Katja Gueinzius
Interaction of
Chlamydophila pneumoniae with the innate immune system
Dissertation
Universität Konstanz
im April 2006
Interaction of
Chlamydophila pneumoniae with the innate immune system
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
an der Universität Konstanz (Fachbereich Biologie) vorgelegt von
Katja Gueinzius aus Eislingen
Tag der mündlichen Prüfung: 9. Juni 2006 Referenten: Prof. Dr. Dr. T. Hartung
Prof. Dr. A. Wendel
für Mäggi für Mäggi für Mäggi für Mäggi
List of publications
Major parts of this thesis are summarized in the following papers:
• Hermann C., Gueinzius K., Oehme A., von Aulock S., Straube E., and Hartung T..
2004. Comparison of quantitative and semiquantitative enzyme-linked immunosorbent assays for immunoglobulin G against Chlamydophila pneumoniae to a microimmunofluorescence test for use with patients with respiratory tract infections. J Clin Microbiol 42: 2476-2479.
• Mueller M., Postius S., Thimm J. G., Gueinzius K., Muehldorfer I., and Hermann C.. 2004. Toll-like receptors 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for the release of monokines. Immunobiology 209: 599-608.
• Gueinzius K., Wittke V., Magenau A., Ferrando-May E., Urbich C., Dimmeler S., and Hermann C.. Endothelial cells are protected against phagocyte-transmitted Chlamydophila pneumoniae infections by laminar shear stress (submitted).
The following papers that are parts of this thesis are in preparation:
• Gueinzius K., Hamann L., Schumann R. R., and Hermann C.
The NOD 3020insC polymorphism enhances cytokine induction by Chlamydophila pneumoniae and represents a risk factor for atherosclerosis (manuscript in preparation).
• Gueinzius K., Dehus O., and Hermann C.
Immune modulation by Chlamydophila pneumoniae - a mechanism of persistence?
(manuscript in preparation).
• Gueinzius K., Morath S., Maass M., and Hermann C.
Isolation of the immune active components of Chlamydophila pneumoniae
Further contributions to publications:
• von Aulock S., Schröder N. W. J., Gueinzius K., Traub S., Hoffmann S., Graf K., Dimmeler S., Hartung T., Schumann R. R., and Hermann C.. 2003. Heterozygous Toll-like receptor 4 polymorphism does not influence lipopolysaccharide-induced cytokine release in human whole blood. J Infect Dis 188: 938-943.
• von Aulock S., Schröder N. W. J., Traub S, Gueinzius K., Lorenz E., Hartung T., Schumann R. R., and Hermann C.. 2004. Heterozygous Toll-Like receptor 2 polymorphism does not affect lipoteichoic acid-induced chemokine and inflammatory responses. Infect Immun 72: 1828-1831.
• von Aulock S., Rupp J., Gueinzius K., Maass M., and Hermann C.. 2005. Critical investigation of the CD14 promoter polymorphism: Lack of a role for in vitro cytokine response and membrane CD14 expression. Clin Diagn Lab Immunol 12: 1254- 1256.
• von Aulock S., Deininger S., Draing C, Gueinzius K., Dehus O., Wittke V., and Hermann C.. Gender difference in cellular cytokine induction capacity upon stimulation with lipopolysaccharide or lipoteichoic acid (submitted).
Acknowledgement
The work presented in this thesis was carried out between January 2003 and April 2006 at the Chair of Biochemical Pharmacology at the University of Konstanz under the supervision of Prof. Dr. Dr. Thomas Hartung and Dr. Corinna Hermann.
I am grateful to Prof. Dr. Dr. Thomas Hartung for entrusting me with this project, for his support, motivation and confidence, for providing the excellent facilities and for enabling me to attend conferences.
Special thanks go to my supervisor Dr. Corinna Hermann for innovative guidance, invaluable advice, her enthusiasm and for her friendship.
I thank Prof. Dr. Albrecht Wendel for welcoming me into the group; his continuous encouragement and interest is greatly appreciated.
I am indepted to the “Frauenförderung” and the “Center for Junior Research Fellows” of the University of Konstanz for the financial support of this thesis.
I especially thank my lab colleagues Dr. Sonja von Aulock, Sebastian Bunk, Mardas Daneshian, Oliver Dehus, Susanne Deininger and Christian Draing for stimulating discussions and mental support, Dr. Siegfried Morath for introducing me into the field of preperative chemistry and Shahjahan Shaid for his assistance and for always making me smile. I thank the skilful team of technicians Leonardo Cobianchi, Annette Haas, Margarete Kreuer-Ullmann and Tamara Rupp for keeping the lab running and Gudrun Kugler for her organizational input. I would like to thank all co-authors for their valuable contributions to this work. Sincere thanks are given to all my current and former colleagues of the “Lehrstuhl Wendel” for creating a great working atmosphere and for an unforgettable time in- and outside the lab.
Finally, I want to thank my family Elsi, Klaus and Gunnar Gueinzius for their perpetual support and Thilo for delicious catering especially during my busy times, his overwhelming faith in me and for always being at my side.
Table of Contents
1 Introduction ... 1
1.1 Chlamydiaceae... 1
1.2 Immune recognition... 2
1.3 Atherosclerosis ... 5
2 Aims of the study ... 9
3 Toll-like receptors 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for the release of monokines... 10
3.1 Abstract... 10
3.2 Introduction ... 11
3.3 Materials and Methods... 12
3.4 Results ... 16
3.5 Discussion... 21
3.6 Acknowledgements... 23
4 Investigation of immune modulation by Chlamydophila pneumoniae and characterization of its immune stimulatory principle ... 24
4.1 Introduction ... 24
4.2 Materials and Methods... 26
4.3 Results and Discussion... 32
5 Comparison of quantitative and semiquantitative enzyme-linked immunosorbent assays for immunoglobulin G against Chlamydophila pneumoniae to a microimmunofluorescence test for use with patients with respiratory tract infections ... 46
5.1 Abstract... 46
5.2 Introduction ... 47
5.3 Materials and Methods... 48
5.4 Results ... 49
5.5 Discussion... 52
5.6 Acknowledgements... 54
6 Endothelial cells are protected against phagocyte-transmitted Chlamydophila pneumoniae infections by laminar shear stress ... 55
6.1 Abstract... 55
6.2 Introduction ... 56
6.3 Materials and Methods... 57
6.4 Results ... 60
6.5 Discussion... 64
6.6 Acknowledgement... 66
7 Summarizing Discussion... 67
8 Summary... 74
9 Zusammenfassung... 76
10 References... 78
1 Introduction
1.1 Chlamydiaceae
Chlamydophila pneumoniae represents the most frequent bacterial infection worldwide. It belongs together with Chlamydia trachomatis and Chlamydophila psittaci to the human pathogenic species of the family Chlamydiaceae. C. trachomatis is an exclusively human pathogen and was identified as the cause of trachoma, an acute eye infection often leading to blindness, in the 1940s. Since then, C. trachomatis has been recognized as a major cause of sexually transmitted and perinatal infections (77, 241). C. psittaci infects many mammalian and avian species, but only the bird infecting strains can occasionally also be transmitted to humans, causing severe pneumonia and a systemic illness known as psittacosis (90).
