- Chair Nutrition and Pharmacology (HNE) (1)
- EPS-2 (1)
- HNE Nutrition and Pharmacology (1)
- Laboratory of Nematology (1)
- Nutritional Biology and Health (1)
Plant Biotechnology meets Immunology : plant-based expression of immunologically relevant proteins
Wilbers, R.H.P. - \ 2015
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Arjen Schots; Geert Smant. - Wageningen : Wageningen University - ISBN 9789462574335 - 229
plantenbiotechnologie - immunologie - planten - eiwitten - farmaceutische eiwitten - interleukine 10 - ontstekingsremmers - biologische activiteit - cytokinen - genexpressie - transforming growth factor - wormen - recombinant eiwitten - glycoproteïnen - plant biotechnology - immunology - plants - proteins - pharmaceutical proteins - interleukin 10 - antiinflammatory agents - biological activity - cytokines - gene expression - helminths - recombinant proteins - glycoproteins
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.
n-3 Fatty acid derived endocannabinoids: a new link between fish oil and inflammation
Balvers, M.G.J. - \ 2012
Wageningen University. Promotor(en): Renger Witkamp, co-promotor(en): K.C.M. Verhoeckx. - S.l. : s.n. - ISBN 9789461731159 - 206
omega-3 vetzuren - meervoudig onverzadigde vetzuren - visoliën - cannabinoïden - ontsteking - anti-inflammatoire eigenschappen - ontstekingsremmers - omega-3 fatty acids - polyenoic fatty acids - fish oils - cannabinoids - inflammation - antiinflammatory properties - antiinflammatory agents
Inflammatory processes are critical components of many illnesses, and dietary n-3 fatty acids have been shown to contribute to a reduction of the inflammatory status, both in in vivo and in vitro studies. The mechanisms underlying the modulation of inflammation are not completely understood, but it is clear that dietary n-3 fatty acids alter the eicosanoid metabolome profile, resulting in increased levels of n-3 fatty acid eicosanoids, whereas eicosanoids from other fatty acids are decreased. Until now, the anti-inflammatory properties of n-3 fatty acids had not been linked to an interaction with endocannabinoids/N-acyl ethanolamides (NAEs) levels before. This thesis describes a series of studies on the link between dietary fatty acids, endocannabinoids/NAEs, and inflammation.
Previous research indicated that dietary fatty acids alter the profile of endocannabinoids/NAEs rather than just affecting single compounds such as arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) as suggested before, and therefore a method based on liquid chromatography coupled to mass spectrometry (LC-MS/MS) to quantify a broad range of endocannabinoids/NAEs was developed. This method was used to demonstrate that n-3 fatty acids are converted to their endocannabinoid derivates by adipocytes in vitro. These n-3 derived NAEs, docosahexaenoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA), were shown to have anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated adipocytes by reducing interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) excretion. Further studies showed that serum free fatty acid levels and plasma NAE levels are correlated under both fasting and post-prandial conditions in women, and demonstrated that plasma AEA and oleoyl ethanolamide (OEA) correlated with body mass index (BMI).
Considering the complexity of endocannabinoid and eicosanoid metabolism, it is likely that their concentrations are dynamic over time and tissue-specific during inflammation. So far, most studies had focused on limited numbers of endocannabinoids and eicosanoids in restricted numbers of tissues or plasma, and the effect of inflammation on DHEA and EPEA levels had not been studied before. Therefore, an animal study was conducted which investigated in detail the time-dependent effects of i.p. LPS on the levels of lipid derived mediators (endocannabinoids/NAEs and eicosanoids) in plasma, liver, ileum and adipose tissue in mice fed with a diet rich in fish oil. The results demonstrated that both DHEA and EPEA levels were increased after LPS treatment, but also time- and tissue dependent effects were observed. Based on these data, another study was performed which investigated the combined effect of different fish oil diets and inflammation on the profiles of endocannabinoids and eicosanoids using the same multi-compartment targeted lipidomics approach. The data indicated that that dietary n-3 fatty acids and inflammation alter both the endocannabinoid and eicosanoid metabolomes towards higher levels of n-3 derived metabolites at the expense of metabolites derived from other fatty acids. Multivariate data analysis revealed that under normal conditions the diet groups were primarily separated based on decreased levels of other than n-3 derived metabolites. However, during inflammation, the separation was primarily explained by increases in n-3 derived compounds. Finally, additional analyses demonstrated that plasma and erythrocytes contain significant levels of esterified NAEs. The esterified levels were approximately 20-60 fold higher than the free NAE levels, and their profiles resembles the free NAE profiles.
In conclusion, (dietary) n-3 fatty acids increased the levels of DHEA and EPEA, and these metabolites displayed anti-inflammatory properties. Although the n-3 fatty acids are likely to be converted to a variety of other metabolites, the work in this thesis suggests that ‘fish oil-derived’ endocannabinoids are a new link between fish oil and its anti-inflammatory properties. Further research is needed to relate nutrition-based modulation of endocannabinoid profiles to more specific effects on health and disease.