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Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco.

Bortesi L, Rossato M, Schuster F, Raven N, Stadlmann J, Avesani L, Falorni A, Bazzoni F, Bock R, Schillberg S, Pezzotti M - BMC Biotechnol. (2009)

Bottom Line: The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively.Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines.The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scientific and Technologic Department, University of Verona, Strada Le Grazie 15, Verona, Italy. luisa.bortesi@univr.it

ABSTRACT

Background: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.

Results: Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.

Conclusion: Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.

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Activation of STAT3 phosphorylation by murine and viral IL-10 produced in tobacco plants. STAT3 phosphorylation (STAT3-YP) was assessed by immunoblot analysis on protein extracts of macrophage cells treated with increasing doses of plant-derived IL-10. (a) J774 cells were cultured for 15 min in the presence of eluate from wild type tobacco plants (lane 1; protein amount equivalent to the 150 ng/ml sample of the mIL-10-producing tobacco line), 20 ng/ml of commercial mIL-10 produced in insect cells (lane 2) or increasing concentrations of plant-derived mIL-10 (lanes 3–6); (b) J774 cells were cultured for 15 min in the presence of the eluate from purification of wild type tobacco plants (lane 1; protein amount equivalent to the 500 ng/ml sample of the viral IL-10-producing tobacco line), 150 ng/ml of commercial vIL-10 (lane 2) or increasing concentration of plant vIL-10 (lanes 3–6). Total cell extracts (50 μg) were separated by SDS-PAGE and immunoblots were performed as described in the Experimental Procedures section. One experiment representative of two is shown. The blots were scanned on the Odyssey Infrared Imaging System at 700 and 800 nm. The relative STAT3-YP levels, as quantified by the Odyssey software and normalized for the total STAT3, are reported below each panel.
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Figure 5: Activation of STAT3 phosphorylation by murine and viral IL-10 produced in tobacco plants. STAT3 phosphorylation (STAT3-YP) was assessed by immunoblot analysis on protein extracts of macrophage cells treated with increasing doses of plant-derived IL-10. (a) J774 cells were cultured for 15 min in the presence of eluate from wild type tobacco plants (lane 1; protein amount equivalent to the 150 ng/ml sample of the mIL-10-producing tobacco line), 20 ng/ml of commercial mIL-10 produced in insect cells (lane 2) or increasing concentrations of plant-derived mIL-10 (lanes 3–6); (b) J774 cells were cultured for 15 min in the presence of the eluate from purification of wild type tobacco plants (lane 1; protein amount equivalent to the 500 ng/ml sample of the viral IL-10-producing tobacco line), 150 ng/ml of commercial vIL-10 (lane 2) or increasing concentration of plant vIL-10 (lanes 3–6). Total cell extracts (50 μg) were separated by SDS-PAGE and immunoblots were performed as described in the Experimental Procedures section. One experiment representative of two is shown. The blots were scanned on the Odyssey Infrared Imaging System at 700 and 800 nm. The relative STAT3-YP levels, as quantified by the Odyssey software and normalized for the total STAT3, are reported below each panel.

Mentions: All the anti-inflammatory effects of IL-10 require the activation of signal transducer and activator of transcription 3 (STAT3), which acts downstream of the IL-10 receptor in both mouse and human cellular models [11]. We therefore tested the ability of plant-derived mIL-10 and vIL-10 to phosphorylate the Tyr705 residue of STAT3. To exclude possible interference from plant proteins co-purifying with the recombinant IL-10 molecules, equal amounts of wild type leaf material were subjected to the same purification procedure on a Ni-NTA column, and the eluate was used as a control in all biological activity assays. Stimulation of the mouse macrophage cell line J774 with different doses of plant-derived mIL-10 and vIL-10 triggered STAT3 tyrosine phosphorylation within 15 min and in a dose-dependent manner (Figure 5). The concentration of plant-derived IL-10 required to elicit STAT3 tyrosine phosphorylation to a degree comparable to that observed in response to commercial mIL-10 (20 ng/ml) and vIL-10 (150 ng/ml) was 50 ng/ml for the murine protein (Figure 5a) and 500 ng/ml for the viral protein (Figure 5b). STAT3 phosphorylation resulted specifically from the presence of recombinant IL-10, since control eluate from wild type plant extracts had no detectable effect even at the highest dose tested (Figure 5).


Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco.

