Nijmegen Centre for Molecular Life Sciences Annual Report 2006 NCMLS
Postal address 259 NCMLS P.O. Box 9101 6500 HB Nijmegen The Netherlands Visiting address Geert Grooteplein 28 6525 GA Nijmegen T: +31 (0)24 361 07 07 F: +31 (0)24 361 09 09 E: info@ncmls.ru.nl I: www.ncmls.nl Editing: Dr. Margaret Mullally, Judith Burgers Design: Final Design Photography: Theo Hafmans Printed by: Thieme MediaCenter, Nijmegen 2 NCMLS
With respect to the quality of scientific research, the year 2006 has been an extremely successful one for the Nijmegen Centre for Molecular Life Sciences (NCMLS). It is therefore with great pleasure that I Foreword With respect to the quality of scientific research, the year 2006 has been an extremely successful one for the Nijmegen Centre for Molecular Life Sciences (NCMLS). It is therefore with great pleasure that I present our annual report 2006, providing an overview of the NCMLS achievements in both science and education. Two new professors have been appointed to the chairs of Biomolecular Chemistry and Biochemistry of Integrated Systems respectively. Within our scientific NCMLS community a number of principal investigators were awarded for their excellence. Besides a number of TOP grants and a Vici award from NWO, the prestigious EURYI prize was awarded from the European Science Foundation. Furthermore, two individual career awards, the Beatrix Foundation Jubilee Award for research on mitochondrial disease and the Spinoza award for excellent scientific contributions in the field Immunology and Cell Biology were bestowed upon two of our most prominent researchers. Following the recommendation of the KNAW committee during the renewal of the recognition of the NCMLS as a graduate school we have restructured the theme, Cell growth and differentiation into two sub-themes, Genetic and epigenetic pathways of disease and Chemical and physical biology. We thus expect to achieve a more focused approach, providing more critical mass within these research sub-themes. An important issue for the upcoming period is to further the international profile of the NCMLS in Europe. Since the start of our NVAO accredited honours MSc research programme, Molecular Mechanisms of Disease (MMD), last year, we have attracted 12 excellent students in the field of molecular life sciences and molecular medicine. Besides the MMD, a renewed PhD programme gives impetus to the NCMLS as a recognised graduate school. This year the Board of directors of the UMC confirmed their commitment to research by approving plans to build a second research building. Given the expansion of NCMLS, this is a fantastic opportunity to reallocate groups, fortifying strategic research themes, building even stronger interactions with local researchers. The new building will also facilitate a life sciences incubator and a novel Science meets Business concept will be developed. NCMLS aims at having strong ties with industrial partners and as such participates in national programmes including Top Instituut Pharma, the BioMedical Materials Program and the Center for Translational and Molecular Medicine. Our 2006 annual report highlights some of the younger researchers within the various research themes of our centre and their contribution to the understanding of the molecular and cellular basis of disease. Wishing you an enjoyable read, Carl Figdor Scientific Director NCMLS Annual Report 2006 Carl Figdor Foreword 3
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Table of contents Page Foreword 3 Nijmegen Centre for Molecular Life Sciences 7 Research 8 Research Themes 8 Societal Impact 10 Awards 10 Technology Platforms 11 The NCMLS as Graduate School 12 MSc Molecular Mechanisms of Disease 12 International PhD programme 13 Members of NCMLS 14 Selected Research Highlights NCMLS 2006 15 Theme 1: Infection, immunity and tissue repair 15 Theme 1a Infection and autoimmunity 16 Theme 1b Immune regulation 20 Theme 1c Tissue engineering and pathology 28 Theme 2: Metabolism, transport and motion 33 Theme 2a Energy and redox metabolism 34 Theme 2b Membrane transport and intracellular motility 38 Theme 3: Cell growth and differentiation 45 Theme 3a Genetic and epigenetic pathways of disease 46 Theme 3b Chemical and physical biology 52 Scientific publications 2006 57 Annual Report 2006 Table of contents 5
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The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals Nijmegen in the Centre interests forof Molecular curiositylife driven Sciences research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. Governing Body Prof. Dr. Carel van Os (Director of Research, Radboud University Nijmegen Medical Centre); Prof. Dr. Gerard Zielhuis (from July 2006) Prof. Dr. Dirk Ruiter (Dean of Radboud University Nijmegen Medical Centre) Prof. Dr. Sjoerd Wendelaar-Bonga (Dean of Faculty of Science, Radboud University Nijmegen); Prof. Dr. Jan Kuijpers (from October 2006) Management Team Prof. Dr. Carl Figdor (Scientific Director, NCMLS) Prof. Dr. Jan van Hest (Faculty of Science) Dr. Margaret Mullally (Assistant Scientific Director, NCMLS) Prof. Dr. Jan Smeitink (Radboud University Nijmegen Medical Centre) Research Council Prof. Dr. Gosse Adema (Theme leader) Prof. Dr. Rene Bindels (Sub-theme leader) Prof. Dr. Han Brunner (Chair of Science committee, UMCN) Prof. Dr. Carl Figdor (Scientific Director, NCMLS) Prof. Dr. Jan van Hest (Member of Management team) Prof. Dr. Martijn Huynen (CMBI representative, member of MMD committee) Prof. Dr. Nine Knoers (representative of PhD committee and top centre Genetic and Metabolic disease) Prof. Dr. Han van Krieken (representative top centre Oncology) Prof. Dr. Jos van der Meer (representative of top centre Infectious disease) Prof. Dr. Joost Schalkwijk (Sub-theme leader; advisor to post-doc platform) Prof. Dr. Jan Smeitink (Member of Management team) Prof. Dr. Henk Stunnenberg (Theme leader) Prof. Dr. Bé Wieringa (Theme leader) Dr. Margaret Mullally (Assistant Scientific Director, NCMLS, member of Management team) Scientific Advisory Committee Prof. Dr. Hans Clevers (chairperson), Hubrecht Laboratory, Utrecht, The Netherlands Prof. Dr. Tony Mikos, Rice University, Houston, US. Prof. Dr. Paola Ricciardi-Castagnoli, University of Milano-Bicocca, Italy Prof. Dr. Rosario Rizzuto, University of Ferrara, Italy. Prof. Dr. Luis. Serrano, EMBL Heidelberg, Germany Prof. Dr. Gert-Jan van Ommen, Leiden University Medical Centre (LUMC), The Netherlands Prof. Dr. John Walker, MRC Dunn, Cambridge, UK. The NCMLS was first recognised as a graduate school in 1995 and again in 2000 by the Royal Netherlands Academy of Arts and Sciences (KNAW). A request for re-accreditation was under review at the KNAW in 2005. Mission: Understanding the cellular basis of disease. The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals in the interests of curiosity driven research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. The NCMLS brings together researchers in several groups at the Radboud University Nijmegen Medical Centre and the Faculty of Science. There is a particular emphasis on the relationship between fundamental and translational research. The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals in the interests of curiosity driven research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. Annual Report 2006 Nijmegen Centre for Molecular Life Sciences 7
