis H37Ra. Splenocytes and lymphocytes were restimulated with 100 μg/mL bovine CII for 72 h at 37 °C under a 5% CO2 − 95% air environment. Cell suspensions were prepared in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich Co) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, and 5.5 mM 2-ME. IL-17, IFN-γ, IL-2, and IL-10 concentrations were measured in the culture supernatant by ELISA using the Quantikine ELISA kit (R&D Systems).
Measurement of type II collagen-specific immunoglobulin titers. A total of 10 μg/mL of CII in PBS was coated overnight at 4 °C onto 96-well plates (Nalge Nunc). After washes with washing buffer (0.05% Tween 20 in PBS), the blocking solution, including 1% bovine serum albumin in PBS, was applied for 1 h. After washing, 100 μL of diluted serum (1:50) was added, and the plates were incubated for 1 h at room temperature. After further washing, horseradish peroxidase-conjugated anti-mouse IgG, IgG1, and IgG2a in blocking solution was added, and the plates were incubated for 1 h at room temperature. After washing, tetramethylbenzidine was added, and optical density was read at 450 nm using a microplate reader.
Statistical analysis. In the CIA experiments, disease incidence was evaluated with the chi-square test and the severity score by the Turkey’s t-test. All values are expressed as means ± S.D. or S.E. We analyzed for significance by using Student’s t-test for two groups or by Turkey’s t-test for multiple groups. P values less than 0.05 were considered significant (SPSS).
Results and discussion Three tandem repeats of APLs (APL4, APL6, and ALP7) or CII epitope (CII256–271) peptides were inserted into the C-terminal highly variable region of the acidic subunit of three types of glutelin precursors, GluA-2, GluB-1, and GluC, that were linked upstream to three different rice endosperm-specific promoters (GluB-1, 16 kDa prolamin, and 10 kDa prolamin) to increase accumulation levels, according to the method described by Wakasa et al.18) (Fig. 1). Three different glutelin expression cassettes
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Fig. 1. The binary vector construct consisted of selection marker gene and expression gene cassettes. Notes: CSP, callus-specific promoter; mALS, mutated acetolactate synthase coding region; 10 K T, 10 kDa prolamin terminator; 16 K P, 16 kDa prolamin promoter; GluB1, modified GluB-1 coding region; 16 K T, 16 kDa prolamin terminator; GluB1 P, GluB-1 promoter; GluA2, modified GluA-2 coding region, GluB1T, GluB-1 terminator; 10 K P, 10 kDa prolamin promoter; GluC, modified GluC coding region; LB, left border; RB, right border; 3 × peptides, three tandem repeated CII256-271, APL4, APL6 or APL7.
containing individual APL peptides were transferred into a single binary vector and introduced into the rice genome. When the individual APL4, ALP6, APL7, and CII256-271 peptides were expressed as fusion proteins with three glutelins under the control of three different rice endosperm-specific promoters together (Fig. 2), they accumulated at levels of 0.93 mg/g seed, 3.69 mg/g seed, 1.35 mg/g seed, and 1.08 mg/g seed as three tandem repeated peptide, respectively20) (Supplementary Fig. S1; see http://dx.doi.org/ 10.1080/09168451.2014.936349). They were deposited in the protein-storage vacuole (PB-II) in a manner similar to that of endogenous glutelins. To investigate the prophylactic effects of APL transgenic rice in DBA/1 J mice, APL4, APL6, APL7 transgenic rice or non-transgenic rice seeds were orally administered once a day for 14 days, followed by an intradermal challenge with the CII antigen (Fig. 3(A)). As shown in Fig. 3(B) and (C), the highest improvement in the clinical score and incidence of arthritis was observed in mice fed APL7 transgenic rice. The incidence of mice treated with APL7 transgenic rice was the lowest among four groups, but there was no significant difference (Fig. 3(C)). The severity of inflammation and erosion in joints was markedly less in CIA mice treated with APL7 transgenic rice than in those treated with APL4, APL6, and non-transgenic rice (Fig. 3(D)). To confirm the efficacy of APL7 transgenic rice, CIA mice fed inherent CII256–271 transgenic rice or nontransgenic rice seeds were compared with mice fed APL7 transgenic rice (Supplementary Fig. S2(a); see http://dx.doi.org/10.1080/09168451.2014.936349). As expected, the clinical score was lower (Supplementary Fig. S2(b); see http://dx.doi.org/10.1080/09168451. 2014.936349) and the onset of CIA was slower in mice fed APL7 transgenic rice than in those fed control rice (Supplementary Fig. S2(c); see http:// dx.doi.org/10.1080/09168451.2014.936349). These results confirmed the prophylactic potential of orally administered transgenic rice seeds expressing APL7 peptides in CIA.