C. pneumoniae was initially isolated from a child’s conjunctiva during a trachoma vaccine trial in Taiwan in 1965 (89) and classified as a new species of the genus Chlamydia (now Chlamydophila) in 1989 (67, 88). C. pneumoniae is ubiquitous and the prevalence of antibodies increases markedly from the age of five, where the first infections and seroconversions occur, to 50% by the age of 20, and to 75% seropositives in the elderly (271). The pathogen is spread via airosol droplets through close personal contact and leads mainly to mild infections of the upper respiratory tract (100). It accounts for 10% of community acquired pneumonia and 5% of pharyngitis, bronchitis and sinusitis (149), but most infections have an asymptomatic course (86). Nevertheless, persistent C.
pneumoniae infections are supposed to contribute to the development of chronic inflammatory diseases like asthma (97, 130), neurological disorders (250, 281) and most prominently atherosclerosis (13, 160, 238). All members of the family Chlamydiaceae are Gram-negative, obligate intracellular parasites, which lack own ATP synthesis (147). It is believed, that the chlamydial ancestors first entered their eukaryotic host cells 2000 million years ago just at the same time point when eukaryotes and mitochondrial ancestors established symbiosis (59, 145).
The life cycle of Chlamydiaceae is marked by an unique biphasic developmental cycle with morphologically distinct infectious and reproductive forms, i.e. elementary (EB) and reticulate bodies (RB). The EB is the electron-dense, infectious form with no metabolic activity, typically 0.2-0.4 µm in diameter (99). EBs can be considered as environmentally resistant structures, adapted to survive in extracellular space and to transmit the infection
to new hosts. Having crossed the hosts mechanical barriers, the EBs attach to the host cells and are taken up via endocytosis. The precise mechanism of uptake, as well as the molecules and receptors required for an effective attachment remain to be elucidated (150, 219, 276). Inside the host cells, EBs remain in membrane-bound vacuolar ‘inclusions’, which are resistant to lysosomal fusion (8). Within the inclusions the EBs transform into RBs, which are metabolically active and can therefore be treated with membrane permeable antibiotics like macrolides (152, 153). RBs are up to 1.5 µm in diameter, take up nutrients from the host cell like the essential amino acid tryptophan, which is required for full chlamydial development and undergo multiple rounds of binary fission (27, 102).
Two to three days after host cell infection, the RBs differentiate back into EBs, which are released through cytolysis or by a process of exocytosis or extrusion of the whole inclusion leaving the host cell intact (99). In vitro, after antibiotic or IFNγ treatment, or after depletion of tryptophan, the RBs can differentiate into aberrant forms, which are larger in size and which show reduced metabolic activity and no replication (116). These aberrant forms are considered as intracellular state of persistence (12). This intracellular persistent state is assumed to occur also in vivo, in lungs or blood vessels, causing an essentially silent, chronic infection (101, 246). During co-evolution only a few exceptional bacteria were capable to establish persistent infections, which entails circumventing host defence mechanisms (57, 92, 127, 280).
1.2 Immune recognition
Facing the continuous daily invasion of microorganisms, the human organism requires sophisticated and efficient defence mechanisms to protect from infections. In this context, the initial recognition of invading bacteria by the innate immune system is crucial for the subsequent induction of inflammatory responses, activation of the adaptive immune system and elimination of the pathogen.
1.2.1 Pattern associated molecular patterns
The innate immune system is designed to recognize highly conserved motifs unique to microorganisms that are not associated with human cells. These motifs are termed
“pathogen associated molecular patterns” (PAMPs). The main immune stimulatory
lipopolysaccharide (LPS) (189, 224) and lipoteichoic acid (LTA) (111, 185), respectively.
Other parts of the bacterial cell wall like peptidoglycan (PGN) and its breakdown products the muropeptides as well as lipoproteins (114, 254, 255, 259, 279), heat shock proteins, especially heat shock protein 60 kD (hsp60) (270) and bacterial DNA (143), also represent potent stimuli for activation of the innate immune system.
The basic structure of LPS of Gram-negative bacteria consists of a polysaccharide moiety attached to a lipid anchor, named lipid A (226). The polysaccharide moiety is made up of a core and a polymer of repetitive oligosaccharide molecules, called O-antigen. The O- antigens are variable among a bacterial genus and often also between bacterial strains and determine the serological specificity. The part of the LPS molecule, which has been shown to be responsible for cytokine induction, is the lipid A, which is highly conserved, but variations in the amount and design of fatty acids occur between species (37, 72, 225, 256). The counterpart to LPS in Gram-positive bacteria is the LTA, which is anchored in the cytosolic membrane and reaches through the murein sacculus (68). LTA is made up of a lipophilic glycolipid anchor with typically two fatty acids linked to a hydrophilic glycerophosphate backbone, which is substituted with D-alanine and N-acetyl- glucosamine (186). It has been shown that for biological activity like cytokine induction, the LTA anchor is indispensable, but the backbone with its substitutions is further increasing potency (54, 187).
1.2.2 Pattern recognition receptors
Innate immune cells, especially monocytes, are well equipped with pattern recognition receptors (PRR), which enable them to recognize different PAMPs. The most prominent PRR are the Toll-like receptors (TLR) (2, 4, 159) and the recently identified NOD (nucleotide-binding oligomerisation domain) proteins (39, 83).
NOD proteins, including NOD1 and NOD2, are cytosolic proteins that have been initially thought to recognize LPS (125), but it has been shown recently that they enable intracellular recognition of muropeptides, which are usually co-extracted with LPS during purification (40, 80). NOD1 and NOD2 consist of three distinct domains, a C-terminal one with a leucine-rich repeat (LRR), which is critical for ligand recognition, a central nucleotide binding site which mediates oligomerization and one or two N-terminal caspase recruiting domains (CARD), which are involved in regulation of apoptosis and activation of inflammatory responses (122, 212). NOD1 is expressed in virtually all tissues (123) and
recognizes muropeptides that contain meso-diaminopimelic acid (40), a component of PGN. Meso-diaminopimelic acid is common for Gram-negative bacteria but also occurs in a few Gram-positive bacteria. NOD2, which is expressed only in monocytes (94) and Paneth cells (155), is a PRR for muramyl dipeptide (MDP) (81), a major constituent and typical breakdown product of PGN, which is present in almost all bacterial cell walls. Only recently, a frame shift mutation in the NOD2 gene 3020insC, which leads to the truncation of the terminal LRR and thus to a protein which cannot detect MDP, has been associated with the occurrence of Crohn’s disease suggesting a major role of NOD2 in intestinal immunity (81, 126).