Within the NCMLS, all research and education is linked to the study of molecular life sciences in relation Research to disease. The centre focuses on three thematic research areas, which are further divided into sub-themes. Research Themes Within the NCMLS, all research and education is linked to the study of molecular life sciences in relation to disease. The centre focuses on three thematic research areas, which are further divided into sub-themes. Theme 1: Infection, immunity and tissue repair Infection and autoimmunity Immune regulation Tissue engineering and pathology The immune system has the dual task of eliminating pathogens and eradicating incipient tumours, while preventing auto-reactive responses harmful to the host. In maintaining this balance, there is a complex interplay between immune and tissue cells and many stimulatory and inhibitory circuits operate simultaneously. Outcomes are further shaped by genetic and environmental factors. Deregulation of this intricate balance is associated with human diseases, ranging from inflammatory and autoimmune disorders to cancer, infection and transplantation disorders. In each case, prolonged deregulation can initiate a cascade of molecular events ultimately leading to tissue damage and destruction. Tissue engineering is a relatively new field of research aimed at repairing or replacing damaged tissues by implanting smart synthetic biomatrices or stem cells. Immune control is intrinsically involved both in tissue acceptance and in preventing autoimmune attacks on engineered tissues. A multi-disciplinary approach (molecule-mouse-patient) is taken to define the molecular basis of immune regulatory circuits, events that trigger or fuel immune-related disorders and infectious diseases, and tissue pathology & regeneration as well as stem cell behaviour & differentiation. Theme 2: Metabolism, transport and motion Energy and redox metabolism Membrane transport and intracellular motility The study of disease at the molecular level but in the context of the macromolecular world of cellular organelles, the intact cell, or organs and tissues in the entire organism is central to the NCMLS. For example, intrinsic genetic problems or extrinsic factors causing cellular energy deprivation, ion and metabolite and water transport failure, toxic accumulation of intermediates, or ischemia and anoxia caused by cerebro-vascular obstruction are related to a range of diseases, including cancer, neuropathy and myopathy, degenerative disorders like Alzheimer s and Parkinson or ischemic/anoxic organ failure. In addition, conditions such as obesity and type II diabetes or some aspects of ageing, have a direct connection to metabolism and molecular transport and motion. Energy and redox metabolism is also often linked to membrane transport and intracellular motility. Metabolites such as ATP, produced in key pathways like glycolysis and mitochondrial respiratory complexes are consumed as fuel or are needed as, for example, as co-factors for, drug-transporters or the acto-myosin motor and sliding machinery involved in cell movements. Defects in metabolic signalling are often involved in renal disease, cardiomyopathy or brain and muscle disorders due to defects in the production or assembly of ATPases, water channels, or the mitochondrial machinery. Theme 3: Cell growth and differentiation Genetic and epigenetic pathways of disease Chemical and physical biology The fate of all cells lies in the fine balance between growth and differentiation. If this balance is disturbed, uncontrolled growth and deregulated cellular development can lead to disease. Studying the molecular processes that underlie growth and differentiation is pivotal to a basic understanding of the causes of many diseases and malfunctions. Multilevel analysis is used to study the functional blueprint of all cellular decisions. Our research activities aim to: Unravel the molecular basis of cell behaviour, which emanates from the genetic and epigenetic code contained in the nucleus in the context of health and disease (e.g. cancer, developmental disorders, mental handicap, cognitive impairments, neurodegenerative disorders and age-related bone diseases). Elucidate protein structure and protein-protein interactions within cellular signaling pathways that control cell proliferation and differentiation. Exploit the potential of molecular chemistry to modify, design and mimic proteins and their building blocks with the purpose to modulate and analyze their activities and properties in the cellular environment. 8 Research NCMLS
NCMLS researchers continue to collaborate at the local, national and international level. The research school is allied with the Institute for Molecules and Materials (IMM), providing a solid platform for integrating the neu- Renown researchers within this theme participate in interdisciplinary research. They are engaged in basic research as well as technology development for subsequent diagnostic and therapeutic approaches and translational research. Examples include microarray-based genomic profiling (ArrayCGH, SNP arrays), expression profiling arrays, wholegenome ChIP-on-chip technology to study epigenetic profiles and target sites of protein (complexes) such as ER and p53 bound to their chromosomal sites of action, proteomics platforms (high accuracy and high throughput mass spectrometry). The integration with high-profile bioinformatics groups is an invaluable asset to all of these activities. The applications of these experimental approaches are manifold and research across the NCMLS benefit from these state-of-the-art technological advancements. Collaboration NCMLS researchers continue to collaborate at the