MBL and L-ficolin levels and genotypes in community-acquired pneumonia MBL and L-ficolin: levels and genotypes in community-acquired pneumonia Thesis, Utrecht University, The Netherlands Printed by Wöhrmann Print Service, Zuthpen ISBN 978-90-8570-734-9 Printing of this thesis was financially supported by the Infection & Immunity Center Utrecht, the Netherlands Society of Medical Microbiology (NVMM) and the Netherlands Society for Microbiology (NVvM), Bayer HealthCare, BD Diagnostics, DiaSorin, Gilead Sciences Netherlands, Mediphos Medical Supplies, Merck Sharp & Dohme, Meridian Biosciences Europe and Pfizer. MBL and L-ficolin levels and genotypes in community-acquired pneumonia MBL en L-ficolin spiegels en genotypen bij thuis opgelopen longontsteking (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 21 april 2011 des ochtends te 10.30 uur door Bjorn Lars Herpers geboren op 16 februari 1974 te Schaesberg Promotoren: Prof.dr. J. Verhoef Prof.dr. J.A.G van Strijp Co-promotoren: Dr. B.M. de Jongh Dr. G.T. Rijkers CONTENTS Glossary 8 Chapter 1 11 General introduction Chapter 2 49 Coding and non-coding polymorphisms in the lectin pathway activator L-ficolin gene in 188 Dutch blood bank donors Chapter 3 59 Hemolytic assay for the measurement of functional human mannose-binding lectin: A modification to avoid interference from classical pathway activation Chapter 4 67 Mannose-binding lectin genotypes in susceptibility to community- acquired pneumonia Chapter 5 81 Deficient mannose-binding lectin-mediated complement activation despite mannose-binding lectin-sufficient genotypes in an outbreak of Legionella pneumophila pneumonia Chapter 6 93 Acute-phase responsiveness of mannose-binding lectin in community-acquired pneumonia is highly dependent upon MBL2 genotypes Chapter 7 105 L-ficolin acts as an acute phase reactant in community-acquired pneumonia Chapter 8 117 Discussion Chapter 9 131 Summary / Samenvatting Chapter 10 135 Curriculum vitae Chapter 11 139 Dankwoord Chapter 12 143 List of publications GLOSSARY Adaptive immunity Those cells, molecules and mechanisms of the immune system that act on danger signals in an antigen-specific manner. The adaptive immune system recognizes pathogens through specific antigenic epitopes associated with a pathogen. Responses in adaptive immunity are shaped over time by repeated challenge, becoming more targeted and they posses mechanisms of memory of earlier immune responses by memory cells. It is the counterpart of innate immunity. Allele A particular DNA sequence of a gene. When a gene is polymorphic, the genetic variants are called alleles. Complement A system of more than 30 fluid phase and cell-associated components, which act in a cascade-like network that is involved in opsonization and lysis of microbes, cellular recruitment and activation, and immune clearance of immune complexes. The complement system is part of the innate immune system and can be triggered by components of both innate and adaptive immunity. Ficolins A group of proteins that share the structure of a collagenous triple helix and a fibrinogen-like C-terminal domain. They are found in a wide range of species. Human ficolins L-ficolin, H-ficolin and M- ficolin resemble MBL in structure and function. FCN2 The gene encoding human L-ficolin. Genotype The genetic constitution. Since humans are diploid and carry two copies of each gene on two homologous chromosomes, the genotype refers to the combination of two alleles or haplotypes. Haplotype A combination of co-occurring SNPs that are located at different sites on a gene. Haplotypes represent the fact that genetic variations at different sites of a gene are linked because they are situated on the same DNA fragment. Heterozygous Carrying different alleles of a gene on the two homologous chromosomes. Homozygous Carrying identical alleles of a gene on both homologous chromosomes. Innate immunity Those cells, molecules and mechanisms of the immune system that act on danger signals in a non-specific, generic manner. The innate immune system recognizes pathogens through evolutionary conserved general patterns associated with these pathogens. Responses in innate immunity are fixed, as they are not further shaped by repeated challenge and they do not posses mechanisms of memory of earlier immune responses. It is the counterpart of adaptive immunity. MASP MBL-associated serine protease. MASPs bind to MBL and ficolins. With their serine protease domains, MASP-2 and MASP-1 enzymatically cleave complement components C4 and C2, setting of the complement cascade. MASP-3 and the splicing variants Map44 and Map19 do not show this enzymatic activity and are thought