To date, ten members of the TLR family have been identified in humans. TLR are transmembrane receptors, expressed primarily on leukocytes but also on some other cells such as endothelial or epithelial cells. While most of the TLR are expressed on the cell surface, TLR3 and TLR9 are located intracellularly (156). The individual TLRs recognize distinct structural components of pathogens and their stimulation directly results in signaltransduction, induction of gene expression and inflammatory responses (3). TLR3 has been shown to recognize dsRNA of viruses (7), whereas TLR9 recognizes unmethylated CpG motifs of bacterial as well as viral DNA (108). The prototypical LPS from enterobacteria is recognized by TLR4 together with MD-2 and the glycosylphosphatidylinositol anchored CD14 (214, 251), while in general, TLR2 mediates responses to LTA (157), lipoproteins (188) and PGN (61). However, some LPS exist, which are, in comparison to the classical LPS from enterobacteria, characterised by differences in the lipid A moiety, like that from Bacteroides fragilis, Leptospira interrogans or Porphyromonas gingivalis that are known to be relatively weak immune stimuli and to act via TLR2 (64, 163). Nevertheless, it is currently discussed, whether these initial findings were due to contaminating lipoproteins, since more recent work also reports TLR4 dependence of these LPS (50, 53). Furthermore, for immune activation by hsp60, which has been reported to depend on TLR2 and TLR4 (252, 264), recent evidence suggests that some of the reported cytokine effects may be due to contaminating LPS (262).
1.2.3 Immune recognition of Chlamydiaceae
All chlamydial species are Gram-negative and are supposed to express a family-specific LPS epitope. So far, the LPS of C. trachomatis and C. psittaci have been isolated, but only in the case of C. trachomatis the LPS structure has been analyzed (106, 233). The
chlamydial Lipid A is highly hydrophobic due to the presence of unusual, long-chain fatty acids (142, 220) and its immune stimulatory potential is 100- to 1000-fold weaker compared to LPS from classical enterobacteria (106, 121). The TLR-dependence of C.
trachomatis LPS has not been clarified so far, since it was first described to be TLR4 dependent (216), but a recent study by Erridge et al. demonstrated that it is TLR2 dependent (64). C. trachomatis and C. pneumoniae are closely related bacteria and it is assumed that the structure of the O-antigen is comparable between the species, but no information on the lipid A structure is available so far. To date, the role of the chlamydial LPS in chlamydial infections is not clear, since both TLR2 and TLR4 are necessary for full immune activation in response to C. pneumoniae and C. trachomatis (47, 51, 195, 216, 217, 283). In addition, chsp60 has been suggested to be a potent inducer of inflammatory responses in endothelial cells and macrophages and a stimulator of vascular smooth muscle cell proliferation via TLR4 (25, 240). Moreover, there are also hints for TLR independent target cell activation (198), suggesting the existence of additional receptors.
Since C. pneumoniae are obligate intracellular bacteria, the cytoplasmatic NOD proteins, which are implicated in intracellular pattern recognition (35, 124), qualify as candidates for the recognition of C. pneumoniae. Only recently, it was shown that NOD1 plays a dominant role in C. pneumoniae-induced interleukin (IL)-8 release in endothelial cells (203). However, it is not clear, which structure of C. pneumoniae may interact with the NOD proteins. NOD proteins have so far been associated with recognition of different types of PGN building blocks (40, 80, 81). Although recent studies suggest a functional PGN pathway in Chlamydiaceae (112, 173), a clear cut biochemical evidence for the chlamydial PGN-synthesis or presence of PGN in Chlamydiaceae is missing (41, 70).
However, Chlamydiaceae are sensitive to antibiotics that inhibit PGN-synthesis (191). This phenomenon has been referred to as the “chlamydial PGN paradox”.
1.3 Atherosclerosis
Persistent infections with C. pneumoniae are discussed as risk factors for the pathogenesis of numerous chronic inflammatory diseases, initially not thought to have an infectious origin, notably atherosclerosis (30, 161).
1.3.1 Pathogenesis of atherosclerosis
Atherosclerosis and its complications are the leading cause of death in the Western world (174). The development of atherosclerotic lesions, which can be already detected in childhood, is marked by the accumulation of low-density lipoproteins (LDL) in so-called
“fatty streaks”. The endothelial vasodilator dysfunction with an imbalance of NO and O2 is thereby considered to be the first detectable alteration of the vessel wall (282). The mature atherosclerotic lesion, the atheroma, is an inflammatory site composed of a lipid-rich necrotic core, modified vascular endothelium, extensively proliferated smooth muscle cells, foamy monocytes/macrophages, lymphocytes, cholesterol and a variety of inflammatory mediators. Although advanced lesions can impede blood flow, myocardial infarctions and strokes result from an acute occlusion that can be due for example to the formation of a thrombus, which can form in response to rupture or erosion of the plaques (230). A variety of risk factors are known to contribute to the development of atherosclerosis. These include genetic and life style factors such as elevated LDL cholesterol levels, high blood pressure, stress, smoking and obesity (274). Interestingly, atherosclerotic lesions show a focal distribution independent of any of these risk factors and occur exclusively at vessel curves and branches, the so called “lesion-prone-regions”. It is supposed that changes in the laminar blood flow (shear stress), which can be reduced, absent or turbulent at sites like curves and branches, make the vessel more susceptible to minimal trauma, which is regarded as one of the first steps in atherogenesis (36, 42). Atherosclerosis is, according to the “response to injury”-theory of Russel Ross, considered as an inflammatory disease (230). Infections represent the most potent stimulus of inflammation and a role of infectious agents in atherogenesis has been discussed for decades (49, 65, 215).
Currently, infections with C. pneumoniae are in the focus of these discussions (161).