Bortesi L, Rossato M, Schuster F, Raven N, Stadlmann J, Avesani L, Falorni A, Bazzoni F, Bock R, Schillberg S, Pezzotti M - BMC Biotechnol. (2009)

Activation of STAT3 phosphorylation by murine and viral IL-10 produced in tobacco plants. STAT3 phosphorylation (STAT3-YP) was assessed by immunoblot analysis on protein extracts of macrophage cells treated with increasing doses of plant-derived IL-10. (a) J774 cells were cultured for 15 min in the presence of eluate from wild type tobacco plants (lane 1; protein amount equivalent to the 150 ng/ml sample of the mIL-10-producing tobacco line), 20 ng/ml of commercial mIL-10 produced in insect cells (lane 2) or increasing concentrations of plant-derived mIL-10 (lanes 3–6); (b) J774 cells were cultured for 15 min in the presence of the eluate from purification of wild type tobacco plants (lane 1; protein amount equivalent to the 500 ng/ml sample of the viral IL-10-producing tobacco line), 150 ng/ml of commercial vIL-10 (lane 2) or increasing concentration of plant vIL-10 (lanes 3–6). Total cell extracts (50 μg) were separated by SDS-PAGE and immunoblots were performed as described in the Experimental Procedures section. One experiment representative of two is shown. The blots were scanned on the Odyssey Infrared Imaging System at 700 and 800 nm. The relative STAT3-YP levels, as quantified by the Odyssey software and normalized for the total STAT3, are reported below each panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Activation of STAT3 phosphorylation by murine and viral IL-10 produced in tobacco plants. STAT3 phosphorylation (STAT3-YP) was assessed by immunoblot analysis on protein extracts of macrophage cells treated with increasing doses of plant-derived IL-10. (a) J774 cells were cultured for 15 min in the presence of eluate from wild type tobacco plants (lane 1; protein amount equivalent to the 150 ng/ml sample of the mIL-10-producing tobacco line), 20 ng/ml of commercial mIL-10 produced in insect cells (lane 2) or increasing concentrations of plant-derived mIL-10 (lanes 3–6); (b) J774 cells were cultured for 15 min in the presence of the eluate from purification of wild type tobacco plants (lane 1; protein amount equivalent to the 500 ng/ml sample of the viral IL-10-producing tobacco line), 150 ng/ml of commercial vIL-10 (lane 2) or increasing concentration of plant vIL-10 (lanes 3–6). Total cell extracts (50 μg) were separated by SDS-PAGE and immunoblots were performed as described in the Experimental Procedures section. One experiment representative of two is shown. The blots were scanned on the Odyssey Infrared Imaging System at 700 and 800 nm. The relative STAT3-YP levels, as quantified by the Odyssey software and normalized for the total STAT3, are reported below each panel.
Mentions: All the anti-inflammatory effects of IL-10 require the activation of signal transducer and activator of transcription 3 (STAT3), which acts downstream of the IL-10 receptor in both mouse and human cellular models [11]. We therefore tested the ability of plant-derived mIL-10 and vIL-10 to phosphorylate the Tyr705 residue of STAT3. To exclude possible interference from plant proteins co-purifying with the recombinant IL-10 molecules, equal amounts of wild type leaf material were subjected to the same purification procedure on a Ni-NTA column, and the eluate was used as a control in all biological activity assays. Stimulation of the mouse macrophage cell line J774 with different doses of plant-derived mIL-10 and vIL-10 triggered STAT3 tyrosine phosphorylation within 15 min and in a dose-dependent manner (Figure 5). The concentration of plant-derived IL-10 required to elicit STAT3 tyrosine phosphorylation to a degree comparable to that observed in response to commercial mIL-10 (20 ng/ml) and vIL-10 (150 ng/ml) was 50 ng/ml for the murine protein (Figure 5a) and 500 ng/ml for the viral protein (Figure 5b). STAT3 phosphorylation resulted specifically from the presence of recombinant IL-10, since control eluate from wild type plant extracts had no detectable effect even at the highest dose tested (Figure 5).

Bottom Line: The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively.Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines.The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scientific and Technologic Department, University of Verona, Strada Le Grazie 15, Verona, Italy. luisa.bortesi@univr.it

ABSTRACT

Background: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.

Results: Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.

Conclusion: Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.

Show MeSH
Related in: MedlinePlus