Projects per year
The incidence of inflammatory disorders in industrialized countries has dramatically increased over the last decennia, which is believed to result from a change in life-style. Treatment of these inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. For now, many inflammatory disorders are treated with broad-acting immunosuppressive drugs or monoclonal antibodies that specifically target pro-inflammatory molecules of the immune system. An alternative therapeutic approach would be to use immunomodulatory proteins that are naturally involved in re-establishing immune homeostasis. This thesis describes the plant-based expression of a variety of immunomodulatory cytokines that may be used as biopharmaceutical proteins in the future. Furthermore, this thesis contains a pioneering chapter on the plant-based expression of immunomodulatory helminth-secreted glycoproteins.
In Chapter 2 we describe the plant-based expression of the immune-regulatory cytokine human transforming growth factor β1 (TGF-β1). By co-expressing human furin with latent TGF-β1 we were able to engineer the post-translational proteolytic processing of TGF-β1, which enabled the production of biologically active TGF-β1. In Chapter 3 we reveal that aggregation is a major production bottleneck for the anti-inflammatory cytokine interleukin-10 (IL-10). By protein engineering we were able to prevent aggregation and created a biologically active fusion protein of IL-10. In Chapter 4 we express biologically active IL-22 in plants. We reveal that, in contrast to current literature, its activity is independent of the presence of N-glycans or their composition. This chapter further reveals that plants offer a powerful tool to allow investigation into the role of N-glycans in protein folding and biological activity of glycoproteins. In Chapter 5 we further explore the potential of glyco-engineering in plants by engineering helminth-like N-glycans. We produce large quantities of two major egg antigens from Schistosoma mansoni and successfully engineer Lewis X, LDN and LDNF N-glycan structures. These plant biotechnological research lines are a showcase for the potential of engineering proteins as well as post-translational modifications in plants with special emphasis on N-glycan engineering. Altogether, the results presented in the first four chapters reveal the remarkable flexibility of plants as a production platform for recombinant proteins. It showcases the potential of engineering proteins as well as post-translational modifications in plants, but it especially highlights the engineering of tailor made N-glycans in plants. This, combined with the speed of transient expression by means of agroinfiltration, makes transient expression in Nicotiana benthamiana a powerful tool to study the role of N-glycans on glycoprotein function.
In parallel to these plant biotechnological research lines, we also developed an in vitro model system based on mouse bone marrow-derived cells to study immunological responses. We used this model to obtain clues on why IL-10 therapy has not been as successful as previously anticipated. In Chapter 6 we have set-up biological activity assays based on bone marrow-derived cells and reveal that IL-10 activity is dependent on both IL-10R1 and IL-10R2, but not IL-10R2-associated signalling via Tyk2. We also show that interactions between IL-10R1 and IL-10R2 (both intracellular and extracellular) reduce cellular binding of IL-10, but are crucial to initiate IL-10 mediated signalling. Furthermore, we observed that macrophages and dendritic cells respond differently to IL-10. This was further investigated in Chapter 7 where we reveal that GM-CSF (the cytokine used to differentiate dendritic cells) is responsible for negatively regulating early IL-10-mediated responses. Strikingly, GM-CSF does not strongly affect the IL-10-induced activation of the transcription factor STAT3. Instead, GM-CSF induces strong constitutive phosphorylation of GSK-3β, a signalling component downstream of the PI3K/Akt pathway. These immunological chapters give novel insights on the mechanism of initiating IL-10-induced signalling and on the possible integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.
|Qualification||Doctor of Philosophy|
|Award date||23 Oct 2015|
|Place of Publication||Wageningen|
|Publication status||Published - 2015|
- plant biotechnology
- pharmaceutical proteins
- interleukin 10
- antiinflammatory agents
- biological activity
- gene expression
- transforming growth factor
- recombinant proteins