local, national and international level. The research school is allied with the Institute for Molecules and Materials (IMM), providing a solid platform for integrating the neurosciences and/or nanoscience with molecular life sciences. Furthermore, the incorporation of the Centre for Molecular and Biomolecular Informatics (CMBI) within the NCMLS furthers the multidisciplinary approach to solving research problems, forming links with the Netherlands Bioinformatics Centre (NBIC). The NCMLS also has associations with the Dutch Programme for Tissue Engineering (DPTE) and the Netherlands Proteomics Centre. The NCMLS contributes to the Top Instituut Pharma and has several both academic and industrial partners in this context (see below). In addition, the NCMLS contributes to the Center for Translational and Molecular Medicine (www.ctmm.nl). A CTMM taskforce has been set up in Nijmegen in which the NCMLS takes a leading role. Furthermore, the NCMLS actively contributes to The European Molecular Imaging Platform, integrated in its associated disciplines and covering types of imaging From Molecule to Man. This platform shows great potential for early detection of disease and monitoring of treatment of disease states. Besides the NCMLS, the platform represents research groups from a number of other institutes and research groups, i.e., Fc Donders, Molecules and Materials (IMM), and the Radboud University Nijmegen Medical Centre (UMC St Radboud). A platform website will soon be launched (www.molecule2man.eu). International collaboration is evident in several collaborative projects and publications. Also, an increasing number of foreign PhD students and post-docs are now working in the NCMLS. In addition, the NCMLS graduate school has established formal contacts with four international institutes in the context of the MSc programme: Molecular Mechanisms of Disease. Annual Report 2006 Collaboration 9
Various members of the NCMLS obtain funding through national and international patient-oriented non-profit Societalorganizations impact such as Awards the Kidney Foundation, Dutch Cancer Society, the Diabetic Foundation, or the Rheumatoid Societal impact Various members of the NCMLS are funded by national and international patient-oriented non-profit organizations, such as the Kidney Foundation, Dutch Cancer Society, the Diabetic Foundation, and the Rheumatoid Arthritis Foundation. In addition, NCMLS members have advisory functions or are board members within these organizations or at the Royal Netherlands Academy of Arts and Sciences (KNAW). Moreover, clinical groups (Berden, Netea, Punt, de Witte, Knoers, Kullberg, Smeitink), which are in daily interaction with patients or their relatives, inform patient organizations and are involved in public and strategic policy. Several examples of translational research are being developed within the NCMLS and between the NCMLS and its collaborators. Awards Prof. Dr. Carl Figdor (Dept. of Tumor Immunology) was awarded the prestigious Spinoza prize by NWO for his excellent scientific contributions in the field Immunology and Cell Biology. Dr. Joost Hoenderop (Dept. of Physiology) received the Euryi Award from the European Science Foundation for his research project Regulation of the epithelial calcium channel by extracellular calciotropic factors. The Veni, Vidi and Vici grants are awarded by the NWO as part of the Vernieuwingsimpuls (Renewal impulse) and is supported by the Ministry of Science, Education and Culture (OCW), and the Royal Academy of Sciences and the Arts (KNAW). A number of these grants were awarded to members of the NCMLS and will be the basis of important future research. VICI award Dr. Peter Deen (Dept. of Tumor Immunology). Molecular regulation of the renal water homeostasis. VIDI awards Dr. Jolanda de Vries (Dept. of Tumor Immunology). Visualization of migrating therapeutic cells. VENI awards Dr. Marieke Coenen (Dept. of Human Genetics). Why does my medicine not work? Appointments Prof. Dr. Ger Pruijn has been appointed to the chair of Biomolecular Chemistry Prof. Dr. Roland Brock has been appointed to the chair of Biochemistry of Integrated Systems Prof. Dr. Jan Smeitink (Dept. of Pediatrics) has been given the Princess Beatrix Foundation Jubilee Award for research on mitochondrial disease. Dr. Mihai Netea (Dept. of Internal Medicine) was appointed a member of The Young Academy by the KNAW. 10 Societal Impact / Awards NCMLS
A GMP facility with clean rooms is used for translational research e.g. immunotherapeutic cell therapy, Technology stem cell transplantation Platforms and gene therapy. In 2004 a total of 65 patients were treated in NCMLS translational research studies Technology Platforms The NCMLS groups its research facilities under five well-defined platforms. Animal models Animal models have proven to be of great importance to molecular life scientists. The available disease-related models include those for arthritis, cancer, kidney disease, tissue engineering, heart transplantation, neural disorders, metabolic disorders, osteoporosis, haematopoiesis, fungal and bacterial septicaemia and malaria (P. falciparum). A behavioural testing battery (comprising mice & rats) is also available to investigate the functional consequences of gene environment interactions and for developing transgenic/knock-out models. Molecular imaging Imaging at the molecular level is an essential tool for life scientists. Electron microscopy and other high-resolution instruments such as Atomic Force Microscopy are available within a state-of-the-art facility. Furthermore, confocal laser scanning microscopy, flow cytometry and other fluorescent microscopic techniques are combined to perform dynamic measurements of fluorescent GFP-based tagged proteins (such as FRET & FRAP) and intracellular metabolites. Magnetic resonance facilities for in vivo NMR and MRI of animals and humans (7 Tesla) are also available. Translational research and cellular therapy Genomics and proteomics DNA sequencing as well as micro-array technology for gene expression profiling are now basic tools used by molecular life scientists. In 2004, novel microfluidic-based quantitative PCR became available to perform high throughput quantitative RT-PCR. A state-of-the-art proteomics facility has also been launched with, for example, 2D-electrophoresis, SELDI- TOF and Mass spectrometry (MALDI-TOF, FT-MS,nLC-MS/MS). Bioinformatics The Centre for Molecular and Biomolecular Informatics (CMBI) is the Dutch national centre for computational molecular sciences and is affiliated with the Faculty of Science. This centre is fully integrated within the NCMLS. The CMBI pursues a research programme with topics ranging from computational small-molecule chemistry to bioinformatics. The centre facilities, including a helpdesk databases and software packages are available to external scientists. The CMBI is also involved in organising courses and tutorials to support these scientists, in addition to maintaining www-based servers to give scientists rapid access to commonly used bioinformatics and small-molecule databases and information systems. A GMP facility with clean rooms is used for translational research e.g. immunotherapeutic cell therapy, stem cell transplantation and gene therapy. In 2004 a total of 65 patients were treated in NCMLS translational research studies. Annual Report 2006 Technology Platforms 11