Fig. 2. Accumulation of four kinds of recombinant peptide as glutelin fusion protein. Notes: (A) Accumulation scheme of modified glutelin in rice seed cells. Black box indicates three-tandem repeated recombinant peptides (CII256-271, APL4, APL6, or APL7). Recombinant peptides accumulate as part of glutelin acidic subunit in protein body II (PB-II) in rice endosperm cells. (B) SDS-PAGE (upper panel) and immunoblot analysis using anti-tandem repeated CII antibody (lower panel). This antibody can react with tandem repeated CII256-271, APL4, APL6, or APL7 peptides.
To elucidate the mechanisms underlying the antiCIA effects of APL7 transgenic rice, we analyzed the effects of APL7 transgenic rice on the production of inflammatory cytokines. IL-10 production was significantly higher in the spleen (Fig. 4(A)) and inguinal lymph nodes (iLN) (Fig. 4(B)), in CIA mice treated with APL7 transgenic rice than in those treated with CII256–271 transgenic rice or non-transgenic rice, but was not in the mesenteric lymph nodes (mLN) (Fig. 4(C)). Furthermore, IFN-γ was slightly increased in the spleen and iLN due to the treatment with APL7 transgenic rice. However, IL-17 could not be detected and the production of IL-2 was varied in these tissues. These results demonstrated that the RA-ameliorating effects of APL7 transgenic rice were mediated through the production of IL-10. Previous reports showed that APL7 (K264A) peptide (405 μg/mouse) suppressed the IL-17 producing antigen specific CD4+ T cells isolated from splenocytes of CIA at day 12 after first immunization with CII in vitro.9)
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Fig. 3. Effect of the oral administration of transgenic rice seeds containing APLs on the development of CIA. Notes: (A) Schematic illustration showing the treatment schedule. APL4, APL6, and APL7 transgenic rice or non-transgenic rice was orally administrated to mice (n = 8 for ALP4, n = 7 for APL7, and n = 6 for APL6 and non-transgenic rice) for 2 weeks, which were then immunized with the CII antigen. Clinical score (B) and incidence of arthritis (C). Data are mean ± SE. (D) Hematoxylin and eosin-stained sections of the hind paws of mice obtained 60 days after the first immunization. Region between arrows indicate cellular infiltrations. Data are representative for four tissue samples. Bars = 70 m.
On the other hand, as shown here it is important to note that oral administration of transgenic rice containing glutelin fusion protein with APL7 peptide (720 μg/mouse), which corresponds to 108 μg/ mouse as APL7 peptide, could suppress the development of arthritis in CIA even under the harsh conditions in the gastrointestinal environment of the digestive tract. The efficacy of APL7 transgenic rice through an oral route suggests that not only mucosal, but also systemic unresponsiveness (immune tolerance) to the CII antigen may be induced via the production of IL-10, resulting in the suppression of arthritis.
As described above, an in vitro analysis revealed the induction of IL-10 from splenic and iLN cells in APL7-treated mice cultured with the CII antigen. Therefore, we examined whether the effects induced by the treatment with APL7 transgenic rice depended on the differentiation of regulatory T cells. The treatment with APL7 transgenic rice did not affect the population of CD4+CD25+Foxp3+ regulatory T cells (Fig. 5(A)). Moreover, when the levels of the activation marker of CD4+ T cells were measured in CIA mice treated with APL7 transgenic rice, the expression of CD44 in the spleen, iLN, and mLN remained unchanged (Fig. 5(B)). Furthermore, the levels or types
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Fig. 4. Increased production of IL-10 by CII-reactive T cells in CIA mice treated with APL7-transgenic rice. Notes: Mice were treated with APL7 or control rice for 2 weeks before first immunization with the CII antigen. On day 35 after the first immunization, mice were euthanized and the spleens (A), iLNs (B), and mLNs (C) were cultured with CII for 72 h. IFN-γ, IL-2, IL-10, and IL-17 concentrations in the culture supernatant were measured by ELISA. Representative data of three independent experiments. Data are the mean ± SD of four samples per group. *P < 0.05, N·S. – not significantly, N.D. – not detected.