1.3.2 Association of Chlamydophila pneumoniae and atherosclerosis
The first hint of a link between C. pneumoniae infection and atherosclerosis came from a serological study of Pekka Saikku and coworkers in Finland in 1988 (237), where a statistically significant correlation between C. pneumoniae antibody levels and the occurrence of acute myocardial infarction was reported. This first study was followed by many others during the last 15 years. However, the results of the seroepidemiological studies were highly controversial. About half of the published results confirmed the initial finding of Saikku et. al., while several others failed (239, 258, 273). Only recently, the validity of serological assays for serodiagnosis has been identified as a
crucial factor. The current gold standard in C. pneumoniae serodiagnosis is the microimmunofluorescence test (MIF), which is a time-consuming method, only reliable if performed by an experienced operator. Multicenter trials have shown large interlaboratory variations (206). Furthermore, differences in antigen preparation and the lack of standardisation result in highly variable assay sensitivities and specificities (109, 117, 169). However, in the early nineties, C. pneumoniae were directly detected in vascular tissue and since then, it has frequently been found in diseased blood vessels by electron microscopy, PCR and immunohistochemistry (146, 148, 162, 245). Notably, C.
pneumoniae has never been detected in healthy arteries (84, 146). It also became clear, that the direct detection results correlate very poorly with serology (167). Furthermore, three independent groups successfully cultured C. pneumoniae, which were isolated from atheromas (128, 164, 221). The most convincing results that C. pneumoniae infection contributes to the development of atherosclerosis came from animal models. In several studies using hyperlipidaemic mouse strains (29, 118) or New Zealand white rabbits (69, 154, 193), intranasal infection with C. pneumoniae accelerated plaque development and organisms could be recovered from lesions. In vitro, all cells which are implicated in the pathogenesis of atherosclerosis like monocytes/macrophages, endothelial cells and smooth muscle cells can be infected by C. pneumoniae, leading to cell activation, i.e.
expression of adhesion molecules and release of pro-inflammatory cytokines (73, 222).
Taken together, there is considerable evidence that live C. pneumoniae are present in atheromatous tissue, even if the presence does not correlate with serology. Furthermore, today it can hardly be proven by serodiagnosis if a chronic or persistent infection has established, since reliable diagnostic markers are lacking. In addition, it is not clear how and when C. pneumoniae actually gain access to the vasculature and which role they might play in the pathogenesis of atherosclerosis.
1.3.3 Possible contribution of Chlamydophila pneumoniae to atherosclerosis
A key challenge is now to understand at a molecular level how C. pneumoniae may contribute to the pathogenesis of atherosclerosis. First, C. pneumoniae may cause the initial injury, inducing the atherosclerotic process. Second, the organism may accelerate the progression of pre-existing disease. Third, it may contribute to a disease complication, such as plaque rupture and myocardial infarction. Finally, it cannot be excluded that they might simply be an innocent bystander.
A proposed model of the pathogenesis has been described as follows (133). The pathogen probably accesses the vasculature during local infection of the respiratory tract. Since free C. pneumoniae EB have not been detected in the circulation so far, infected leucocytes may serve as vectors to disseminate the infection from the lung to other susceptible tissues including arteries (17, 19, 182). C. pneumoniae are capable to infect monocytes, but the ability of the organism to replicate in matured human macrophages is very limited (1, 275). A recent publication by Ger van Zandbergen and co-workers also suggests polymorphonuclear neutrophils (PMN) as a vector system for systemic distribution of C.
pneumoniae (265). PMN are potent phagocytes abundant in the blood, which are quickly recruited to the original site of infection. Once C. pneumoniae have been delivered to the vasculature, they might induce the expression of adhesion molecules and the production of inflammatory cytokines in vascular cells. For example, infected endothelial cells augment the expression of adhesion molecules that may promote leucocyte adherence, migration and intimal inflammation (136, 184). Smooth muscle cells respond to infected endothelial cells with proliferation (43, 249) and with the release of cytokines such as IL-6 which may alter atheroma biology to direct infection (178). Furthermore, the release of TNF, IL-1 and IL-6 by infected macrophages may also promote lesion progression (199).
Two other key events in atherogenesis are the transformation of macrophages into fat- laden foam cells after uptake of LDL and the oxidation of lipoproteins at the site of lesion development, resulting in tissue damage (230). It has been shown in vitro, that chlamydial LPS is a major trigger of LDL uptake (131). Furthermore, it was shown that monocyte- mediated LDL oxidation is enhanced in the presence of chsp60, which is abundantly expressed by persistent Chlamydia and which has been also identified within human atheromatous tissue (12, 132). Beside LDL oxidation, chsp60 also promotes the expression of matrix metalloproteinases by macrophages, which may weaken atherosclerotic plaques so that they rupture more readily and myocardial infarctions can occur (141).
In conclusion, viable C. pneumoniae, which are actually present in the atheroma, are unlikely to be simply harmless bystanders, but it is still not clear at which stage of atherogenesis they arise first and for which complications they can be truly taken into account.
2 Aims of the study
The respiratory pathogen Chlamydophila pneumoniae occurs world-wide with a seroprevalence of ≥70%. Beside acute infections, the association of intracellular persisting C. pneumoniae with various chronic inflammatory diseases, in particular atherosclerosis, has raised most interest. To understand the mechanisms of persistence, immune activation and inflammation induced by C. pneumoniae, as well as its respective avoidance strategies need to be clarified.
The aim of the first part of the thesis was to investigate the involvement of different pattern recognition receptors as well as immune modulatory activities by C. pneumoniae and to isolate and characterize its immune stimulatory principle.
• To investigate the role of TLR2 and TLR4 in vivo, a murine model was established reflecting the natural course of infection. For this purpose, the infection was monitored by serology and by determination of the bacterial burden by PCR.
• Beside TLR2 and TLR4, the intracellular NOD proteins qualify as recognition receptors for C. pneumoniae. Therefore, the impact of different polymorphisms in the NOD2 gene on C. pneumoniae-induced cytokine release was examined.
• The immune modulatory potential of C. pneumoniae was further elucidated in comparison to Gram-negative bacteria and to classical LPS from enterobacteria.
• The isolation of immune active structures of C. pneumoniae has been carried out by butanol extraction and chromatography.
C. pneumoniae can be found in atherosclerotic lesions, but not in healthy tissue. Reliable serodiagnostic assays which are easy to perform and to analyze, are a prerequisite for seroepidemiological studies to prove the association with atherosclerosis. The aim of the second part of this thesis was to evaluate the sensitivity and specificity of new semi- quantitative ELISAs and to investigate possible routes of dissemination from the lung to the vessel wall:
• The reliability of assays for C. pneumoniae serodiagnosis was evaluated in comparison to the gold standard MIF.
• It was investigated whether PMN might qualify as carriers for C. pneumoniae, transmitting the infection from the lung to endothelial cells. In addition, the protective effect of shear stress on prevention of endothelial cell infection was investigated.