The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where students The NCMLS follow as Graduate a tailor-made School research programmes shoulder to shoulder with professionals. The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where students follow a tailor-made research programmes shoulder to shoulder with professionals. The regulation of cellular processes is crucial for human development, and maintenance of health throughout life. It is clear that cellular malfunction affects common multi-factorial diseases such as diabetes, immune and inflammatory disorders, renal disease, cardiovascular, metabolic and neurodegenerative diseases as well as obesity and certain forms of cancer. In the fight against such diseases, the Nijmegen Centre for Molecular Life Sciences (NCMLS) Graduate School which is part of Radboud University Nijmegen and Radboud University Nijmegen Medical Centre plays a key role. A major goal of the NCMLS is to translate basic knowledge generated from biomedical research into clinical application, in order to improve diagnostics and develop new treatments. All MSc and PhD students are registered junior members of the research school and have the corresponding responsibilities and privileges. Tailor-made tuition Students are guided throughout the practical training period by a supervisor and throughout the entire programme by a mentor, who stimulates them to explore their abilities and develop general research competencies, including reflection. Together with these coaches, students draw up a personal training and supervision plan. Such a broad and interdisciplinary approach to research is particularly important in the international scientific arena. Excellent career prospects There is considerable demand for specialists in fundamental molecular biology and cell biology as well as in its application to the treatment of diseases such as cancer, autoimmune and inflammatory disorders, and metabolic and neurodegenerative disorders. Our MSc enables students to move rapidly into an international PhD programme, giving them a more mature perspective and a broader range of experimental approaches than is possible within standard MSc programmes. They are also prepared for further training as a PhD-level researcher. Graduates are exempted from certain elements of the NCMLS PhD programme. For example, the graduate course is taught during the MSc phase and the main practical project can be incorporated into a PhD project, thus shortening the PhD period. Education is focussed is on the domain of molecular life sciences related to disease and in particular in three main fields related to molecular medicine, cell biology and translational research. Programmes are aligned along the three research themes. The dedicated research programmes within the Graduate school are briefly highlighted as follows: MSc Molecular Mechanisms of Disease The NCMLS offers an exclusive Masters programme in Molecular Mechanisms of Disease, which is taught by top researchers and clinicians. This programme is accredited by the NVAO, the Dutch-Flemish accreditation organisation and a member of ECA, a European consortium for accreditation. A unique and challenging programme This ground-breaking programme translates disease-related basic research in cellular and molecular biology into clinical experimental research in patients. Designed to meet the needs of talented students with the drive, motivation and ambition to push forward their scientific careers, it represents a unique opportunity to develop a research project and build up an international research network. This extremely competitive programme provides a sound balance of theory and practice. We enrol just 30 students per year, each of whom is allocated a personal mentor and research supervisor. This selective approach guarantees excellence, especially during the research training period. According to one of our international partners the prestigious Mayo Clinic in the USA, the programme offers a focused combination of didactic and practical components suited to optimally synthesize current discoveries, conceptual breakthroughs and technological advancement mandatory for the effective education of the new vanguard of multidisciplinary investigators. The MSc programme The programme, which starts each September, is modular in structure, lasts 24 months and is worth 120 European credits. Its main goal is to prepare talented, motivated students for independent research projects, which provide the basis for continuing to take a PhD degree. There is a strong international component and an emphasis on a multidisciplinary approach to answering research questions related to molecular mechanisms of disease. The general modules are taught mainly in the first year, while the focus in the final year is on individual project work leading to a MSc thesis. Translational research and technology platforms Students are exposed to Translational research, which links basic science and the treatment of disease. This is crucial when studying the molecular mechanisms of the following diseases: autoimmune and inflammatory disorders (such as rheumatoid arthritis), renal disease, neurodegenerative diseases, metabolic disorders, microbial infection 12 The NCMLS as Graduate School NCMLS