of anti-CII IgG antibodies such as IgG, IgG1, and IgG2a were not significantly altered (Supplementary Fig. S3; see http://dx.doi.org/10.1080/09168451.2014. 936349). Since the number of CD4+CD25+Foxp3+ regulatory T cells was not changed by the treatment with APL7 transgenic rice, some Foxp3 negative IL-10 producing regulatory T cells including type 1 regulatory T (Tr1) cells21,22) or lymphocyte activation gene-3 (LAG3) regulatory T cells (LAG3+ Treg) may be implicated in the suppression of autoimmunity.23) The
precise mechanisms responsible for the production of IL-10 by CD4+ T cells due to the intake of APL7 transgenic rice should be examined in future studies. In conclusion, the prophylactic treatment with APL7 transgenic rice attenuated the clinical inflammation associated with CIA by inducing novel IL-10 producing CD4+CD25− T cells. Furthermore, the oral administration of transgenic rice seeds containing APL7 may provide a novel therapeutic strategy for controlling RA arthritis as an antigen-specific immunotherapy.
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Fig. 5. Effect of the APL7 transgenic rice treatment on regulatory T cells and CD4+ T cells in CIA mice. Notes: Mice were treated with APL7 or control rice and then challenged with the CII antigen. The spleens, iLNs, and mLNs were harvested on day 35 and stained with CD25, Foxp3 (A), and CD44 (B). (A) A bar graph showing the proportion of CD4+CD25+Foxp3+ cells in CD4+ T cells from the spleen, iLN, and mLN, which was obtained from flow cytometric analysis for the expression of CD25 and Foxp3 was gated for CD4+ T cells. (B) Mean fluorescent intensity (MFI) of CD44 expression in CD4+ cells in the spleen, iLN, and mLN. Data are the mean ± SD of 4 mice per group. N·S. – not significantly.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.936349.
Acknowledgments This work was supported by the Agri-Health Translational Research Project from the Ministry of Agriculture, Forestry, and Fisheries of Japan and the Research Program for Intractable Diseases, Health and Labor Sciences Research Grants from the Ministry of Health, Labor, and Welfare, Japan, and the Ministry of Education, Culture, Sports, Science, and Technology.
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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis a
b
a
a
a
Mana Iizuka , Yuhya Wakasa , Hiroto Tsuboi , Hiromitsu Asashima , Tomoya Hirota , Yuya a
a
a
Kondo , Isao Matsumoto , Takayuki Sumida & Fumio Takaiwa
b
a
Faculty of Medicine, Department of Internal Medicine, University of Tsukuba, Tsukuba, Japan b
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan Published online: 10 Jul 2014.
To cite this article: Mana Iizuka, Yuhya Wakasa, Hiroto Tsuboi, Hiromitsu Asashima, Tomoya Hirota, Yuya Kondo, Isao Matsumoto, Takayuki Sumida & Fumio Takaiwa (2014) Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis, Bioscience, Biotechnology, and Biochemistry, 78:10, 1662-1668, DOI: 10.1080/09168451.2014.936349 To link to this article: http://dx.doi.org/10.1080/09168451.2014.936349
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 10, 1662–1668
Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis Mana Iizuka1, Yuhya Wakasa2, Hiroto Tsuboi1, Hiromitsu Asashima1, Tomoya Hirota1, Yuya Kondo1, Isao Matsumoto1, Takayuki Sumida1 and Fumio Takaiwa2,* 1
Faculty of Medicine, Department of Internal Medicine, University of Tsukuba, Tsukuba, Japan; 2Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan
Received February 20, 2014; accepted May 5, 2014
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http://dx.doi.org/10.1080/09168451.2014.936349
Rheumatoid arthritis is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We generated transgenic rice seeds expressing three types of altered peptide ligands (APL) and the T cell epitope of type II collagen (CII256–271). When these transgenic rice and non-transgenic rice seeds were orally administrated to DBA/1 J mice once a day for 14 days, followed by immunization with CII, the clinical score of collagen-induced arthritis (CIA) was reduced and inflammation and erosion in the joints were prevented in mice fed APL7 transgenic rice only. IL-10 production against the CII antigen significantly increased in the splenocytes and iLN of CIA mice immunized with the CII antigen, whereas IFN-γ, IL-17, and IL2 levels were not altered. These results suggest that IL-10-mediated immune suppression is involved in the prophylactic effects caused by transgenic rice expressing APL7. Key words:
altered peptide ligands; autoreactive T cells; rheumatoid arthritis; transgenic rice; type II collagen