3 Toll-like receptors 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for the
release of monokines
Markus Mueller, Stefan Postius*, Jean G. Thimm*, Katja Gueinzius, Inge Muehldorfer*, and Corinna Hermann
Biochemical Pharmacology, University of Konstanz, Germany
*ALTANA Pharma AG, Konstanz, Germany
Immunobiology (2004); 209: 599-608
3.1 Abstract
Activation of immune cells by Chlamydophila pneumoniae in vitro has been shown to be TLR2-dependent, but TLR4 is also involved to a minor extent. To investigate the role of TLR2 and TLR4 in vivo, a murine model of C. pneumoniae infection was established. Mice were infected intranasally with a low inoculum of 106 C. pneumoniae elementary bodies (EB) and spreading of bacteria was monitored by real-time PCR. The bronchoalveolar lavage (BAL) showed maximal bacterial load on the day of infection and the lung two days later. By day 95, C. pneumoniae were eradicated completely. In serum, anti-C.
pneumoniae IgG became detectable on day 18 by microimmunofluorescence test. The course of infection was mild with no apparent symptoms, lack of acute phase response and no induction of TNFα and IL-6 in BAL, lung supernatants or blood. Infection of TLR2-/- and C3H/HeJ mice revealed no differences in clearance of bacteria and serological responses compared to wild-type controls, even if a dose of 107 EB was used. Intracellular replication of C. pneumoniae in the lungs was proven by the efficacy of antibiotic treatment. These findings indicate that in vivo TLR2 and TLR4 are not important for the development of antibodies and elimination of C. pneumoniae.
3.2 Introduction
C. pneumoniae represents an emerging Gram-negative pathogen identified only 14 years ago (88), which occurs world-wide with a seroprevalence of 70% in the adult population (86, 158, 236). It is assumed that C. pneumoniae account for 10% of community acquired pneumonia (6, 85, 98), but most infections are asymptomatic (86, 96) and therefore many people are not aware they have been infected by C. pneumoniae. Due to the obligate intracellular localization of C. pneumonia, they are difficult to treat with antibiotics and often persist in the host organism (78, 96, 152, 153).
During infection, the cells of the innate immune system represent the first barrier against invading pathogens. Primarily conserved bacterial structures, the so called pathogen associated molecular patterns are recognized by specific receptors present on immune cells. The family of toll-like receptors (TLR) has been identified as key receptors involved in pathogen recognition and has been intensively investigated and characterized during the last years(2, 159). TLR4 mediates immune responses to Gram-negative bacteria initiated via recognition of their lipopolysaccharide (LPS), a major constituent of the outer Gram-negative membrane (15, 16, 267). In contrast, TLR2 has been shown to be activated by compounds of Gram-positive bacteria like lipoteichoic acid (157), peptidoglycan (243) and lipoproteins from borrelia (114), treponema (204) and mycoplasma (71).
C. pneumoniae represents a Gram-negative pathogen and carries a LPS; therefore a role of TLR4 in alerting immune responses might be assumed. However, in vitro studies have demonstrated a role of TLR2 in recognition of C. pneumoniae (44, 197, 217), but TLR4 also seems to be involved to some extent (25, 44, 217, 240). Most in vivo models investigate the association of C. pneumoniae with atherosclerosis (28, 31, 118, 172, 180, 181). Only a few reports address immune defence mechanisms. Here, an important role of CD8+T-cells and IFNγ-dependent reactions for protection against C. pneumoniae has been shown (207f, 208, 231, 232, 269). However, so far the role of the TLRs has not been investigated in vivo. Therefore, we established a murine model reflecting the natural course of infection together with monitoring methods for the determination of antibody titers and a highly sensitive, quantitative real-time PCR for the detection of C. pneumoniae burden. The role of TLR2 and TLR4 was investigated with TLR2-deficient (TLR2-/-) and C3H/HeJ mice, which express a non-functional TLR4 receptor.
3.3 Materials and Methods
3.3.1 Chlamydophila pneumoniae propagation and isolation
Since C. pneumoniae can infect humans via aerosols, C. pneumoniae propagation as well as all following studies were carried out in safety cabinets and using personal air filters. C.
pneumoniae HK isolate (a clinical respiratory isolate, generously provided by Prof. E.
Straube, National Consultative Laboratory for Chlamydia, Institute of Medical Microbiology, University of Jena, Germany) was used in this study. Cultivation of C. pneumoniae was performed by intracellular growth of the bacteria in the human epithelial cell line HEp-2 (ATCC CCL-23) (227), as described by Rodel et al. (228). The multiplication was monitored using the Kallestad Pathfinder system (Sanofi Diagnostics Pasteur, Redmond, WA, USA). In order to exclude Mycoplasma contamination, cell cultures and chlamydial stocks were routinely tested by Mycoplasma PCR ELISA (Roche Diagnostics GmbH, Mannheim, Germany). The number of C. pneumoniae was quantified by real-time PCR (see below). For this purpose, C. pneumoniae infected HEp-2 cells were harvested, the cell debris was separated by low speed centrifugation (10 min x 500g) and DNA was prepared with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
3.3.2 Infection of mice and organ preparation
Four week old, specified pathogen-free female Swiss Webster mice, C3H/HeN mice, C3H/HeJ mice (Charles River Laboratories, Sulzfeld, Germany), TLR2 wild-type mice (TLR2+/+) and TLR2-/- mice, generated by homologous recombination by Deltagen (Menlo Park, CA, USA) and kindly provided by Tularik (South San Francisco, CA, USA) were fed ad libitum with Altromin 1314 (Altromin, Lage an der Lippe, Germany) and kept at 20-22°C, 50-60% air humidity and at a 12 hour day-night cycle. The genetic background of the TLR2 mice was B57BL/6. All TLR2 mice were bred in the animal facility of the University of Konstanz, Germany. They were inoculated intranasally with 106 or 107 EB of C.
pneumoniae HK under light ether anesthesia to induce hyperventilation. One drop (50 µl) of inoculum was delivered onto the nostrils.
3.3.3 Kinetics
At 15 min as well as on days two, four, 11, 18, 33 and 95 after infection, Swiss Webster mice (5 mice per group) were anesthetized (Trapanal i.p. (100 mg/kg), ALTANA Pharma, Konstanz, Germany), opened along the midline of the abdomen and the abdominal aorta was cannulized and perfused thoroughly for 1 min with approximately 5 ml sterile PBS (PAA Laboratories, Cölbe, Germany) with 1,6 mg/ml EDTA (Sarstedt, Nümbrecht, Germany) to remove blood from the organs. The blood was collected from the vena cava and the respective dilution was calculated by determination of the hemoglobin content (Hemoglobin test kit, Sigma, Munich, Germany). BAL fluids were obtained by transtracheal lavage technique. The BAL was immediately stored at -70°C. Samples of lung, aorta, brain, bladder, spleen and liver were obtained. All organs were weighed and homogenized (except in case of the lung where a specimen for histopathology was removed) in a sterile homogeniser (Medicon, 50 µM Dako Cytometation, Hamburg, Germany) using a Medimachine (Dako Cytometation) with 1 ml sterile ice cold PBS. After homogenization the tissue was filtrated through a 50 µM mesh Filcon sterile syringe (Dako Cytometation) to avoid clotting and stored at –70°C.
