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PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/65650 Please be advised that this information was generated on 2021-10-07 and may be subject to change. MENINGOCOCCAL DISEASE Tom Sprong Het onderzoek in dit proefschrift werd mogelijk gemaakt door een beurs van de Nederlandse Organisatie voor Wetenschappelijk Onderzoek, beurs nummer 920-03-176 Colofon Thesis Radboud University Nijmegen Medical Centre, with Summary in Dutch Meningococcal Disease © Tom Sprong, Nijmegen, 2009 All rights reserved ISBN: 978-909-02-3865-4 Printing: Print Partners Ipskamp Cover design and Lay out: Martijn Prins Cover art: Kasper Peter Sprong: ‘impression of meningococci in blue’ MENINGOCOCCAL DISEASE Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus, prof. mr. S.C.J.J. Kortmann, volgens het besluit van het college van decanen in het openbaar te verdedigen op woensdag 4 februari 2009 om 15.30 uur precies door Tom Sprong geboren op 20 september 1975 te s´Hertogenbosch Promotor: Prof. dr. J.W.M. van der Meer Copromotor: Dr. ir. M. van Deuren Manuscriptcommissie: Prof. dr. R. de Groot Prof. dr. J.G. van der Hoeven Prof. dr. T.W. Kuijpers Paranimfen: Drs. N. Sprong Drs. W.C.H. Jacobs CONTENTS 1. Introduction and outline of the thesis 9 2. Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response 15 3. Neisseria meningitidis can induce pro-inflammatory cytokine pro-duction via pathways independent from CD14 and toll-like receptor 4 33 4. Complement activation and complement-dependent inflammation by Neisseria meningitidis are independent of lipopolysaccharide 49 5. Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria. 67 6. N. meningitidis lipid A mutant lipopolysaccharides (LPS) function as LPS antagonists in humans by inhibiting toll-like receptor 4 (TLR4) dependent cytokine production 85 7. Mannose binding lectin enhances IL-1beta and IL-10 induction by non-lipopolysaccharide (LPS) components of Neisseria meningitidis 99 8. Inhibition of C5a-induced inflammation with preserved C5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis 115 9. Deficient alternative complement pathway activation due to factor D deficiency by two novel mutations in the Complement Factor D gene in a family with meningococcal infections 141 10. Mannose-binding lectin is a critical factor in systemic complement activation during meningococcal septic shock 159 11. PCR-Restriction Fragment Length Polymorphism method to detect the X/Y polymorphism in the promoter site of the mannose-binding lectin gene 173 12. The double-edged sword of complement activation during sepsis. 179 13. Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability 193 14. Macrophage migration inhibitory factor (MIF) in meningococcal septic shock and experimental human endotoxemia. 207 15. PTX3 and C-reactive protein in severe meningococcal disease 223 16. Mannose-binding lectin (MBL) and meningococcal disease, a case-parent study 237 17. Innate cytokine production capacity and clinical manifestation of meningococcal disease 253 18. Summary and General Discussion 269 Nederlandse Samenvatting en Discussie 279 Dankwoord - word of thanks 290 Curriculum Vitae 293 List of publications 294 Awards and Presentations on national and international conferences 298 Introduction and outline of the thesis 1 Meningococci are new pathogens. Evolutionary speaking brand-and-shining-new pathogens, which emerged probably as recently as 200 years ago. They appeared when harmless Neisserial commensals in our nasopharynx obtained the ability to cause invasive disease, possibly due to accidentally acquiring a polysaccharide capsule from another bacterium. In contrast, our immune systems are old. Very old, probably nearly as old as multicellular life itself. In spite if its age however, our immune system is perfectly fit to deal with the multitude of micro-organisms that threaten us from our environment. So, how does our ancient immune system battle a new pathogen such as Neisseria meningitidis? Some people, often previously perfectly healthy, young children, may be left utterly defenseless. When they become infected, and get meningococcal septic shock, they develop rapidly progressive disease that kills one out of five patients, leaving another third with severe sequelae. Fortunately, only a minority of people will acquire invasive meningococcal disease. Also, not all people exhibit the same type of illness, there are multiple clinical manifestions associated with meningococcal infections. In sharp contrast to the dreadful