Rheumatoid arthritis (RA) is a common autoimmune disease primarily manifesting as chronic synovitis that leads to cartilage destruction, bone erosion, periarticular decalcification, and ultimately impaired joint function. Although the pathogenesis of RA remains unclear, there is sufficient evidence to suggest the involvement of T cells in the inflammatory process, such as the infiltration of T cells, especially CD4+ T cells, in RA joints.1–3) Type II collagen (CII) is an abundant extracellular matrix protein present in joint cartilage, and plays a critical role in the smooth movement of joints. CII is one of the target autoantigens in RA and its reactive T
cell clones have been established in vitro from the synovial T cells of RA.4,5) Altered peptide ligands (APL) are peptides that have substitutions in amino acid residues at T cell receptor contact sites.6,7) We reported previously that peripheral blood mononuclear cells from HLA-DRB1*0101 Japanese patients with RA were highly reactive to the 256–271 peptide of CII, and also that designed APLs suppressed the T cell response to its immunodominant epitope (CII256–271, GEPGIAGFKGEQGPKG).8) APL6 (G262A) and APL7 (K264A), in particular, were shown to have a potential inhibitory effect on the T cell response.9) Antagonistic APLs can inhibit the function of limited T cell populations; therefore, they may be useful as an antigen-specific therapy for autoimmune diseases. It has been reported in animal model of RA that repeated oral administration of CII peptide antigen induces immune tolerance, resulting in the suppression of collagen-induced arthritis (CIA).10–12) Plants are an ideal production platform for high value products such as biopharmaceuticals.13,14) In addition to the scalability, safety, and cost-effectiveness commonly associated with plant production, seeds are especially advantageous in terms of high productivity and stability. Rice seeds represent a potential candidate for the development of plant-derived edible drugs because even artificial products can be easily accumulated, and products bioencapsulated in specific organelles called protein bodies (PBs) can be protected from harsh conditions and proteolysis in the gastrointestinal tract.15,16) In the present study, we developed a transgenic rice plant expressing the APLs of CII256–271 in its rice seeds, which were orally administered to DBA/1 J mice to examine their prophylactic effects against a CII challenge. Furthermore, arthritis-suppressive immune mechanisms were investigated using mice in which prophylactic effects were observed.
*Corresponding author. Email: [email protected] Abbreviations: CII, collagen type II; CIA, collagen-induced arthritis; CSP, callus specific promoter; FBS, fetal bovine serum; iLN, inguinal lymph nodes; IL, interleukin; INF, interferon; mALS, mutated acetolactate synthase; mLN, mesenteric lymph nodes; PBS, phosphate buffered saline; RA, Rheumatoid arthritis. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Transgenic rice for prophylaxis of rheumatoid arthritis
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Materials and methods Generation of transgenic rice expressing APLs and the T cell epitope of CII in seeds. DNA fragments encoding the three-tandem repeat of the T cell epitope of collagen type II (CII256–271) or its analog peptides (APL4 [G262D; as a control of APLs], APL6 and APL7) were inserted into the C-termini of the acidic subunit of modified glutelins (GluA-2, GluB-1, and GluC). Three expression cassettes, each composed of modified glutelins containing individual peptides directed by the endosperm-specific glutelin 2.3 kb GluB-1, and 0.8 kb 10 kDa prolamin and 0.9 kb 16 kDa prolamin promoters,17) were transferred into the binary vector CSP::mALS 43 GWII18) using the Multisite Gateway system (Invitrogen), and subsequently introduced into the rice genome of low glutelin mutant a123 (cv. Koshihikari variety) via Agrobacterum-mediated transformation.19) Transgenic rice plants were selected based on their resistance to the herbicide Bispyribac and grown in a closed greenhouse. Homozygous transgenic lines were used for this study. At least four dry seeds for each independent transgenic rice line were ground to a fine powder using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan). Total seed protein was extracted using a buffer containing 50 mM Tris–HCl (pH6.8), 4% SDS, 8 M urea, 5% 2mercaptoethanol (2-ME), 20% glycerol, and 0.01% bromophenol blue. The expression of the APLs or CII peptides in mature seeds was detected immunologically using a rabbit antibody raised against the CII256-271 peptide (GEQGPKGEPGI) derived from the consensus region of the three-tandem repeated CII256-271, APL4, APL6, and APL7.
