Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts--oral administration protects against development of insulitis in non-obese diabetic mice

Abstract

Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit-human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB-green fluorescent protein (CTB-GFP) or interferon-GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing beta-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few beta-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing beta-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T(1) progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases.

Figure 1

Southern analysis of cholera toxin B subunit–human proinsulin (CTB-Pins) tobacco transformants. (a) Schematic representation of expected results in Southern analysis. Full lines indicate expected fragment for wild-type (4.2 kb) and transformed (6.35 kb) genomes; broken line indicates the probe hybridization sites. (b) Southern analysis of second regenerants. 5CP lines 13, 14, M, O and P were derived from independent transformation events; WT, wild-type.

Figure 2

Regeneration and Southern analysis of cholera toxin B subunit–human proinsulin (CTB-Pins) lettuce transformants. (a, b) Schematic diagrams of lettuce transformation vector and integration cassette, integration site and resulting transplastome. Full lines indicate the sizes of the integration cassette (2.4 kb) and expected fragments for the wild-type (3.75 kb) and transformed (6.15 kb) genomes in Southern analysis; broken line indicates the probe hybridization sites. (c) Southern analysis of second regenerants. L100 and L101, independent transplastomic lines; WT, wild-type. (d) Primary regeneration in lettuce without formation of callus.

Figure 3

Production of transplastomic Latuca sativa and confirmation of maternal inheritance. (a) Lettuce plants propagated by rooting of nodal cuttings were transferred to soil in the glasshouse. Plants matured with no apparent aberrant phenotype (b) and produced normal inflorescences (c). Flowers heads opened (d), and seeds were harvested. T₁ seeds were plated on half-strength Murashige and Skoog (MS) medium containing 50 mg/L spectinomycin, together with wild-type seed (e). T₁ plants flourished and were transferred to the glasshouse (f).

Figure 4

Protein analyses. Western blots prepared from wild-type (WT) and transplastomic lettuce and tobacco were probed with human proinsulin monoclonal antibody. (a) Total protein (~20 µg) extracted from lettuce leaves was loaded into wells for each sample. In lane 6, L101-5, ~10 µg of total soluble protein (TSP) was loaded. (b) Total protein (~10 µg) extracted from tobacco leaves was loaded into wells. (c) Cholera toxin B subunit–human proinsulin (CTB-Pins) in plant samples was detected by polyclonal anti-CTB and quantified by comparison with known quantities of CTB standard. Lanes 1–3, bacterial CTB (25, 50, 100 ng). Lanes 4–6, transplastomic tobacco lines 5CP-13, 5CP-14, 5CP-M (~6 µg of TSP in each lane). Lanes 7 and 8, transplastomic lettuce lines L-100 and L-101 (~36 µg TSP in each lane). (d) Plot of integrated density values (IDVs) for quantitative analysis from standard curve. Broken line, data points; full line, trend line. (e) Estimation of CTB-Pins in tobacco and lettuce leaves using spot densitometry CTB immunoblots. (f) Tobacco and lettuce transformants were assayed for GM₁ binding. 5CP, CTB-Pins tobacco; L101 and L101, CTB-Pins lettuce; BSA, negative control (bovine serum albumin).

Figure 5

(a) Histochemical staining of pancreatic sections. (i) Haematoxylin and eosin staining of a section of the pancreas (showing an islet, isl) of a mouse receiving cholera toxin B subunit–human proinsulin (CTB-Pins) for 7 weeks (n = 7); scale bar, 50 µm. Lymphocytes are seen outside the islet (arrow). (ii) Arrows indicate the borders of an islet in the pancreas of a mouse receiving CTB–green fluorescent protein (CTB-GFP) (n = 5; control group). Blue dots show lymphocytic infiltration of the islet. (iii) A large islet with severe lymphocytic infiltration in a mouse receiving untransformed plant leaf material (UN-Tr; n = 3). (iv) Severe lymphocytic infiltration in a mouse receiving interferon–GFP (IFN-GFP) (n = 5). (b) Scoring (S) of the degree of insulitis according to the severity of the lymphocytic infiltration of the pancreatic Langerhans islets. Score 1, no or pre-islet infiltration; 2, minimal infiltration; 3, moderate infiltration; 4, severe infiltration; 5, more than 80% of the islets infiltrated. All sections were scored in a blind manner. (c) Graphic representation of insulitis scoring in untransformed, IFN-GFP, CTB-GFP and CTB-Pins plant-treated groups; bars represent standard deviation (*P < 0.05). n indicates the number of animals in each treatment group.

Figure 6

Assessment of insulin production and apoptosis in pancreatic β-cells. (a) Insulin immunoreactivity in Langerhans islets of a mouse receiving cholera toxin B subunit–human proinsulin (CTB-Pins). (b) Caspase-3 immunostaining in the same section is shown in the red channel. (c) Merged picture of (a) and (b). (d) A view of the pancreas showing the remnant of a large Langerhans islet in a mouse receiving untransformed plant leaf material. (e) Caspase-3 immunoreactivity in the same section taken in the red channel. (f) Merged picture of (d) and (e).

Figure 7

Immunostaining for cytokine production. (a) Interleukin-10 (IL-10) immunoreactivity in the pancreas of three mice administered untransformed plant leaf material (i–iii). Blood vessels (BV) and Langerhans islets (isl) are indicated. (iv–vi) Islets of mice receiving cholera toxin B subunit–human proinsulin (CTB-Pins). Small arrows indicate perivascular infiltration of IL-10-expressing lymphocytes. Large arrows indicate IL-10-positive lymphocytes inside or around the islets. (b) Interleukin-4 (IL-4) immunoreactivity in the pancreas of mice receiving interferon–green fluorescent protein (IFN-GFP), CTB-GFP or CTB-Pins plant leaf material. Small arrows indicate position of the islets.

Figure 8

Determination of immunoglobulin (Ig) and glucose levels. (a) Serum levels of IgA, IgG2a and IgG1 in non-obese diabetic (NOD) mice receiving cholera toxin B subunit–human proinsulin (CTB-Pins)-expressing plant leaf material. Control groups receiving untransformed plant material (Un-Tr) or interferon–green fluorescent protein (IFN-GFP)-expressing leaf material are also shown. (b) Blood and urine glucose levels in various groups of NOD mice after oral administration of CTB-Pins (6 weeks post-treatment). Un-Tr, n = 1; IFN-GFP, n = 1; CTB-GFP, n = 2; CTB-Pins, n = 2. Using Student's t-test, P value is less than 0.05. Bars, standard deviation. n indicates the number of animals in each treatment group.