to have a regulatory role. MBL Mannose-binding lectin. MBL is a multimeric pattern recognition receptor circulating in plasma that binds to invading pathogens and activates the complement system. Proteins similar to MBL are found in a wide range of species. MBL2 The gene encoding MBL. Multimeric Consisting of multiple subunits, called monomers. Higher order multimers consist of many monomeric subunits, while lower order multimers or oligomers consist of a few. Polymorphism The occurrence of more than one allele of a gene within a population, representing genetic diversity in a population. SNP Single nucleotide polymorphism (SNP, pronounced as “snip”). A DNA sequence variation consisting of only one variant nucleotide. 1 General introduction General introduction Lung immune defenses To resist the constant challenge of potentially infectious agents, the lungs posses innate and adaptive immune defense mechanisms that prevent colonization and clear invading pathogens (Figure 1).1 Structural defenses form the first barrier for invading pathogens. The anatomical design of the nose causes such an airflow that large particles (>5µm) are caught on the nasal mucosa before entering the lower airways. Smaller particles can be trapped in the mucus overlying the respiratory epithelium. By the coordinated movement of the underlying cilia, mucus and particles are removed mechanically by forcing them towards the mouth. The cough and sneeze reflexes also help to remove depositions from the airways.2 If pathogens succeed in passing through physical and mechanical barriers, the innate immune system is called upon. Soluble antimicrobial molecules including the various components of the complement system, alveolar macrophages, neutrophils, natural killer cells and dendritic cells are active in the first minutes of colonization and invasion. They restrain infection by their intrinsic antimicrobial activity and the regulation of inflammation and activation of the adaptive immune response. Soluble enzymes like lysozyme and peroxidase posses antibacterial activity. Defensins are secreted by epithelial cells and neutrophils and posses activity against Gram-positive and Gram-negative bacteria, mycobacteria, fungi and viruses.3 They bind to the microbial cell membrane, forming pores that lead to lysis of the cells. Pentraxins such as C-reactive protein (CRP), serum amyloid P and PTX3 bind to microbial structures, thus opsonizing microbes for phagocytosis and complement mediated lysis.4 The complement system consists of more than 30 fluid phase and cell-associated components, which act in a cascade-like network that plays a pivotal role in innate immunity, as it is involved in opsonization and lysis of microbes, cellular recruitment and activation, and immune clearance of immune complexes. The complement system can be triggered by components of innate and adaptive immunity.5,6 The most well-known activator of complement in innate immunity is mannose-binding lectin (MBL), a plasma protein belonging to the collectin family. Collectins are oligomeric proteins with a collagenous region and C-type lectin domains, capable of binding saccharide moieties on microbial surfaces.7 The collectins surfactant proteins A and D are found in high concentrations in the alveoli of the lungs, functioning as opsonins and agglutinins of pathogens in the lower airways. MBL not only opsonizes microbes for phagocytosis, but subsequently can activate the complement system as well. The ficolins share some structural and functional properties with MBL. Instead of recognizing microbial structures through a C-type lectin domain, they possess a fibrinogen-like domain binding to acetylated moieties also found on microbes.8,9 Binding of ficolins leads to opsonization and complement activation as well.10 The complement system, as well as its activators MBL and the ficolins, will be discussed in more detail below. Besides soluble components, the innate immune system also encompasses cellular elements. In the lungs, alveolar macrophages and neutrophils can kill invading microbes via phagocytosis and subsequent production of reactive oxygen species and lytic General introduction | 13 invading pathogen immune system upper airway lysozyme peroxidase direct pathogen death lactoferrin defensins neutrophils phagocytosis lower airways macrophages reactive oxygen species defensins lysosomal enzymes direct pathogen death complement activation collectins ficolins complement activation pentraxins induction of adaptive immune response dendritic cells T cells B cells pathogen evasion and survival clearance of pathogen Figure 1 | Pulmonary defences against invading pathogens