Mice with CIA. DBA/1 J mice were purchased from the Charles River Laboratory (Yokohama, Japan). They were maintained in specific pathogen-free conditions in the laboratory animal resource center. The Ethics Review Committee of Tsukuba University approved the study and all experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals of the same university. Mice were immunized intradermally with 100 μg bovine type II collagen (CII: Collagen Research Center) in Complete Freund’s adjuvant (CFA; Difco). Each mouse received a booster dose of 100 μg CII intraperitoneally on day 21. Mice were observed at 3-day intervals and evaluated for the severity of arthritis by scoring each paw. The score 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 in the entire paw and joints). The score used for each animal was the sum of the scores in all four paws. Treatment protocol for CIA. Transgenic rice seed powder (80 to 135 mg) containing CII256–271, APL4, APL6, or APL7 was adjusted to administer almost same amount of recombinant protein as glutelin fusion protein (700–800 μg) per mouse, which include 100–120 μg as
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APL peptide. On the other hand, 100 mg of non-transgenic rice seed powder was administered to each mouse as a control. Rice seed powder suspended in 500–750 μL of phosphate buffered saline (PBS) was orally administrated to DBA/1J mice once a day for 2 weeks, which were then immunized with the CII antigen to induce the arthritis. Measurement of the ex-vivo CII-specific T cell response. DBA/1 J mice were immunized intradermally with 100 μg bovine CII emulsified in CFA containing 250 μg of inactivated Mycobactrium tuberculosis H37Ra. Splenocytes and lymphocytes were restimulated with 100 μg/mL bovine CII for 72 h at 37 °C under a 5% CO2 − 95% air environment. Cell suspensions were prepared in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich Co) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, and 5.5 mM 2-ME. IL-17, IFN-γ, IL-2, and IL-10 concentrations were measured in the culture supernatant by ELISA using the Quantikine ELISA kit (R&D Systems).
Measurement of type II collagen-specific immunoglobulin titers. A total of 10 μg/mL of CII in PBS was coated overnight at 4 °C onto 96-well plates (Nalge Nunc). After washes with washing buffer (0.05% Tween 20 in PBS), the blocking solution, including 1% bovine serum albumin in PBS, was applied for 1 h. After washing, 100 μL of diluted serum (1:50) was added, and the plates were incubated for 1 h at room temperature. After further washing, horseradish peroxidase-conjugated anti-mouse IgG, IgG1, and IgG2a in blocking solution was added, and the plates were incubated for 1 h at room temperature. After washing, tetramethylbenzidine was added, and optical density was read at 450 nm using a microplate reader.
Statistical analysis. In the CIA experiments, disease incidence was evaluated with the chi-square test and the severity score by the Turkey’s t-test. All values are expressed as means ± S.D. or S.E. We analyzed for significance by using Student’s t-test for two groups or by Turkey’s t-test for multiple groups. P values less than 0.05 were considered significant (SPSS).
Results and discussion Three tandem repeats of APLs (APL4, APL6, and ALP7) or CII epitope (CII256–271) peptides were inserted into the C-terminal highly variable region of the acidic subunit of three types of glutelin precursors, GluA-2, GluB-1, and GluC, that were linked upstream to three different rice endosperm-specific promoters (GluB-1, 16 kDa prolamin, and 10 kDa prolamin) to increase accumulation levels, according to the method described by Wakasa et al.18) (Fig. 1). Three different glutelin expression cassettes
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Fig. 1. The binary vector construct consisted of selection marker gene and expression gene cassettes. Notes: CSP, callus-specific promoter; mALS, mutated acetolactate synthase coding region; 10 K T, 10 kDa prolamin terminator; 16 K P, 16 kDa prolamin promoter; GluB1, modified GluB-1 coding region; 16 K T, 16 kDa prolamin terminator; GluB1 P, GluB-1 promoter; GluA2, modified GluA-2 coding region, GluB1T, GluB-1 terminator; 10 K P, 10 kDa prolamin promoter; GluC, modified GluC coding region; LB, left border; RB, right border; 3 × peptides, three tandem repeated CII256-271, APL4, APL6 or APL7.
