Plant Biotechnology meets Immunology
Plant-based expression of immunologically relevant proteins
Ruud H. P. Wilbers
Thesis committee
Promotor Prof. Dr J. Bakker Professor of Nematology Wageningen University
Co-promotors Dr A. Schots Associate professor, Laboratory of Nematology Wageningen University
Dr G. Smant Assistant professor, Laboratory of Nematology Wageningen University
Other members Prof. Dr H.F.J. Savelkoul, Wageningen University Prof. Dr H. Steinkellner, University of Natural Resources & Life Sciences, Vienna, Austria Prof. Dr M. Yazdanbakhsh, Leiden University Medical Center Prof. Dr H.J. Bosch, Utrecht University and Plant Research International, Wageningen
This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences
Plant Biotechnology meets Immunology
Plant-based expression of immunologically relevant proteins
Ruud H. P. Wilbers
Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 23 October 2015 at 1.30 p.m. in the Aula.
Ruud H. P. Wilbers Plant Biotechnology meets Immunology – Plant-based expression of immunologically relevant proteins, 230 pages
PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summaries in English and Dutch
ISBN 978-94-6257-433-5
Table of contents
Chapter 1 ! General Introduction 9
Chapter 2 ! Plant-based expression of active human transforming growth factor-β1 requires co-expression of furin 29
Chapter 3 ! 3D domain swapping causes extensive multimerisation of human interleukin-10 when expressed in planta 51 ! Chapter 4 ! The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity 77 ! Chapter 5 ! Engineering of plants for the expression of helminth glycoproteins with their native N-glycan structures 107 ! Chapter 6 ! IL-10R2 mediated conformational changes of IL-10R1 are required to initiate IL-10 signalling 137
Chapter 7 ! GM-CSF negatively regulates early IL-10 mediated responses 163 ! Chapter 8 ! General Discussion 185
Summary 215 Samenvatting 219 Acknowledgements 223 Curriculum vitae 227 Publications 228 Educational Statement of the Graduate School ‘Experimental Plant Sciences’ 229
General Introduction x ______
The human body is able to defend itself against viruses, pathogenic bacteria and fungi or multicellular parasites, such as helminthic worms. The complex set of cells that forms the immune system defends our body against pathogens and parasites. Immune cells travel throughout our body using the cardiovascular and lymphatic system. Our immune cells are specialised in the recognition of foreign (non-self) compounds and organisms, but are also capable of distinguishing different types of pathogens or parasites. Upon recognition, a specific arm of the immune system is triggered to efficiently eradicate the pathogen or parasite that is encountered. After the infection is cleared the immune system returns to its resting state. Yet, in order to function properly an intricate balance of the immune system is required as uncontrolled immune responses can result in tissue damage.
Different layers of defence The first line of defence against invading pathogens or parasites is a layer of epithelial cells in those organs that are in direct contact with the outside world, such as skin, lungs and intestine. The epithelial layer that encounters the widest variety of non-self compounds and organisms is the intestinal epithelium (Figure 1). Intestinal epithelial cells are highly specialised cells as they serve as gatekeepers of our intestinal tract. They form a physical barrier to prevent the entry of pathogens and parasites, while these cells simultaneously facilitate the uptake of nutrients and water from our food. The protective function of the intestinal epithelium is in the first place mediated by the tight assembly of epithelial cells, which does not allow the entry of microorganisms (microbes). Secondly, specialised intestinal epithelial cells, so-called goblet cells, secrete mucins into the intestinal lumen. These mucins form a mucus layer and are another physical barrier that prevents attachment of pathogens to the epithelial cells. Finally, normal intestinal epithelial cells as well as specialised Paneth cells secrete large amounts of anti-microbial peptides that reside in the mucus layer [1]. When invading pathogens or parasites breach this first line of defence, the immune system relies on the rapid detection of these foreign compounds and organisms. Cells of the innate immune system are responsible for the early detection of such organisms and do so by recognizing specific structures shared by classes of microbes (so-called “pathogen-associated molecular patterns” or PAMP’s). These structures include double stranded RNA from viruses, unmethylated DNA from bacteria or complex lipids and carbohydrates that are not found on host cells. The recognition of PAMP’s is a defence response that can be found in all classes of plant and animal life. The cell types that establish innate immunity in vertebrate animal species are phagocytes (such as neutrophils, macrophages and dendritic cells) and natural killer (NK) cells. NK cells do not recognize foreign microbes, but are specialised in recognizing virus-infected cells or tumour cells. On the other hand, dendritic cells, macrophages and various other immune cells are
11 Chapter 1 ______ equipped with pattern recognition receptors (PRR’s), which are proteins that are able to recognize specific PAMP’s. PRR’s can be found as membrane-bound receptors on the cell surface, but also inside the cell (either cytoplasmic or membrane-bound). PRR’s can be further distinguished in functionality as they function as signalling receptors or receptors that mediate uptake into the cell by a process called endocytosis (or both). Toll-like receptors (TLR’s) constitute one class of membrane-bound PRR’s that recognize different PAMP’s and are key to activate innate immune cells upon recognition of non-self compounds. To date, over 10 mammalian TLR’s have been identified and each receptor is required for the recognition of PAMP’s from different classes of pathogens [2]. For instance, TLR4 can bind lipopolysaccharide (LPS) from gram-negative bacteria, whereas TLR3 is able to detect viral RNA inside the cell. Upon recognition of PAMP’s by TLR’s an activation signal is transduced into the cell that triggers the production of cytokines. Cytokines are signalling molecules of the immune system. They can change the activation state of immune cells or attract other immune cells to the site of infection. One cytokine commonly produced upon infection is tumour necrosis factor (TNF- ) and is a key cytokine that drives inflammation. C-type lectin receptors (CLR’s) are another class of PRR’s and are specialised in the recognition of carbohydrate structures (self and non-self), such as fungal -glucans or complex carbohydrate structures found on helminth-secreted proteins [3-5]. Whereas TLR’s have a minor role as endocytosis receptors, the main role of CLR’s is to facilitate endocytosis of foreign compounds or microbes. C-type lectin receptors often collaborate with TLR’s and have been shown to modulate TLR-induced signalling [3, 6]. Nevertheless, endocytosis and/or phagocytosis (internalization of large particles or microbes) are key steps in shaping the immune response against extracellular pathogens, parasites or their secreted compounds. Internalization leads to the formation of vesicles (endosomes or phagosomes) that are transported into the cell where they fuse with lysosomes. Lysosomes are intracellular vesicles that are enriched with compounds that enable the killing of microbes and degrade foreign compounds. All combined, innate immune responses function as a broad-acting mechanism by which short-term immunity can be provided against microbes. The innate immune system is often sufficient to eliminate intruders, but their responses are limited by the diversity of molecular patterns that can be recognized. Also, innate immune cells respond the same way upon repeated exposure to microbes and are not able acquire memory. The adaptive immune system is a third layer of defence, which is only found in vertebrate species. The adaptive immune system is able to recognise a diverse array of foreign compounds in a highly specific manner, however it requires time to respond. Furthermore, the adaptive immune system also has the ability to acquire memory and
12 General Introduction x ______
Viruses and other Extracellular Commensal bacteria Extracellular parasites intracellular pathogens bacteria and fungi and dietary factors and allergens
anti-microbial dendritic cell peptides intestinal sIgA epithelium IL-23
IL-12 IL-6 TGF- IL-4 TGF- mast cell