manifestation of meningococcal septic shock, there even are some patients that present with chronic 'benign' meningococcal bacteraemia. This is a disease in which patients can have meningococci in their blood for weeks and still recover completely with simple antibiotic treatment. Although great progression has been made previously, many children are still killed or severely disabled by meningococcal infections. We still cannot prevent children from being infected. Therefore, we need to study pathogenesis. What if we could turn - using our knowledge of pathogenesis - the clinical course of fulminant meningococcal septic shock into benign meningococcal bacteraemia?! The present thesis studies the interplay between meningococci and the innate immune system. The focus is on the role of cytokines and the complement system. The questions are straightforward and simple: what happens during invasive disease, how does it happen and are there ways to stop it from happening? The answers are complex but will increase our knowledge, hopefully leading to new and better treatments for this dreadful disease. Lipopolysaccharide (LPS) has always been regarded as the principle cytokine inducing element of N. meningitidis and other Gram-negative bacteria, and LPS is also thought to be important for the activation of complement. In chapters 2 - 4 we made use of a genetically modified meningococcus that was deficient for LPS, addressing the question how important LPS versus non LPS components of 12 1 meningococci are in the induction of cytokines and the activation of complement. Cytokines are crucial mediators of the inflammatory response during meningococcal disease. Excessive production of these cytokines is held responsible for the development of tissue damage and shock. The relative contribution of lipopolysaccharide (LPS) and non-LPS of Neisseria meningitidis to the pro- inflammatory cytokine response in human cells and to mortality in a murine model of meningococcal sepsis is addressed in chapter 2. In chapter 3 we dealt with the question which role CD14 and TLR4 play in the induction of cytokines by LPS or non LPS components of meningococci. The complement system is crucial in the initial defense against Neisseria meningitidis. In contrast, uncontrolled activation in meningococcal sepsis contributes to the development of tissue damage and shock. Whether complement activation and complement dependent inflammation in whole blood by meningococci is dependent or independent of LPS is studied in chapter 4. For E. coli LPS it has been reported that lipoproteins inhibit cytokine production, opening new options for future therapies. However, meningococcal LPS is structurally different from E. coli LPS and intact Gram-negative bacteria are composed of more cytokine inducing elements then solely LPS. In chapter 5 we questioned whether lipoproteins also neutralize meningococcal LPS or complete Gram-negative bacteria. Another way to inhibit cytokine production by LPS is to use LPS antagonist molecules. Recently, novel variant LPS species derived from N. meningitidis have been created with a lipid A portion consisting of five or four fatty acids in the lipid A part of the LPS. Because penta- or tetra-acylated LPS from other bacterial species is reported to be antagonistic, in chapter 6 we explored whether these meningococcal LPS variants show antagonism for LPS induced TLR4 dependent cytokine production. A bridge between cytokine production and the complement system is found in chapter 7, where we study the influence of mannose binding lectin (MBL), the key molecule of the lectin pathway of complement activation on cytokine production by non-LPS components of meningococci. In chapters 8 - 12 we addressed in vitro, in a whole blood model, as well as in vivo, in patients with meningococcal disease, the issue how complement is activated systemically during meningococcal septic shock. In addition, we also investigated ways to stop the consequences of systemic activation of complement. Chapter 8 13 we asked whether an anti-C5a molecule is able to inhibit complement dependent inflammation in whole blood. In addition, in this chapter, the mechanism by which complement is activated by meningococci is studied. In chapter 9, we report a family deficiency for complement factor D with meningococcal infections and addressed the question to what extent factor D deficiency affects complement activation by meningococci. In chapter 10 we dealt with the question to what extent the alternative and lectin pathways of complement