containing individual APL peptides were transferred into a single binary vector and introduced into the rice genome. When the individual APL4, ALP6, APL7, and CII256-271 peptides were expressed as fusion proteins with three glutelins under the control of three different rice endosperm-specific promoters together (Fig. 2), they accumulated at levels of 0.93 mg/g seed, 3.69 mg/g seed, 1.35 mg/g seed, and 1.08 mg/g seed as three tandem repeated peptide, respectively20) (Supplementary Fig. S1; see http://dx.doi.org/ 10.1080/09168451.2014.936349). They were deposited in the protein-storage vacuole (PB-II) in a manner similar to that of endogenous glutelins. To investigate the prophylactic effects of APL transgenic rice in DBA/1 J mice, APL4, APL6, APL7 transgenic rice or non-transgenic rice seeds were orally administered once a day for 14 days, followed by an intradermal challenge with the CII antigen (Fig. 3(A)). As shown in Fig. 3(B) and (C), the highest improvement in the clinical score and incidence of arthritis was observed in mice fed APL7 transgenic rice. The incidence of mice treated with APL7 transgenic rice was the lowest among four groups, but there was no significant difference (Fig. 3(C)). The severity of inflammation and erosion in joints was markedly less in CIA mice treated with APL7 transgenic rice than in those treated with APL4, APL6, and non-transgenic rice (Fig. 3(D)). To confirm the efficacy of APL7 transgenic rice, CIA mice fed inherent CII256–271 transgenic rice or nontransgenic rice seeds were compared with mice fed APL7 transgenic rice (Supplementary Fig. S2(a); see http://dx.doi.org/10.1080/09168451.2014.936349). As expected, the clinical score was lower (Supplementary Fig. S2(b); see http://dx.doi.org/10.1080/09168451. 2014.936349) and the onset of CIA was slower in mice fed APL7 transgenic rice than in those fed control rice (Supplementary Fig. S2(c); see http:// dx.doi.org/10.1080/09168451.2014.936349). These results confirmed the prophylactic potential of orally administered transgenic rice seeds expressing APL7 peptides in CIA.
Fig. 2. Accumulation of four kinds of recombinant peptide as glutelin fusion protein. Notes: (A) Accumulation scheme of modified glutelin in rice seed cells. Black box indicates three-tandem repeated recombinant peptides (CII256-271, APL4, APL6, or APL7). Recombinant peptides accumulate as part of glutelin acidic subunit in protein body II (PB-II) in rice endosperm cells. (B) SDS-PAGE (upper panel) and immunoblot analysis using anti-tandem repeated CII antibody (lower panel). This antibody can react with tandem repeated CII256-271, APL4, APL6, or APL7 peptides.
To elucidate the mechanisms underlying the antiCIA effects of APL7 transgenic rice, we analyzed the effects of APL7 transgenic rice on the production of inflammatory cytokines. IL-10 production was significantly higher in the spleen (Fig. 4(A)) and inguinal lymph nodes (iLN) (Fig. 4(B)), in CIA mice treated with APL7 transgenic rice than in those treated with CII256–271 transgenic rice or non-transgenic rice, but was not in the mesenteric lymph nodes (mLN) (Fig. 4(C)). Furthermore, IFN-γ was slightly increased in the spleen and iLN due to the treatment with APL7 transgenic rice. However, IL-17 could not be detected and the production of IL-2 was varied in these tissues. These results demonstrated that the RA-ameliorating effects of APL7 transgenic rice were mediated through the production of IL-10. Previous reports showed that APL7 (K264A) peptide (405 μg/mouse) suppressed the IL-17 producing antigen specific CD4+ T cells isolated from splenocytes of CIA at day 12 after first immunization with CII in vitro.9)
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Fig. 3. Effect of the oral administration of transgenic rice seeds containing APLs on the development of CIA. Notes: (A) Schematic illustration showing the treatment schedule. APL4, APL6, and APL7 transgenic rice or non-transgenic rice was orally administrated to mice (n = 8 for ALP4, n = 7 for APL7, and n = 6 for APL6 and non-transgenic rice) for 2 weeks, which were then immunized with the CII antigen. Clinical score (B) and incidence of arthritis (C). Data are mean ± SE. (D) Hematoxylin and eosin-stained sections of the hind paws of mice obtained 60 days after the first immunization. Region between arrows indicate cellular infiltrations. Data are representative for four tissue samples. Bars = 70 m.