3.3.4 Antibiotic treatment
Swiss Webster mice (n=5) were inoculated intranasally with C. pneumoniae HK as described above. Antibiotic treatment was started two days after inoculation with C.
pneumoniae. A mixture of 20 mg x kg-1 rifampicin (Sigma) and 20 mg x kg-1 azithromycin (Pfizer, Karlsruhe, Germany) were administered by lavage thrice at 7 a.m., 1 p.m. and 7 p.m. for four days. All substances were dissolved in 1% tylose (Merck, Darmstadt, Germany) and applied in a volume of 25 ml x kg-1. Mice were killed at day seven after infection and serum, BAL and lung were obtained for further analysis.
3.3.5 Role of TLR
Swiss Webster mice, C3H/HeN mice, C3H/HeJ mice, TLR2+/+ and TLR2-/- mice (n=5) were inoculated intranasally with C. pneumoniae HK as described above. At day 2 or 18 after infection mice were killed and BAL and lung were obtained for further analysis.
3.3.6 Histopathology
Histopathology was performed blinded to the infection conditions at the laboratory of Dr. K.
Tuch (ALTANA Pharma, Hamburg, Germany). Lung tissue from infected and control mice was removed as described above and immediately fixed in 8% MOPS-buffered formalin, pH 7.4. Specimens were embedded in paraffin, sectioned and stained with hematoxylin and eosin.
3.3.7 DNA preparation
DNA extraction was carried out using the DNeasy Tissue Kit (Qiagen) according to the manufacturer’s protocol with slight modifications: 40 µl of proteinase K were incubated with 260 µl buffer ATL and 100 µl organ homogenate over-night at 55°C. Then 400 µg RNAse and 400 µl buffer AL were added and the sample was incubated at 70°C for 10 min. After addition of 400 µl 100% ethanol, 650 µl of the sample were loaded onto the column in two steps and centrifuged. After two wash steps the DNA was eluted using 2x 100 µl AE buffer. For extraction of DNA from BAL, 210 µl ATL buffer, 150 µl BAL and 10 µg carrier RNA (Qiagen), the latter of which was used to improve the DNA yield, were mixed and incubated for 3 h at 55°C. No RNAse digestion was performed and the DNA was eluted using 100 µl AE.
3.3.8 Real-time PCR
Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche).
Primer and probe sequences were selected from the 16S rRNA of C. pneumoniae (Accession no. U68426). The primers resulted in an amplification product of 640 bp.
Primer sequences were for the forward primer 5’-ATGTGGATGGTCTCAACCCCAT-3’ and for the reverse primer 5’-GGCGCCTCTCTCCTATAAATAGG-3’ (Thermo Hybaid/Interactiva Division, Ulm, Germany). Probe sequences were 5’- ACCTCACGGCACGAGCTGACGA-3’ for the fluorescein labelled probe and 5’-AGCCATGCAGCACCTGTGTATCTGTCC-3’ for the LC Red640 labelled probe (TIB MOLBIOL Syntheselabor, Berlin, Germany). Real-time PCR was done in 20 µl with 0.5 µM of each primer, 0.1 µM of each probe, 2 mM MgCl2, 2 µl FastStart DNA Master Hybridization Probes (Roche) and 5 µl DNA (40 ng) template. Thermal cycling was
cycles) with an annealing temperature of 65°C and an elongation time of 26 sec.
Fluorescence was measured at the end of each annealing phase.
Comparison of the selected primers (BLAST search) with the GenBank sequences (National Center for Biotechnology Information, NCBI, Microbial Genome Database, MBGD)(257) revealed no cross-binding activity. We also tested different bacterial strains including Salmonella typhimurium (ATCC 15277), Staphylococcus aureus (ATCC strain 12598), Escherichia coli (E. coli, K-12 strain JM-109, a kind gift from Dr. G. Grütz, Charité, Berlin, Germany), Borrelia burgdorferi strain sensu stricto (a kind gift from R. Oehme, LGA Stuttgart, Germany), Eubacterium acidaminophilum (DSM strain 3595), Methanospirillum hungatei (a kind gift from Prof. B. Schink, University of Konstanz, Germany), and samples of murine DNA prepared from BAL, blood, lung, aorta, brain, bladder, spleen and liver to exclude cross-reactivity in the assay. Furthermore, to control for false-negative and false- positive results, a positive and a negative control were included in each run. The amount of EB in the samples was determined using chlamydial DNA as standard. The concentration of the standard DNA was calculated on the basis that the chlamydial genome consists of 1.2 million base pairs weighing 1.3 fg.
3.3.9 Isolation and stimulation of murine bone marrow cells
C3H/HeN mice, C3H/HeJ mice, TLR2+/+ and TLR2-/- mice were put under terminal pentobarbital anaesthesia (Narcoren, Merial, Halbergmoos, Germany). Bone marrow cells were isolated from the femurs by rinsing with 10 ml ice-cold PBS and were transferred to siliconized glass tubes. After centrifugation, cells were resuspended in medium (RPMI 1640, PAA Laboratories) containing 10% FCS (Roche) and transferred to 96-well cell culture plates (5x 105 cells/well, Greiner, Frickenhausen, Germany). Murine bone marrow cells were then stimulated with C. pneumoniae, LPS from Salmonella abortus equi (Sigma) or LTA from Staphylococcus aureus (prepared in house according to Morath et al. (185)) and incubated for 24 h at 37°C and 5% CO2.
3.3.10 Serology
Serology was performed at the laboratory of Dr. U. Brunner (Konstanz, Germany). Serum antibodies were detected by MIF using glass slides with EBs from the C. pneumoniae isolate TWAR-183 (Labsystems, Helsinki, Finnland, purchased from Merlin, Bornheim, Germany). Murine Ig were detected with fluorescein-conjugated goat antibodies, anti- murine IgM was purchased from Biozol (Eching, Germany) and anti-murine IgG from Sifin
(Berlin, Germany). Titers of <1:8 were regarded as negative because of non-specific background reactions.
3.3.11 ELISA
The cytokines tumour necrosis factor α (TNFα), interleukin 6 (IL-6) and interleukin 10 (IL- 10) were determined in BAL, blood and supernatants of lung homogenates by ELISA based on commercial antibody pairs and standards (TNFα antibodies were purchased from R&D, Wiesbaden, Germany, IL-6 antibodies and IL-6 and TNFα standards from Pharmingen, Hamburg, Germany). IL-10 was measured by OPT-EIATM mouse IL-10 Set (BD Biosciences, San Diego, CA, USA). Serum amyloid A (SAA) was determined in blood using a kit from Biosource (Solingen, Germany). The detection limits of the ELISAs were 39 pg/ml, 13 pg/ml, 10 pg/ml and 120 ng/ml for TNFα, IL-6, IL-10 and SAA, respectively.
