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