On the other hand, as shown here it is important to note that oral administration of transgenic rice containing glutelin fusion protein with APL7 peptide (720 μg/mouse), which corresponds to 108 μg/ mouse as APL7 peptide, could suppress the development of arthritis in CIA even under the harsh conditions in the gastrointestinal environment of the digestive tract. The efficacy of APL7 transgenic rice through an oral route suggests that not only mucosal, but also systemic unresponsiveness (immune tolerance) to the CII antigen may be induced via the production of IL-10, resulting in the suppression of arthritis.
As described above, an in vitro analysis revealed the induction of IL-10 from splenic and iLN cells in APL7-treated mice cultured with the CII antigen. Therefore, we examined whether the effects induced by the treatment with APL7 transgenic rice depended on the differentiation of regulatory T cells. The treatment with APL7 transgenic rice did not affect the population of CD4+CD25+Foxp3+ regulatory T cells (Fig. 5(A)). Moreover, when the levels of the activation marker of CD4+ T cells were measured in CIA mice treated with APL7 transgenic rice, the expression of CD44 in the spleen, iLN, and mLN remained unchanged (Fig. 5(B)). Furthermore, the levels or types
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Fig. 4. Increased production of IL-10 by CII-reactive T cells in CIA mice treated with APL7-transgenic rice. Notes: Mice were treated with APL7 or control rice for 2 weeks before first immunization with the CII antigen. On day 35 after the first immunization, mice were euthanized and the spleens (A), iLNs (B), and mLNs (C) were cultured with CII for 72 h. IFN-γ, IL-2, IL-10, and IL-17 concentrations in the culture supernatant were measured by ELISA. Representative data of three independent experiments. Data are the mean ± SD of four samples per group. *P < 0.05, N·S. – not significantly, N.D. – not detected.
of anti-CII IgG antibodies such as IgG, IgG1, and IgG2a were not significantly altered (Supplementary Fig. S3; see http://dx.doi.org/10.1080/09168451.2014. 936349). Since the number of CD4+CD25+Foxp3+ regulatory T cells was not changed by the treatment with APL7 transgenic rice, some Foxp3 negative IL-10 producing regulatory T cells including type 1 regulatory T (Tr1) cells21,22) or lymphocyte activation gene-3 (LAG3) regulatory T cells (LAG3+ Treg) may be implicated in the suppression of autoimmunity.23) The
precise mechanisms responsible for the production of IL-10 by CD4+ T cells due to the intake of APL7 transgenic rice should be examined in future studies. In conclusion, the prophylactic treatment with APL7 transgenic rice attenuated the clinical inflammation associated with CIA by inducing novel IL-10 producing CD4+CD25− T cells. Furthermore, the oral administration of transgenic rice seeds containing APL7 may provide a novel therapeutic strategy for controlling RA arthritis as an antigen-specific immunotherapy.
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Fig. 5. Effect of the APL7 transgenic rice treatment on regulatory T cells and CD4+ T cells in CIA mice. Notes: Mice were treated with APL7 or control rice and then challenged with the CII antigen. The spleens, iLNs, and mLNs were harvested on day 35 and stained with CD25, Foxp3 (A), and CD44 (B). (A) A bar graph showing the proportion of CD4+CD25+Foxp3+ cells in CD4+ T cells from the spleen, iLN, and mLN, which was obtained from flow cytometric analysis for the expression of CD25 and Foxp3 was gated for CD4+ T cells. (B) Mean fluorescent intensity (MFI) of CD44 expression in CD4+ cells in the spleen, iLN, and mLN. Data are the mean ± SD of 4 mice per group. N·S. – not significantly.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.936349.
Acknowledgments This work was supported by the Agri-Health Translational Research Project from the Ministry of Agriculture, Forestry, and Fisheries of Japan and the Research Program for Intractable Diseases, Health and Labor Sciences Research Grants from the Ministry of Health, Labor, and Welfare, Japan, and the Ministry of Education, Culture, Sports, Science, and Technology.
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