Feeding transgenic plants that express a tolerogenic fusion protein effectively protects against arthritis

Abstract

Although much explored, oral tolerance for treatment of autoimmune diseases still awaits the establishment of novel and effective vectors. We investigated whether the tolerogenic CTA1(R7K)-COL-DD fusion protein can be expressed in edible plants, to induce oral tolerance and protect against arthritis. The fusion protein was recombinantly expressed in Arabidopsis thaliana plants, which were fed to H-2(q) -restricted DBA/1 mice to assess the preventive effect on collagen-induced arthritis (CIA). The treatment resulted in fewer mice exhibiting disease and arthritis scores were significantly reduced. Immune suppression was evident in treated mice, and serum biomarkers for inflammation as well as anticollagen IgG responses were reduced. In spleen and draining lymph nodes, CD4(+) T-cell responses were reduced. Concomitant with a reduced effector T-cell activity with lower IFNγ, IL-13 and IL-17A production, we observed an increase in IL-10 production to recall antigen stimulation in vitro, suggesting reduced Th1, Th2 and Th17 activity subsequent to up-regulated IL-10 and regulatory T-cell (Treg) functions. This study shows that edible plants expressing a tolerogen were effective at stimulating CD4 T-cell tolerance and in protecting against CIA disease. Our study conveys optimism as to the potential of using edible plants for oral treatment of rheumatoid arthritis.

Keywords: CIA; FoxP3; IL-10; autoimmunity; edible plants; transgenic plants.

Figure 1

Arabidopsis thaliana plant expression of the CTA1(R7K)‐COL‐DD tolerogen. Schematic diagrams of the pGreen transformation vector and integration cassette/construct. Left border (LB) and right border (RB) flank the transfer DNA (t‐DNA) integrated into the nuclear genome of A. thaliana (a). Western blot analysis of transgenic plants expressing CTA1(R7K)‐COL‐DD (1), empty vector pGreen (2), CTA1(R7K)‐DD (3) or untransformed WT (4) using anti‐CTA1‐DD antibodies with positive bands at ~39 kDa (b). Expression levels of CTA1(R7K)‐COL‐DD in the TG plant were estimated by a semiquantitative analysis using recombinant CTA1(R7K)‐COL‐DD (c). Protein expression levels in different parts of TG plants were assessed in leaves (L), stems (S), siliques (P) and inflorescence (F). Protein was extracted with 50 mm Tris buffer (pH 7.5) containing 8 m urea. Extracts were 1 mg biomass/μL and 15 μL of each sample was loaded onto the gel. (d). Furthermore, the following subcellular fractions were examined for their content of CTA1(R7K)‐COL‐DD; soluble cytoplasmic (S), insoluble (IS) and chloroplast (Chl) fractions (e).

Figure 2

Feeding transgenic Arabidopsis thaliana plants to mice protects against CIA disease. DBA/1 mice were divided into groups of ten and fed approximately 70 g of WT or TG A. thaliana according to the experiment protocol (a). After the booster dose of CII in IFA on day 21, the mice were under observation for onset of CIA and scored regularly to determine the progression of disease. AUC valued were calculated for statistical analyses (b–c). Data are shown from one representative experiment and shown as mean ± CI (b) or from five pooled experiments (n = 20–50) and shown as mean ± SEM (c). Statistical significance was determined by a one‐way ANOVA, where * P < 0.05 and **** P < 0.0001

Figure 3

Systemic responses to CIA are reduced in plant fed mice. Serum was collected from all remaining mice at the end of the experiment. CII‐specific antibody levels of the different IgG subclasses were determined by ELISA, and values are shown as mean log₁₀‐titres ±SD from one representative experiment (n = 4–5) (a). Levels of MMP‐3 (b) and IL‐6 (c) in serum are given as individual values and the mean ± SEM from pooled experiments (b–c). Statistical significance was calculated using an unpaired Student's t‐test (a) or a one‐way ANOVA (b–c), where * P < 0.05 and ** P < 0.01 and *** P < 0.001.

Figure 4

Suppressed CD4 T‐cell responses to CIA in mice fed transgenic plants. At the end of the experiment, lymphocytes from spleen or popliteal lymphnodes were isolated from mice that had not yet been euthanized and restimulated with recall COL _259–274 peptide in vitro. Proliferation was assessed after 72 hours by thymidine incorporation (a), and supernatants were collected and analysed for cytokine content (b). Furthermore, FoxP3+ CD4 T cells in peripheral blood were quantified by FACS (c). Proliferation data (a) are summarized from one representative experiment of one (popLN) or four (SP) and shown as mean ± SEM. Relative cytokine production in TG plant fed mice vs WT plant fed mice (b) is pooled data from two to four experiments and shown as mean ± SEM. Representative FoxP3+ CD4 FACS plots (c) are shown and summarized as mean ± SD from one of two independent experiments with similar findings. Statistical significance was measured by Student's t‐test (a–b) or a one‐way ANOVA (c), where * P < 0.05 and ** P < 0.01 and *** P < 0.001.

Figure 5

Feeding tolerance‐inducing plants protects against CIA disease and tissue destruction. When mice were euthanized, before or at the termination of the experiment, joints were collected and stained for haematoxylin and eosin (a). Lymphocyte infiltrates and cartilage destruction were estimated to determine the severity of synovitis, inflammation and bone erosion (b). Data from one of two independent experiments is shown as a scatter dot plot, and statistical significance was determined by a one‐way ANOVA, where * P < 0.05 and ** P < 0.01.