1.1 Lyme borreliosis
Lyme borreliosis (LB), which was first described in the mid-1970's, represents the most frequent vector-borne disease in many European countries (1) and the USA (2).
In endemic regions of Southern Germany an incidence of LB between 50 – 600 per 100.000 inhabitants was found (3, 4). According to recent studies up to 52% of highly exposed individuals in endemic areas of Baden Württemberg are infected with Borrelia burgdorferi (B. burgdorferi) and up to 24% of ticks are infected with this pathogen (5). B. burgdorferi, the causative agent of LB, is a corkscrew-shaped bacterium which belongs to the family of Spirochaetaceae. Infection which occurs via a tick bite either leads to a subclinical stage or results in a range of clinical symptoms divided into three stages: the early localized, the disseminated and the late, chronic stage. Different organs can be affected, including the heart, joints, skin and central nervous system. Diagnosis of LB still represents a major problem since the commercially available and in-house tests do not offer the desirable standardized performance. If infection with B. burgdorferi is not adequately treated, i.e. as early as possible and with the recommended antibiotics, it may lead to a chronic multisystemic disorder, which is difficult to cure.
The most puzzling feature of LB is that B. burgdorferi is often not eradicated even in the presence of an active immune response. Several hypotheses have been put forward to explain persistence of Borrelia in the human host during months and years in spite of active immune cells. In the thesis presented here the question how Borrelia persist in the human host was addressed, providing some evidence that the pathogen modulates the host’s immune response by inducing anti-inflammatory responses and rendering monocytes and macrophages anergic.
1.2 Toll-like receptors
Recently the toll family of highly conserved transmembrane receptor proteins has been identified. The members of this family share a highly homologous cytoplasmic domain, similar to the IL-1-receptor, a very short transmembrane domain, and an extracellular portion consisting of a various number of leucine-rich repeats. They are expressed on immune cells and on tissue cells and represent a critical link between
immune stimulants produced by microorganisms and the initiation of host defense.
Activation of these receptors results in the release of antimicrobial peptides, inflammatory cytokines and costimulatory molecules that initiate adaptive immunity.
Up to now at least 10 different members have been identified, which vary in their ligand specificity, expression patterns and presumably in signal transduction pathways, consequently activating different genes. Toll-like receptor (TLR) 4, which was the first TLR found in humans, was identified as the major LPS recognition receptor. TLR2 is characterized by associating with TLR1 and TLR6 and having the broadest spectrum of ligands, including lipoteichoic acid from Gram positive bacteria, peptidoglycan, zymosan and lipoproteins from Treponema, Mycoplasma and also B.
burgdorferi. However, the role of TLR2 in recognition of other membrane components of Borrelia is not conclusively clear and will be addressed in this thesis.
1.3 Tolerance and cross-tolerance
In its simplest terms “endotoxin tolerance” refers to a hyporesponsive state following a second or additional dose of endotoxin in contrast to the responses observed after an initial exposure to endotoxin. Since its first descriptions in the 1960s extensive research has been undertaken to understand the molecular and cellular background of this phenomenon. The "lipopolysaccharide-tolerant" phenotype of macrophages and monocytes is characterized by reduced TNFα-, IL-1β- and IL-6-release, enhanced cyclooxygenase-2 activation, inhibition of mitogen-activated protein kinase activation, and impaired NFκB-translocation upon re-stimulation with LPS. Similarly as human monocytes and macrophages can become tolerant in vitro, monocytic cells from patients with systemic inflammatory response syndrome and sepsis have many characteristics of endotoxin tolerance. It is postulated that the clinical significance of this desensitization is a natural regulatory mechanism aimed to control an otherwise autodestructive sytemic inflammation. Recently, increasing evidence is coming up showing that other stimuli than LPS including lipoteichoic acid, Staphylococcus aureus, macrophage-activating lipopeptide (MALP) -2, bacterial DNA (CpG), and arabinose-capped lipoarabinomannan (LAM) render macrophages anergic to each other as well as to LPS. Based on these findings the term “cross-tolerance” or
“hetero-tolerance” has been coined to describe tolerance induction between different stimuli. In the present study it was investigated whether Borrelia burgdorferi also has the capacity to desensitize macrophages. Since Borrelia-induced
hyporesponsiveness could represent a mechanism enabling the survival of this pathogen in the host despite the presence of immune cells.
1.4 Granulocyte colony-stimulating factor (G-CSF)
In line with its name the granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor which recruites granulocytes from the bone marrow.
However, since its discovery two decades ago, much more functions of this pleiotropic protein have been described. In addition to controlling production and maturation of neutrophilic granulocytes in the bone marrow it also primes mature granulocytes resulting in an increased oxidative burst or phagocytosis. Furthermore, it exerts pronounced anti-apoptotic effect on these cells. While G-CSF acts on neutrophils as a pro-inflammatory cytokine by augmenting their bactericidal functions, it influences monocytes with its anti-inflammatory properties, reducing their release of pro-inflammatory cytokines such as TNFα, IL-12 and IL-1β. The lymphocytes seem not to be influenced directly by G-CSF, but lacking monocyte factors attenuate IFNγ-formation.
Due to its unique anti-infectious and hematopoietic pharmacological properties on the one hand, and the fact that it is very safe on the other hand, G-CSF has become one of the most prominent endogenous proteins produced biotechnologically in broad clinical use. In this work G-CSF was employed in combination with antibiotics as an immunosupportive treatment to test a novel therapy for late stage LB.
1.5 Aims of the study
Lyme borreliosis (LB) is the most common tick borne disease in European countries and the United states. An incidence of 50 – 600 per 100.000 inhabitants has been reported and up to 24% Borrelia burgdorferi-infected ticks were found in highly endemic areas. Since the first description of LB in the mid 1970s, it is still unclear how Borrelia persist facing the bodies phagocytic and other immune clearance mechanisms. They sometimes even persist in case of antibiotic therapy. Thus, understanding the immunopathology of Borrelia infection represents still a major challenge.
A vaccine, which was only partially effective since it exclusively protected against infection with Borrelia burgdorferi s.s., but not against the two other strains pathogenic for humans in Europe, has been available for about three years.
However, since its withdrawal in spring 2002 no vaccine exists to protect population from this infection. The lack of a vaccine on the one hand and the sometimes unsatisfactory treatment on the other hand, illustrate the need to find new concepts in treatment of this disease. Since a better understanding of immunopathology of LB is important for novel therapeutic interventions, immune avoidance of Borrelia was examined in the first part of the present thesis: In particular the following issues were addressed:
Characterization of the immune response of blood leukocytes from late stage LB patients in comparison to healthy donors
Comparison of Borrelia- and LPS-induced cytokine release in blood from healthy donors
Characterization of Borrelia-induced immunosuppression as a possible immune evasion mechanism
Characterization of Borrelia-induced anergy of blood leukocytes in an in vitro model of immunomodulation and comparison with endotoxin tolerance
Based on the results of the first part, the aim of the second part was to propose a new concept in LB therapy. The procedure was as follows:
Establishing the mouse model of Lyme borreliosis
Testing the effect of immunosupportive treatment with the hematopoietic growth factor G-CSF (Filgrastim) on ankle swelling and bacterial burden in two different Borrelia-infected mouse strains
These studies represented the basis for a clinical trial, in which the combination therapy of antibiotics plus Filgrastim in a late stage LB patient was tested.