3.3.12 Statistics
Statistical analysis was performed using the GraphPad InStat program 3.0 (GraphPad Software, San Diego, USA). Differences between two groups were assessed by unpaired t test. Unpaired samples were assessed by one-way analysis of variance followed by Dunnett’s test. Data were log-transformed to achieve Gaussian distribution. In the figures
*, ** and *** represent p values <0.05, <0.01 and <0.001, respectively.
3.4 Results
Bone marrow derived cells from C3H/HeJ and TLR2-/- mice as well as from their respective wild-types were incubated in the presence of 5x106 EB of C. pneumoniae/ml and IL-6 release was measured. As shown in figure 1, cells from TLR2-/- mice were not able to respond to stimulation with C. pneumoniae, while cells from C3H/HeJ mice were responsive, but cytokine secretion was significantly reduced compared to C3H/HeN cells.
The responsiveness of the C3H/HeJ and of the TLR2-/- cells was confirmed by stimulation with 1 µg/ml LPS and 10 µg/ml LTA, respectively (data not shown). These in vitro data
TLR2 and TLR4 are involved. In order to investigate the role of TLR2 and TLR4 for pathogen recognition and for the course of infection in vivo, we established a murine C.
pneumoniae infection model.
Figure 1 C. pneumoniae inducible cytokine release by TLR-deficient murine bone marrow cells 5x105 bone marrow cells from TLR2+/+, TLR2-/-, C3H/HeN and C3H/HeJ mice were incubated in the presence of 5x106 C. pneumoniae per ml for 24 h. The release of IL-6 was determined in the cell-free supernatants by ELISA. ***represents significance versus wild-type cells. Data are means ± SEM.
To reflect the physiological situation, where most C. pneumoniae infections are asymptomatic, mice were infected intranasally with a very low infectious inoculum of 106 EB of C. pneumoniae (corresponding to 104-105 inclusion forming units) and the course of infection was monitored by real time PCR for 95 days. At 15 min and on days two, four, 11, 18, 33 and 95 of infection the C. pneumoniae load of lung and BAL was determined by real-time PCR, the lung was analyzed by histopathology and the cytokine levels of TNFα, IL-6 and IL-10 were determined in BAL and lung supernatants, as well as TNFα, IL-6, IL- 10 and the acute phase protein SAA in blood. Furthermore, the development of anti-C.
pneumoniae antibodies was monitored by MIF in the serum of the mice. The MIF analysis revealed that the animals produced anti-C. pneumoniae antibodies of the IgM and later the IgG class, indicating a specific immune response (figure 2). IgM was detectable in two animals on day 11 and three animals on day 18, but had vanished in all by day 33.
Accordingly, IgG was detectable first on day 18 in four animals but increased further and was present in all five animals on day 33.
0 100 200 300 400 500 600 700 800 900 1000 1100 1200
TLR2 +/+
TLR2 -/-
C3H/HeN C3H/HeJ
C. pneumoniae 5x106/ml
n = 27 n = 33
***
***
IL-6 [pg/ml]±±±± SEM
Overall, no apparent symptoms of infection were visible. Indeed, neither the cytokines TNFα, IL-6 or IL-10 nor SAA were found at significant levels in serum during the whole course of infection. The levels of these cytokines in BAL and lung supernatants were also below the detection limit at all time points. Histopathology of lung slices revealed that there were no significant signs of interstitial pneumonitis or infiltration of polymorphonuclear leukocytes at any time point after infection. However, C. pneumoniae could be detected in the BAL and lung of all animals for 11 days, in three animals on day 18 and in two animals on day 33 by real-time PCR. Employing a quantitative real-time PCR, the highest numbers of C. pneumoniae were retrieved from the BAL 15 min after infection, which represents the initial inoculum, from the lung two days after infection. C. pneumoniae load then declined steadily, until by day 95, no more C. pneumoniae were detectable (figure 3 a+b).
Figure 3 Time course of C. pneumoniae burden in BAL and lungs of C. pneumoniae-infected mice a) BAL and b) lung were obtained from Swiss Webster mice at 15 min and on day two, four, 11, 18, 33 and 95 after infection with 106 C. pneumoniae EB. DNA was extracted and the bacterial load was determined by real-time PCR at each time-point. Bacterial load of BAL was significantly different (p ≤ 0.01) from non- infected controls at 15 min, on day two and day 11 after infection and for the lung at 15 min, on day two and day four. Bacterial load of the lung was significantly different from day two at day 11-95. Data are means ±
SEM of samples from five mice.
11 18 33
0 10 20 30 40 50 60 70
IgM IgG
days after infection
1/titer
Figure 2 Detection of anti-C.
pneumoniae IgM and IgG in C.
pneumoniae-infected mice
Serum was collected from Swiss Webster mice on day 11, 18 and 33 after infection with 106 C.
pneumoniae EB. IgM and IgG anti-C.
pneumoniae antibodies were determined by MIF. Data are means
± SEM of serum from five mice.
0 5 10 15 20 25 30 35
0 250 500 750 1000
90 95 100 50000
75000 100000
BAL
number of C.p./10 µl eluate
0 5 10 15 20 25 30 35
0 50 100 150 200 250 300 350
90 lung
number of C.p./40 ng DNA
a) b)
To examine the role of TLR2 and TLR4 for C. pneumoniae infection in vivo, C3H/HeJ, TLR2-/- as well as their respective wild-types were infected intranasally with 106 or 107 C.
pneumoniae EB and sacrificed at day 18. Swiss Webster mice served as positive controls for the C. pneumoniae infection. Day 18 was chosen since at this time-point bacteria were still detectable in the lung and BAL, but eradication had already started and effectiveness of eradication could be compared. Surprisingly, under both conditions, i.e. infection with 106 EB or 107 EB, the bacterial load of C. pneumoniae in the lung (figure 4a) or BAL (figure 4b) was not significantly different between the groups and the antibody levels were also comparable (figure 4c). It is thus concluded that eradication of C. pneumoniae takes place independent of recognition via TLR2 or TLR4. After infection with 107 EB a significant amount of IL-6 could be detected in the lung supernatants of all mice and a trend towards reduction of IL-6-release was observed in samples from the TLR2-/- compared to the TLR2+/+ mice. Since this difference was not statistically significant the experiment was repeated and the mice were sacrificed already at day two after infection. The bacterial burden of the lungs of all mice was again at the same level as confirmed by PCR.
HeN HeJ 2+/+ 2-/- SW 1
10 100 1000 BAL
Cp/10µl eluate
HeN HeJ 2+/+ 2-/- SW 1
10 100
Cp/40ng murine DNA lung
HeN HeJ 2+/+ 2-/- SW 1
10 100 1000
1/antibody titer
antibodies
Figure 4 Bacterial burden of BAL and lung and C. pneumoniae antibody titer of C. pneumoniae-infected mice a) BAL and b) lung were obtained from mice at day 18 after infection with 107 C.
pneumoniae EB. DNA was extracted and the bacterial load was determined by real-time PCR.
c) Serum was collected from mice at day 18 after infection with 107 C. pneumoniae EB. Anti-C. pneumoniae IgG antibodies were determined by MIF.