The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where students follow a tailor-made research programmes shoulder to shoulder with professionals. (including malarial infections), viral infections and cancer. Students also become familiar with technology platforms which link basic science, technology and disease such as genomics, proteomics, bioinformatics, cellular therapy, tissue engineering and molecular imaging. Master classes and international experience One of the highlights of the programme is a series of Master classes, which are given by leading authorities from renowned international institutes. These offer valuable insights both within and beyond the chosen specialist topics, explaining the current state of the art in important scientific areas. Students participate in exchanges with a number of collaborating institutes and formal NCMLS partners, including the University of Southern Denmark, the University of Münster, the Rudolf Virchow Center for Experimental Biomedicine at Würzburg (both in Germany), the University of Milano-Bicocco in Italy and the Mayo Clinic in the USA. PhD programme Awards 2006 Winner of best PhD thesis: Dr. Tom Nijenhuis (Dept. of Physiology), thesis entitled Epithelial Ca 2+ and Mg 2+ channels in health and disease. Winner of best student report: Anneke Navis (Dept. of Human Genetics), report entitled Characterisation of RPGRIP1 interacting proteins. International PhD programme The aim of the NCMLS PhD training programme is to provide PhD students with a multi-faceted education in the field of Molecular Life Sciences. NCMLS PhD students therefore follow an interdisciplinary training programme, containing both compulsory and elective components. The elective components allow a section of the NCMLS training programme to be tailor-made to complement the specific specialisation of the individual student. The training programme encourages both practical and theoretical participation in several NCMLS activities. Entrance requirements The training programme is accessible to PhD students from one of the research groups that are related to the graduate school. Students with a NCMLS M.Sc. degree, with a higher professional degree or with a similar degree from a foreign institute can enter the programme. Medicine, (Medical) Biology, Molecular Mechanisms of Disease, Chemistry, Physics, Mathematics, Computer Science and (Bio) Engineering are suitable preparatory fields of study. Because of the heterogeneity of the knowledge and interest of the students, a diverse education programme, in which many subjects will be taught, is provided. The NCMLS certificate Along side practical training, PhD students are given the opportunity to broaden their skills through participation in specialised knowledge transfer courses. A certain number of courses are thought to be necessary in the development and education of each PhD student as independent researchers. Students that complete a full programme of NCMLS courses will be awarded with the NCMLS certificate. Annual Report 2006 The NCMLS as Graduate School 13
Members of NCMLS I: Infection, Immunity and II: Metabolism, transport III: Cell growth and Tissue Repair and motion differentiation (a) Infection and autoimmunity "(a) Energy and redox (a) Genetics and epigenetic metabolism" pathways of disease Schalkwijk* Wieringa#* Stunnenberg#* Berden Blom Brunner Galama Heerschap Cremers Kullberg Huijnen De Jong Lubberts Nijtmans Franke Melchers Smeitink Geurts van Kessel Netea Smits Logie Pruijn Van den Heuvel Lohrum Sauerwein Wevers Martens Van de Berg Willems Roepman Van de Loo Schalken Van der Meer Van Bokhoven Van Kuppeveld Veenstra Van Venrooij Veltman Wesseling (b) Immune regulation (b) Membrane transport and (b) Chemical and physical intracellular motility biology Adema#* Bindels* Van Hest* De Witte Deen Van Delft Dolstra Drenth Gielen Figdor Fransen Hendriks Hilbrands Hoenderop Van Leeuwen Hoogerbrugge Knoers Lubsen Jansen Masereeuw Nolte Joosten De Pont Rowan Van Leeuwen Russel Rutjes Oosterwijk Speller Punt Theuvenet Raijmakers Vriend Van der Reijden De Waal Ruers Van Zoelen (c) Tissue engineering and pathology van Kuppevelt* Jansen Van de Kraan Van Krieken Torensma Van der Vlag * subtheme leader Our people from Faculty of Science 14 Members of NCMLS NCMLS
Annual Report 2006 Theme 1 Infection, immunity and tissue repair
One of the most important challenges of modern medicine is to cope with the increasing impact of the epidemics of obesity, diabetes Mihai Netea and cardiovascular Interleukin-18 Netea, M.G., Joosten, L.A.B., Lewis, E., Jensen, D.R., Voshol, P.J., Kullberg, B.J., Tack, C.J., van Krieken, H., Kim, S.H., Stalenhoef, A.F., Van de Loo, F.A., Verschueren, I., Pulawa, L., Akira, S., Eckel, R.H., Dinarello, C.A., Van den Berg, W., van der Meer, J.W.M. (2006). Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance. Nature Medicine, 12 (6), 650-656. Understanding metabolism and insulin resistance Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance One of the most important challenges of modern medicine is to cope with the increasing impact of the epidemics of obesity, diabetes and cardiovascular complications. These pathological conditions very often occur together in the so-called metabolic syndrome. Resistance to the hypoglycemiant effects of insulin and to the anorexigenic effects of leptin has been shown to be central factors in the pathogenesis of metabolic syndrome. Recently, the release of inflammatory mediators such as cytokines from the adipose tissue has been proposed to be a major contributor to insulin resistance. In the present study it is shown for the first time that surprisingly, interleukin-18 (IL-18), one of these proinflammatory cytokines, has beneficial effects on insulin resistance and metabolic syndrome. The absence of IL-18 in genetically-modified mice leads to increased food intake, obesity, insulin resistance and diabetes, while administration of recombinant IL-18 has the potency to reverse these effects. In addition, the mechanisms responsible for these effects, namely inhibition of food intake at the hypothalamic level and of gluconeogenesis in the liver, are described. The results of this study provide evidence for an important role of IL-18 in energy and glucose homeostasis; lack of endogenous IL-18 results in obesity, insulin resistance and hyperglycemia. Firstly, the energy balance (intake vs. expenditure) is influenced by a newly-described class of mediators called adipocytokines, among which leptin, adiponectin or ghrelin, are the most important. The present study is the first to involve IL- 18 in these important physiological processes. Secondly, glucose concentrations are a consequence of a tightly regulated balance between glucose absorption in the gut, glucose uptake and utilization, glucose release from liver glycogen deposits, and liver gluconeogenesis. While several hormones, such as glucagon, catecholamines, steroids and growth