Expression of murine and viral interleukin-10 in tobacco for immunomodulation of galt for the prevention of autoimmune diseases

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. The ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10 was investigated. To reach high accumulation levels of the transgenes, plastid transformation of the IL-10 genes as well as different targeting strategies of the nuclear encoded recombinant proteins were investigated. Chloroplast transformation turned out not to be a feasible approach for the recombinant production of IL-10, as unsatisfactory accumulation levels were obtained upon expression of both transgenes. For tobacco nuclear transformation, three different subcellular targeting strategies, directing the recombinant protein into the endoplasmic reticulum (ER), cytosol and apoplast, were first assessed in transient expression experiments, and stable transgenic plants were then generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the ER. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization, stability 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. It was therefore demonstrated that tobacco plants are able to correctly process viral and murine IL- 10 into biologically active dimers, 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 evaluation of the immunomodulatory properties and effectiveness of the viral and murine IL-10 in inducing oral tolerance.