Thursday 21 April 2011
MBL and L-ficolin: levels and genotypes in community-acquired pneumonia
Promotor: Prof.dr J. Verhoef and prof.dr J.A.G. van Strijp
Defence: 21 April 2011
Under normal physiological conditions, the pulmonary defense mechanisms described above are capable of clearing the lungs from microbes and particles, keeping the lower respiratory tract sterile. The development of pneumonia therefore represents a breach in the pulmonary defense mechanism either by an extraordinary large inoculum or high virulence of a pathogen, or a defect in the host defense itself.
In this thesis, the interplay between invading pathogens, host defense genes and their encoded proteins are studied in the context of community-acquired pneumonia and two activators of the lectin pathway of complement: MBL and L-ficolin. By studying genotypes, protein levels and functionality in a cohort of hospitalized patients with CAP and in an outbreak of legionellosis, we aim to obtain more insight into the reciprocal effect of these complement proteins and pneumonia on each other, in the light of the genetic background of the patients.
Both MBL and L-ficolin are part of the humoral innate immune system. The innate immune system is designed for immediate activation upon microbial challenge, the first phase of infections like pneumonia. Other components of the innate immune system show a sharp increase or decrease in concentration and/or activity upon microbial encounter, a phenomenon termed the acute phase response. Therefore, this thesis not only investigates steady state conditions such as genetic polymorphisms and etiologic pathogens, but also the dynamics of MBL and L-ficolin levels in the acute phase of pneumonia in light of genetic polymorphisms and the etiology and course of disease.
An introduction is given in the main topics of the thesis. After summarizing the different components of innate and adaptive immunity of the lung, the complement system of innate immunity is further looked into. Routes of activation, its role in inflammation, regulatory mechanisms and deficiencies are discussed. Two initiators of the lectin pathway of complement activation, MBL and L-ficolin, are the subject of investigation of this thesis and their structure, functional properties and genetics appear to be very similar. In this thesis, these two proteins are investigated in the context of pneumonia. A brief summary of CAP and its etiological agents are given.
The first question to be answered was whether the FCN2 gene is polymorphic. For MBL2, polymorphisms were already known. The homology between structure and functionality of MBL and L-ficolin led us to ask whether FCN2 also harbored SNPs possibly influencing protein level and function. With the use of denaturing gradient gel electrophoresis, we identified 10 SNPs, two of which were coding polymorphisms leading to amino acid substitution in the ligand-binding domain of L-ficolin.
This chapter describes the modification of a hemolytic assay for the quantification of MBL activity. The redundancy of activation pathways of the complement system can cause technical problems in vitro when trying to measure a single component of the system. In the hemolytic MBL assay, complement activation by the antibody-mediated classical pathway was shown to interfere with measurements of MBL activity. Modification of the assay by addition of anti-C1q antibodies inhibited this unwanted interference.
In a large clonal outbreak of legionellosis, the influence of MBL2 genotypes as a risk factor for acquiring the disease was studied. Furthermore, the effect of the disease on MBL function in relation to the MBL2 genotype was investigated. Genetic MBL deficiency was not a risk factor for developing legionellosis in this cohort. A large proportion of patients with MBL-sufficient genotypes showed absence of MBL functionality during legionellosis. This was not observed in the control subjects, suggesting that the deficiency in MBL activity in legionellosis is an effect of the disease rather than a risk factor for acquiring it.
In this chapter, the role of MBL2 genotypes in CAP in relation to the causative agent was the subject of study. MBL binds different pathogens with different avidity, and pathogens have different ways to cause infection, for example by entering host cells. Therefore, it was investigated whether MBL2 genotypes were correlated with the type of etiologic pathogen and with outcome. MBL deficiency was not a risk factor for developing pneumonia in this cohort. MBL-deficient genotypes were observed more frequently in viral (co)infections. No correlation was found between pneumococcal pneumonia and genotypes.
We have further studied the genetic polymorphism of MBL2 and the behavior of MBL levels during CAP in relation to these genetic properties. By incorporating genotypes in the analysis of the behavior of MBL during the acute phase of pneumonia, it was shown that MBL is an acute phase reactant. However, MBL levels can only rise during the acute phase response if the genotype allows rapid upregulation of the production. With low-producing promoter genotypes, consumption of MBL during pneumonia outweighs MBL production.
Do FCN2 polymorphisms exert similar effects on L-ficolin levels during pneumonia as shown for MBL in chapter 6? In an analogous study design, it was shown that the FCN2 promoter SNP -986G>A, and to a lesser extend -4A>G influence protein levels in both the acute and convalescent phase, but do not influence acute-phase responsiveness.
The data from the previous chapters are further discussed in the context of recent literature. Hypotheses on cause and effect of the observed behavior of MBL and L-ficolin are proposed together with suggestions for further research on the investigated topics.