Data are medians plus individual values of samples from five mice.
a) b)
c)
Again, the IL-6 levels of the lung supernatants of the TLR2-/- mice showed lower IL-6 levels compared to TLR2+/+, while IL-6 levels were similar in C3H/HeN and HeJ mice (figure 5a).
Similar results were obtained for TNFα release (figure 5b). The levels of the anti- inflammatory cytokine IL-10 were not significantly different between wild-type and TLR- deficient mice (TLR2+/+: 424 ± 51 vs. TLR2-/-: 539 ± 55 pg IL-10/ml; C3H/HeN: 357 ± 75 vs.
C3H/HeJ: 377 ± 79 pg IL-10/ml).
Figure 5 Cytokine levels of lung supernatants of C. pneumoniae-infected mice
Supernatants of lung homogenates were obtained from mice at day two after infection with 107 C.
pneumoniae EB. IL-6 (a) and TNF (b) were determined by ELISA. Data are medians plus individual values of samples from five mice.
The lack of major symptoms further raised the question, whether C. pneumoniae had infected the murine tissue or were simply deposited and eliminated by time. To prove that C. pneumoniae actually infect and replicate intracellularly, we studied the effects of a previously reported effective combination of rifampicin and azithromycin (168). C.
pneumoniae are known to be sensitive to antibiotics only during replication. Treatment started at day two post infection and was carried out p.o. for four days three times daily.
On day seven, the bacterial burden was determined in BAL and lung (figure 6). Compared to untreated controls, bacterial numbers decreased significantly due to antibiotic treatment indicating that intracellular replication rendered them sensitive to antibiotics.
TLR2+/+ TLR2-/- C3H/HeN C3H/HeJ 0
20 40 60 80
TNF [pg/ml]
p= 0.05
TLR2+/+ TLR2-/- C3H/HeN C3H/HeJ 0.0
0.3 0.6 0.9 1.2
p= 0.05
IL-6 [ng/ml]
a) b)
untreated Rif./Az.
0 3 0 0 0 6 0 0 0 9 0 0 0 1 2 0 0 0 1 5 0 0 0 1 8 0 0 0
*
lu n g
number ofC.p./40 ng DNA
untreated Rif./Az.
0 3 0 0 0 6 0 0 0 9 0 0 0 1 2 0 0 0 1 5 0 0 0 1 8 0 0 0
* * B A L
number ofC.p./10 µl eluat
a
b
**
*
3.5 Discussion
In this study a murine model was established in order to investigate the role of TLR2 and TLR4 for C. pneumoniae infection in vivo. This was initiated with a very low infectious inoculum of 106 C. pneumoniae EB, i.e. 104-105 inclusion forming units, via the respiratory tract and the course of infection was monitored both by serology and by bacterial burden of organs. The mild or asymptomatic course of infection in our model appears to reflect the clinic of the human infection. The low potency of C. pneumoniae to elicit an inflammatory cytokine response represents a possible explanation for the often asymptomatic course.
Furthermore, in this model, C. pneumoniae infection in mice appears to occur only in the lungs, with no obvious persistence or spread of bacteria to other organs. Swiss Webster mice were chosen for the establishment of the model, because a homogeneous susceptibility towards intranasal C. pneumoniae infection with less individual variation compared to other mouse strains had been reported(278). The infection-kinetics, self- limitation of infection and development of antibodies as described in the present study were similar to what has been published by others(135, 207, 278). We found that the bacterial load of lung, BAL and antibody development at day 18 after infection was not significantly different between the Swiss Webster and the wild-type mice C3H/HeN and
Figure 6 Effect of antibiotic treatment on C. pneumoniae burden in lung and BAL of C. pneumoniae-infected mice
a) BAL and b) lung were obtained from mice at day seven after infection with C.
pneumoniae. In addition, the different groups of mice were either vehicle treated or received rifampicin/azithromycin. DNA was extracted and the bacterial load was determined by real-time PCR for each time- point. Data are means ± SEM of samples from five mice.
TLR2+/+. This similarity suggests that the different mice strains do not differ to major extent with regard to the time course of eradication of bacteria.
Surprisingly, the course of bacterial infection, bacterial clearance and development of antibodies was not altered in the TLR4- or TLR2-deficient mice compared to the respective wild-types. Mice with impaired TLR function tended to show even lower bacterial numbers, indicating that the clearance is at least not impaired. In contrast, the in vitro stimulated cytokine release was found to be strongly TLR2-dependent and in part TLR4-dependent, which is in line with a report by Prebeck et al. who show that in murine dendritic cells stimulated by C. pneumoniae both TLR2 and TLR4 play different roles for individual cytokines (217). However, in vivo cytokine release was found to be only TLR2-dependent, but cytokine levels were three-fold lower in C3H/HeN-J mice compared to TLR2 strains.
These findings raise the question whether TLR2 and TLR4 are decisive for innate immune recognition and initiation of specific immune responses towards C. pneumoniae in vivo.
There is increasing evidence that TLR2 and 4, which are expressed on cardiovascular cells and in the atherosclerotic plaque are involved in the initiation and the progression of the disease (52). It is tempting to speculate now, that C. pneumoniae, if present in the cardiovascular tissue, might contribute to inflammation by activating TLR2-expressing cells. We are not aware of previous studies investigating the role of TLRs in immune recognition of C. pneumoniae and no in vivo data are available so far. However, a recent study by Darville et al. investigated the role of TLR2 and 4 for inflammatory responses to C. trachomatis infection. In vitro, cells from TLR4-deficient mice showed enhanced cytokine release if infected with C. trachomatis, but during in vivo infection the mice behaved like wild-type mice. While, like in our case, TLR2 played a decisive role for cytokine release after in vitro and in vivo infection, TLR2-deficient mice displayed a similar time-course of infection and even less oviduct pathology than wild-type mice (51). This further supports the hypothesis that dysfunction of a given TLR, which is required for pathogen recognition in vitro, is not necessarily detrimental in vivo. Although several publications have shown that C3H/HeJ mice are highly susceptible to Gram-negative bacterial infections (23, 95, 214), an equal sensitivity has been shown for TLR4 wild-type and TLR4-deficient animals after intravenous or peritoneal infection with Escherichia coli (66, 105). Listeria monocytogenes induces inflammatory responses via TLR2, but mice deficient in TLR2 are not more susceptible than wild-type controls (62). These findings can most probably be explained by receptor redundancy (263), meaning that the induction of