hormone, increase plasma glucose concentrations, insulin has long been the only hypoglycemic hormone. Only recently it has become apparent that other endogenous hormones, such as insulin-like growth factor and adiponectin, also have hypoglycemic effects. The data presented here suggest that also IL- 18 possesses a previously-unknown glucoselowering potential. Our data also provide an IL-18-dependent mechanism of STAT3 modulation, which has been demonstrated by others to control insulin and leptin resistance, impaired glucose tolerance and obesity. The paradox of hyperglycemia in IL-18-/- mice and of the high concentrations of IL-18 in the circulation of patients with type 2 diabetes mellitus may signify an attempt of the organism to counteract hyperglycemia, or could be a consequence of reduced sensitivity of these patients to the effects of IL-18, similar to insulin resistance. In conclusion, this study describes a new mechanism of modulation of energy metabolism and insulin resistance, which is influenced by IL-18. These findings open new avenues for understanding this complex pathological condition and for designing new therapeutic strategies. Figure: Obese IL-18-/- mice. Deficiency of interleukin- 18 in mice leads to hyperphagia, obesity and insulin resistance 16 Mihai Netea Understanding metabolism and insulin resistance NCMLS
Interleukin (IL)-32 is a proinflammatory cytokine originally described as a transcript termed NK4, present in activated Leo Joosten natural killer (NK) cells Interleukin-32 Joosten, L.A.B., Netea, M.G., Kim, S.H., Yoon, D.Y., Oppers-Walgreen, B., Radstake, T.R.D., Barrera, P., van de Loo, F.A.J., Dinarello, C.A., van den Berg, W.B. (2006). IL-32, a proinflammatory cytokine in rheumatoid arthritis. Proc Nat Acad Sci, 103 (9), 3298-3303. Understanding rheumatoid arthritis Interleukin-32, a novel pro-inflammatory cytokine in rheumatoid arthritis. Interleukin (IL)-32 is a proinflammatory cytokine originally described as a transcript termed NK4, present in activated natural killer (NK) cells and T lymphocytes. In the search for IL-18 inducible genes independent of IL-12 or IL-15 costimulation, IL-18 induced several expected proinflammatory genes in the human lung epithelial cell line A549. However, there was a high level of expression in a transcript termed NK4 with no known function. Upon expression of the recombinant form of the NK4 transcript, it became clear that NK4 encoded for a protein with many of the characteristics of proinflammatory cytokines. For these reasons, the name was changed to IL-32. Although IL-32 was first reported as a transcript in IL-2 activated NK and T cells, it appears that epithelial cells and macrophages are a dominant and widespread source. IL-32 is prominently induced by IFN-γ in several cell types. In addition, IL-32 itself stimulates proinflammatory cytokine and chemokine production, such as TNFα and IL-8, via the activation of NF-κB and p38 MAP kinase, and it is recently shown that it induces active IL-1β release through a caspase-1- dependent mechanism. Therefore, IL-32 is a novel proinflammatory cytokine which likely plays an important role in chronic inflammation. Enhanced IL-32 expression is detected in several inflammatory disorders, such as Crohns disease, atherosclerosis and rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease that predominantly affects multiple peripheral joints. Although the exact mechanisms which contribute to the disease pathogenesis are still largely unknown, it is generally well accepted that numerous inflammatory cells such as T cells, B cells, fibroblast-like synoviocytes and antigen presenting cells (APC) and their extensive production of several pro-inflammatory mediators, such as tumor necrosis factor alpha (TNFα) and interleukin-1 (IL-1) are implicated. Histopathologic features of RA synovial tissue encompass infiltration by macrophages and T cells, synovial lining hyperplasia, neoangiogenesis, and pannus formation. Finally, this leads to chronic joint inflammation with concomitant cartilage and bone destruction. In collaboration with Dr. Mihai G. Netea, Department of Infectious Diseases,, and Prof. Dr. Charles A. Dinarello, Division of Infectious Diseases, UCHSC, Denver, Colorado, USA we determined the participation of this novel cytokine IL-32 in the inflammation observed in RA. We subsequently investigated the expression of IL-32 on RA synovial tissue from patients with unaffected joints and with moderate or severe arthritis and examined whether the local expression of IL-32 is related to that of other pro-inflammatory cytokines. We reported that the synovial expression of IL-32 is strongly correlated with that of TNFα and IL-1β, but also with the severity of micro- and macroscopic joint inflammation. In addition, we assessed potential arthritogenic capacity of IL-32 and determined whether IL-32 mediated pro-inflammatory effects is dependent on TNFα, which is a key player in RA. IL-32 itself induced joint inflammation with concomitant mild cartilage damage when injected intra-articularly in murine knee joints. Using TNFα gene deficient mice, we showed that IL-32-driven joint inflammation was dependent of TNFα, but the cartilage damage was TNFα independent. Our findings support the role of IL-32 as a primary proinflammatory mediator in RA. The new cytokine IL-32 drives the local production of proinflammatory cytokine TNFα and represents therefore a potential novel target in RA. Figure: Expression of IL-32 in RA synovial tisue and inflammation provoked by local hil-32γ injection into a murine knee joint. A, RA synovial tissue stained for IL- 32 expression by immunohistochemistry. B. Isotype control antibody. C, Polymixin B (to control potential LPS contamination of ril-32γ) injected murine knee joint, no joint inflammation. H&E, 100x. D, Joint inflammation after i.a. injection of 100 ng ril-32γ. H&E, 100x. E, Note the severe cell influx in joint cavity and synovial tissue. H&E, 200x F, Monocyte/macrophage-like cells at day 4 after IL-32γ injection. H&E, 400x G, No cartilage matrix proteoglycans loss at day 4 after polymixin B injection, visualized by Safranin O staining (200x). H, Mild depletion of cartilage proteoglycans at day 4 after IL-32γ exposure. Annual Report 2006 Leo Joosten Understanding rheumatoid arthritis 17
