Interaction of Chlamydophila pneumoniae with the innate immune system

Abstract

Chlamydophila pneumoniae is an obligate intracellular pathogen which leads to mostly asymptomatic infections of the respiratory tract. After acute infection, C. pneumoniae can establish persistent infections in the host. To be able to prevent persistence, it is necessary to understand how C. pneumoniae are recognized by the innate immune cells and which evasion strategies are used to hide from the immune system. The association of atherosclerosis with persistent C. pneumoniae infections is discussed for decades now. Especially seroepidemiological studies lead to controversial results. In this respect, the validity of assays for serodiagnosis was identified as crucial key factor. Most convincing evidence comes from studies which report the presence of viable C. pneumoniae in the diseased vessel wall, while they were never detected in healthy tissue. However, the route of dissemination from the lung to the vasculature is still unclear. Currently, PMN are discussed as vectors for C. pneumoniae.To investigate the role of TLR2 and TLR4 in vivo, a murine model was established. Mice were infected intranasally with a low dose of 106 C. pneumoniae and the infection was monitored by serology and determination of the bacterial burden in the lung and BAL by quantitative real-time PCR. No differences in clearance of bacteria and serological response were observed between TLR2-/- and TLR4 deficient mice and their respective wild types, but cytokine release was diminished in TLR2-/- and in part inTLR4-deficient mice in vivo and in vitro.DNA samples of 160 healthy subjects were genotyped for the NOD2 3020insC frame shift mutation and 7.5% heterozygous polymorphism carriers were identified. Their C. pneumoniae-induced cytokine release from blood leukocytes was significantly higher compared to wild-types.The cytokine-inducing capacity of C. pneumoniae was shown to be weak in comparison to E. coli and enterococcal LPS and was characterized by a more pronounced release of anti-inflammatory mediators.The presence of an anti-CD14 antibody and the LPS-specific inhibitor LALF completely abrogated the release of pro-inflammatory cytokines by C. pneumoniae, while the anti-inflammatory IL-10 was not affected. Furthermore, prototypic LPS-induced pro-inflammatory cytokine release was impaired by co-incubation with C. pneumoniae, while again IL-10 remained unaffected.Isolation of immune active compounds of C. pneumoniae was carried out by butanol extraction. After separation by HIC a pool of immune active fractions with comparable cytokine inducing properties as whole C. pneumoniae was obtained. Chemical analysis revealed that the pooled fractions are still structurally inhomogeneous but contain a lipopeptide. Analysis by LAL confirmed that a low amount of LPS was present.The ELISAs, SeroCP and SeroCP Quant and the sELISA were evaluated for C. pneumoniae IgG serodiagnosis in comparison to the gold standard MIF. The concordance with MIF was ≥90% for all ELISAs for a collective of 80 patients and the PPVs were all ≥93%, while the NPVs ranged from 68-83%. False-negative results were obtained only from samples with low titers in MIF.Co-incubation of C. pneumoniae-infected PMN with endothelial cells (HUVEC) resulted in transmission of about 10% of the PMN-ingested C. pneumoniae to HUVEC and inclusions were formed. C. pneumoniae harvested from HUVEC retained infectivity since they were still able to multiply in HEp-2 cells. Exposure to laminar shear stress exhibits a protective effect on HUVEC and impedes formation of inclusions after C. pneumoniae infection by PMN.In summary, this thesis emphasizes the high complexity of the interactions of C. pneumoniae with the innate immune system. We have shown that at least three PRR, i.e. TLR2, TLR4 and NOD are engaged in immune recognition and subsequent induction of inflammatory responses induced by C. pneumoniae. Modulations of immune reactions are most probably triggered by the characteristic anti-inflammatory properties of C. pneumoniae which may lead to the asymptomatic course of infection and render them capable to escape host defence and to establish intracellular persistent infections. Moreover, instead of being eradicated after uptake by phagocytosis, C. pneumoniae stay viable and thus can use the phagocytes as shuttle vectors spreading the infection to the vasculature. Thereby, the prevention of vascular infection by laminar shear stress can explain the focal distribution of C. pneumoniae into areas prone to develop atherosclerotic lesions.This thesis elucidates risk factors, pathogenesis, immune activation and evasion as well as diagnostics of the most prominent infectious agent suspected to contribute to atherosclerosis. These aspects are crucial to finally settle the argument, whether myocardial infarction has in part to be considered an infectious disease.