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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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Effect of plant-derived murine IL-10 on LPS-induced SOCS3 mRNA and SOCS3 protein expression. (a) J774 cells were stimulated for 2 h with 100 ng/ml of LPS alone or in combination with commercial mIL-10 (20 ng/ml), or plant mIL-10 (50 and 100 ng/ml). Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Total RNA was extracted and then analyzed for SOCS3 mRNA expression by real time RT-PCR. The graph shows the SOCS3 mRNA levels (mean ± SD) assayed in triplicate and normalized to GAPDH expression. (b) J774 cells were incubated for 18 h in the presence of LPS alone (lane 2) or in combination with 20 ng/ml commercial IL-10 (lane 3) or 50 ng/ml (lane 4) or 100 ng/ml (lane 5) plant derived mIL-10. Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Whole cell extracts (50 μg) were immunoblotted using anti-NH2 terminus SOCS3 antibody (upper panel) and antibodies specific for GAPDH (lower panel), followed by incubation with AlexaFluor 680 goat anti-rabbit and IRDye 800 goat anti-mouse antibody. The relative levels of SOCS3 protein, as quantified by the Odyssey software and normalized for the total GAPDH content, are reported at the bottom of each lane. The data shown in (a) and (b) are representative of two independent experiments. ** p < 0.001.
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Figure 6: Effect of plant-derived murine IL-10 on LPS-induced SOCS3 mRNA and SOCS3 protein expression. (a) J774 cells were stimulated for 2 h with 100 ng/ml of LPS alone or in combination with commercial mIL-10 (20 ng/ml), or plant mIL-10 (50 and 100 ng/ml). Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Total RNA was extracted and then analyzed for SOCS3 mRNA expression by real time RT-PCR. The graph shows the SOCS3 mRNA levels (mean ± SD) assayed in triplicate and normalized to GAPDH expression. (b) J774 cells were incubated for 18 h in the presence of LPS alone (lane 2) or in combination with 20 ng/ml commercial IL-10 (lane 3) or 50 ng/ml (lane 4) or 100 ng/ml (lane 5) plant derived mIL-10. Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Whole cell extracts (50 μg) were immunoblotted using anti-NH2 terminus SOCS3 antibody (upper panel) and antibodies specific for GAPDH (lower panel), followed by incubation with AlexaFluor 680 goat anti-rabbit and IRDye 800 goat anti-mouse antibody. The relative levels of SOCS3 protein, as quantified by the Odyssey software and normalized for the total GAPDH content, are reported at the bottom of each lane. The data shown in (a) and (b) are representative of two independent experiments. ** p < 0.001.

Mentions: It has been reported that suppressor of cytokine signaling 3 (SOCS3) is one of the targets of murine IL-10 [24,25]. Indeed, SOCS3 mRNA and SOCS3 protein expression in lipopolysaccharide (LPS)-stimulated J774 cells is enhanced in the presence of commercial mIL-10 [26]. Similarly, purified plant-derived mIL-10 increased the expression of LPS-induced SOCS3 mRNA (Figure 6a) and SOCS3 protein (Figure 6b). Finally, we verified that the plant-derived mIL-10 and vIL-10 were fully functional by testing their inhibitory activity on the LPS-induced production of tumor necrosis factor alpha (TNFα). The amount of TNFα released into the culture supernatant of J774 cells stimulated with LPS for 18 h was significantly reduced in the presence of plant-derived mIL-10 to the same extent observed with the commercial mIL-10 (Figure 7a). Similarly, the ability of plant derived vIL-10 to inhibit LPS-induced TNFα production was equivalent to that of its commercial counterpart (Figure 7b). Addition of control eluate from wild type plants had no effect on the J774 response to LPS, or the suppressive activity of IL-10 (Figure 7). These results confirmed that the inhibition of TNFα production was dependent on the recombinant cytokines, and not on any co-purified plant proteins.