In order to establish a productive infection, viruses have developed strategies to evade or counteract cellular defence mechanisms. Danny Duijsings Enteroviruses (e.g., ER-Golgi transport Wessels, E., Duijsings, D., Niu, T., Neumann, S., Oorschot, V., de Lange, F., Lanke, K., Klumperman, J., Henke, A., Jackson, C., Melchers, W., van Kuppeveld, F. (2006). A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1. Dev Cell, 11, 191-201. Understanding virus-host interaction How a viral protein blocks ER-to-Golgi transport In order to establish a productive infection, viruses have developed strategies to evade or counteract cellular defence mechanisms. Enteroviruses (e.g., poliovirus and coxsackievirus) are non-enveloped, cytolytic RNA viruses that do not rely on an intact secretory pathway to release their progeny. Instead, these viruses induce a general inhibition of early secretory pathway transport, most likely to suppress cytokine secretion and surface antigen presentation. The viral 3A protein plays an important role in this inhibition of protein transport. Indeed, we showed that a coxsackievirus carrying a 3A protein defective in inhibiting protein transport is less virulent in mice. Virus titers of the mutant virus were significantly lower in the pancreas and heart, which are the target organs of coxsackievirus. Futhermore, the mutant virus caused considerably less pathological damage, whereas the wild type virus caused massive fibrosis and necrosis in the heart. Thus, early secretory pathway transport inhibition by 3A is of importance for the in vivo infectivity of enteroviruses by suppressing both innate and adaptive immune responses. Protein transport between the ER and Golgi is mediated by tubulovesicular transport containers that depend on two coat complexes, COP-II and COP-I. The COP-II coat is recruited to the membrane by the GTPase Sar1, and is involved in membrane deformation and vesicle formation at ER exit sites (ERES), specialized domains at the ER where secretory cargo is concentrated. Following budding, the vesicles fuse to form the ER-Golgi-intermediate compartment (ERGIC). ERGIC containers are transported along microtubules to the cis side of the Golgi complex, where they fuse with Golgi membranes. The COP-I coat acts in retrograde transport within the Golgi and from the Golgi and ERGIC to the ER. COP-I function is also required for ongoing anterograde transport. Recruitment of the COP-I coat requires the membrane association of ADP-ribosylation factor 1 (Arf1), a small GTPase that cycles between an inactive (GDP-bound), cytosolic state and an active (GTP-bound), membrane bound state. Cycling of Arf1 between its GTPand GDP-bound state is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). We identified the mechanism by which the enterovirus 3A protein inhibits protein transport between the endoplasmic reticulum (ER) and the Golgi. Using a combination of biochemical approaches and live cell imaging techniques, we provide evidence that 3A interferes with the activation of the GTPase ADPribosylation factor 1 (Arf1), which regulates the recruitment of the COP-I coat complex to membranes. 3A specifically interacts with the N terminus of the ArfGEF GBF1, and stabilises an abortive complex of GBF1 and Arf1-GDP on membranes, and is similar to the mechanism used by the drug Brefeldin A (BFA). 3A is the first viral protein recognised to interfere with Arf1-mediated COP-I assembly, and provides a valuable tool for dissecting the early steps of the secretory pathway. Figure: (A) 3A redistributes COP-I to the cytosol. BGM cells were transfected with EGFP-3A, fixed and stained with a polyclonal antibody against COP-I. (B) 3A freezes GBF1 on Golgi membranes. The membrane cycling behaviour of EYFP-GBF1 in control cells (blue line) and ECFP-3A-expressing cells (purple line) was determined by FRAP microscopy. (C) Schematic model of the mechanism by which 3A inhibits Arf1 activation. Arf1-GDP and GBF1 are recruited independently to the membrane to catalyse GDP-to-GTP exchange. 3A freezes an intermediate, abortive complex of Arf1-GDP and GBF1 on membranes, thus efficiently blocking GBF1 activity. As a result, no new Arf1-GTP is produced, COP-I recruitment cannot proceed, and ER-to-Golgi transport is blocked. 18 Danny Duijsings Understanding virus-host interaction NCMLS
The epidermis is the outermost layer of the skin that forms the physical barrier against the environment. It also serves as a permeability barrier Tsing Cheng preventing Epidermal differentiation Cheng, T., Hitomi, K., van Vlijmen-Willems, I.M., de Jongh, G.J., Yamamoto, K., Nishi, K., Watts, C., Reinheckel, T., Schalkwijk, J., Zeeuwen, P.L. (2006). Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumainbinding site. A novel clue for the role of cystatin M/E in epidermal cornification. J Biol Chem, 281, 15893-9. Understanding skin barrier formation Protease-antiprotease balance controls stratum corneum formation The epidermis is the outermost layer of the skin that forms the physical barrier against the environment. It also serves as a permeability barrier preventing water loss from our body. During epidermal differentiation, keratinocytes from the basal layer gradually transform into dead corneocytes of the cornified layer. In this process the keratinocytes form the cornified envelope, an insoluble structure of crosslinked proteins that is assembled adjacent to the plasma membrane. Cornification is followed by the degradation of the corneodesmosomes that hold the corneocytes together. As a result the corneocytes eventually flake off from the skin surface, a process called desquamation. This complex process of epidermal differentiation is dependent on interactions between several proteins and enzymes. Disturbance of these protein interactions causes abnormal terminal differentiation, resulting in an impaired barrier function of the epidermis. Cystatin M/E is a cysteine protease inhibitor that is mainly expressed in the epidermis and specifically in the stratum granulosum. Our previous investigations revealed that a null mutation in the mouse cystatin M/E gene causes the murine ichq phenotype, which is characterized by neonatal lethality and abnormalities in cornification and desquamation, demonstrating an essential role for cystatin M/E in epidermal differentiation. In the skin of these mice we observed excessive and premature activity of transglutaminase 3, an enzyme that is responsible for crosslinking of proteins into the cornified envelope, which may explain the disturbed cornification. Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, a lysosomal protease involved in the processing of cathepsins. Although cystatin M/E contains a predicted binding site for lysosomal cysteine proteases, no high affinity binding for any member of this family has been demonstrated to date. Here we report that the cysteine proteases cathepsin L and cathepsin V are inhibited by cystatin M/E through a reactive site that is distinct from the legumain-binding site. Cystatin M/E variants with an essential amino acid mutation in one of the reactive sites show a loss of inhibitory activity against the respective proteases (Figure 1). Using immunofluorescence on normal human skin, we found that cystatin M/E co-localizes with cathepsin L and cathepsin V in the upper layers of the epidermis (Figure 2). In addition, we show that cathepsin L is the elusive enzyme that processes and activates epidermal transglutaminase 3. We conclude that cystatin M/E has an important regulatory function in human epidermal differentiation. Cystatin M/E regulates crosslinking of proteins by transglutaminase 3 in cornified envelope formation through its inhibitory activities against cathepsin L and legumain. In addition, we showed that cystatin M/E was also found in the extracellular space in the stratum corneum, associated with corneodesmosomes, where it was closely associated with cathepsin V. This finding suggests that cystatin M/E could also have a role in the desquamation process as cathepsin V activity is involved in the degradation of corneodesmosomal proteins (Zeeuwen et al., J Invest Dermatol 127:120-128, 2007). Figure 1: Three-dimensional representation of cystatin M/E showing the two inhibitory sites of the protein. The regions of the binding sites for legumain and for the cysteine proteases are marked. The W135A variant was rendered inactive against cathepsin L and cathepsin V but retained legumain-inhibiting activity. Conversely, the N64A variant lost legumain-inhibiting activity but remained active against both cysteine proteases. Figure 2: A) Co-localization of cystatin M/E and cathepsin L in the stratum granulosum of the epidermis of normal human skin. Cathepsin L is also expressed in the deeper layers of the epidermis, but not in the stratum corneum. B) Cystatin M/E and cathepsin V co-localize in the stratum granulosum and the stratum corneum. Annual Report 2006 Tsing Cheng Understanding skin barrier formation 19
Regulatory T-cells (Treg) play a crucial role in maintaining control of effector lymphocytes. This recently (re)-discovered T cell subset is Roger Sutmuller extremely potent in the Treg function Sutmuller, R.P.M., den Brok, M.H.M.G.M., Kramer, M., Bennink, E.J., Toonen, L.W.J., Kullberg, B.-J., Joosten, L.A., Akira, S., Netea, M., Adema, G.J. (2006). Toll-like receptor 2 controls expansion and function of regulatory T cells. J Clin Invest, 116 (2), 485-494. Understanding regulatory T-cells in health and disease Toll-like Receptor 2 controls expansion and function of regulatory T cells Regulatory T-cells (Treg) play a crucial role in maintaining control of effector lymphocytes. This recently (re)-discovered T cell subset is extremely potent in the inhibition of immune reactions. Several studies have shown that in vivo Treg depletion results in autoimmune syndromes like thyroiditis, gastritis, diabetes mellitus, and colitis. How the Treg themselves are regulated or how they suppress immunity is however largely unknown. The fact that they were unable to be expanded in vitro contributed to the difficulty of analyzing them. Pathogen recognition receptors from the Toll like receptor (TLR) family recognize pathogenassociated molecular patterns (PAMPs). These TLRs, expressed by cells of the innate immune system, mediate danger signals crucial for the generation of effective immunity. Recently, TLRs were also found on T-cells, including Treg, suggesting a role for danger signals in the control of Treg function. Treg play a central role in the prevention of autoimmune responses harmful to the host. During acute infection however, Treg may hinder effector T cell activity directed towards the elimination of the pathogenic challenge. In addition, it has been found that Treg prevent tumors from being attacked by tumor specific T cells. We recently found the Treg subset in TLR2-/- mice to be significantly reduced compared with wildtype littermate control mice, indicating a link between Treg and TLR2. We now show that TLR2-ligands but not TLR4- or TLR9-ligands, act directly on purified Treg in a MyD88-dependent fashion. When combined with T-Cell Receptor-stimulation, TLR2-triggering induced Treg proliferation in vitro and in vivo and results in a temporal loss of the suppressive Treg phenotype in vitro by directly affecting the Treg themselves. Importantly, in TLR2-/- recipient mice, adoptively transferred wildtype Treg were neutralized by systemic administration of TLR2-ligand during the acute phase of a Candida albicans infection resulting in a 100-fold reduced Candida outgrowth. This demonstrates that TLR2 also controls the function of Treg in vivo and establishes a direct link between TLRs and the control of immune responses through regulatory T- cells. The current opinion holds that TLR mediated recognition of pathogens results in DC activation and subsequent initiation of T-cell responses. We can now add a direct Treg modulating capacity to TLR-ligands. Applying an acute fungal infection model, we unequivocally demonstrated that TLR2-triggering on the Treg themselves abrogates their suppressive activity in vivo resulting in increased IFN-γ production and decreased fungal outgrowth. We hypothesize that TLR2-ligands provided by a microbial invasion during acute infection, mediate Treg expansion and abrogation of Treg-mediated suppression, thus allowing a potent immune response to occur. However, during post-infection, when the immune system has cleared the pathogen and hence TLR2-ligands are declining, the expanded Treg regain their suppressive activity and could help to restore the immune balance. In addition to microbial derived PAMPs, TLR2- signaling could possibly be induced by endogenous proteins (e.g. Heat Shock Proteins). This would allow the modulation of the Treg compartment in a non-pathogenic, stress induced (anti)-inflammatory environment. A final important implication of our finding is that it has now become possible to culture and expand the Treg in vitro, allowing for the in depth analysis of these cells. This research will increase our understanding of the role of these cells in health and disease. Photo: In vitro proliferation of Treg is induced by Toll-like Receptor stimulation 20 Roger Sutmuller Understanding regulatory T-cells in health and disease NCMLS