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)

Effect of plant-derived murine IL-10 on LPS-induced SOCS3 mRNA and SOCS3 protein expression. (a) J774 cells were stimulated for 2 h with 100 ng/ml of LPS alone or in combination with commercial mIL-10 (20 ng/ml), or plant mIL-10 (50 and 100 ng/ml). Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Total RNA was extracted and then analyzed for SOCS3 mRNA expression by real time RT-PCR. The graph shows the SOCS3 mRNA levels (mean ± SD) assayed in triplicate and normalized to GAPDH expression. (b) J774 cells were incubated for 18 h in the presence of LPS alone (lane 2) or in combination with 20 ng/ml commercial IL-10 (lane 3) or 50 ng/ml (lane 4) or 100 ng/ml (lane 5) plant derived mIL-10. Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Whole cell extracts (50 μg) were immunoblotted using anti-NH2 terminus SOCS3 antibody (upper panel) and antibodies specific for GAPDH (lower panel), followed by incubation with AlexaFluor 680 goat anti-rabbit and IRDye 800 goat anti-mouse antibody. The relative levels of SOCS3 protein, as quantified by the Odyssey software and normalized for the total GAPDH content, are reported at the bottom of each lane. The data shown in (a) and (b) are representative of two independent experiments. ** p < 0.001.
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Related In: Results  -  Collection

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Figure 6: Effect of plant-derived murine IL-10 on LPS-induced SOCS3 mRNA and SOCS3 protein expression. (a) J774 cells were stimulated for 2 h with 100 ng/ml of LPS alone or in combination with commercial mIL-10 (20 ng/ml), or plant mIL-10 (50 and 100 ng/ml). Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Total RNA was extracted and then analyzed for SOCS3 mRNA expression by real time RT-PCR. The graph shows the SOCS3 mRNA levels (mean ± SD) assayed in triplicate and normalized to GAPDH expression. (b) J774 cells were incubated for 18 h in the presence of LPS alone (lane 2) or in combination with 20 ng/ml commercial IL-10 (lane 3) or 50 ng/ml (lane 4) or 100 ng/ml (lane 5) plant derived mIL-10. Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Whole cell extracts (50 μg) were immunoblotted using anti-NH2 terminus SOCS3 antibody (upper panel) and antibodies specific for GAPDH (lower panel), followed by incubation with AlexaFluor 680 goat anti-rabbit and IRDye 800 goat anti-mouse antibody. The relative levels of SOCS3 protein, as quantified by the Odyssey software and normalized for the total GAPDH content, are reported at the bottom of each lane. The data shown in (a) and (b) are representative of two independent experiments. ** p < 0.001.
Mentions: It has been reported that suppressor of cytokine signaling 3 (SOCS3) is one of the targets of murine IL-10 [24,25]. Indeed, SOCS3 mRNA and SOCS3 protein expression in lipopolysaccharide (LPS)-stimulated J774 cells is enhanced in the presence of commercial mIL-10 [26]. Similarly, purified plant-derived mIL-10 increased the expression of LPS-induced SOCS3 mRNA (Figure 6a) and SOCS3 protein (Figure 6b). Finally, we verified that the plant-derived mIL-10 and vIL-10 were fully functional by testing their inhibitory activity on the LPS-induced production of tumor necrosis factor alpha (TNFα). The amount of TNFα released into the culture supernatant of J774 cells stimulated with LPS for 18 h was significantly reduced in the presence of plant-derived mIL-10 to the same extent observed with the commercial mIL-10 (Figure 7a). Similarly, the ability of plant derived vIL-10 to inhibit LPS-induced TNFα production was equivalent to that of its commercial counterpart (Figure 7b). Addition of control eluate from wild type plants had no effect on the J774 response to LPS, or the suppressive activity of IL-10 (Figure 7). These results confirmed that the inhibition of TNFα production was dependent on the recombinant cytokines, and not on any co-purified plant proteins.

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