Matthias Maass

Endovascular presence of viable Chlamydia pneumoniae is a common phenomenon in coronary artery disease.

OBJECTIVES We sought to examine coronary arteries for the presence of viable bacteria of the fastidious species Chlamydia pneumoniae. BACKGROUND The respiratory pathogen C. pneumoniae has been implicated in the pathogenesis of coronary artery disease (CAD). Previous studies have demonstrated an antichlamydial seroresponse to be a cardiovascular risk factor and coronary atheromata to contain chlamydial components in varying proportions. Endovascular demonstration of replicating bacteria is required to provide evidence for an infectious component in CAD and a rationale to discuss antimicrobial therapy. METHODS Myocardial revascularization was performed in 70 patients. Atherosclerotic lesions from 53 coronary endarterectomy and 17 restenotic bypass samples were cultured and subjected to nested polymerase chain reaction (PCR) for C. pneumoniae. Antichlamydial immunoglobulin G (IgG), IgA and IgM was examined by microimmunofluorescence. RESULTS Viable C. pneumoniae was recovered from 11 (16%) of 70 atheromata, and chlamydial deoxyribonucleic acid (DNA) was detected in 21 (30%) of 70 atheromata; 17 nonatherosclerotic control samples were PCR-negative (p < 0.01). Fifteen (28%) of 53 endarterectomy and 6 (35%) of 17 bypass samples were PCR-positive. DNA sequencing of six different PCR products did not reveal differences between coronary isolates and respiratory reference strains, suggesting that common respiratory strains gain access to the systemic circulation. Serologic results did not correlate with direct detection results and did not identify individual endovascular infection. CONCLUSIONS A significant proportion of atherosclerotic coronary arteries harbor viable C. pneumoniae. This finding supports the hypothesis of a chlamydial contribution to atherogenesis. Whether chlamydiae initiate atherosclerotic injury, facilitate its progression or colonize atheromata is unknown. However, the endovascular presence of viable bacteria justifies a controlled clinical investigation of antimicrobial treatment benefit in the therapy and prevention of CAD.

Chlamydia pneumoniae Infection in Circulating Human Monocytes Is Refractory to Antibiotic Treatment

BackgroundRecovery of the intracellular bacterium Chlamydia pneumoniae from atherosclerotic plaques has initiated large studies on antimicrobial therapy in coronary artery disease. The basic concept that antibiotic therapy may eliminate and prevent vascular infection was evaluated in vitro and in vivo by examining the antibiotic susceptibility of C pneumoniae in circulating human monocytes, which are thought to transport chlamydiae from the respiratory tract to the vascular wall. Methods and ResultsBlood monocytes (CD14+) from 2 healthy volunteers were obtained before and after oral treatment with azithromycin or rifampin and then inoculated with a vascular C pneumoniae strain and continuously cultured in the presence of the respective antibiotic. Progress of infection and chlamydial viability was assessed by immunogold-labeling and detection of C pneumoniae–specific mRNA transcripts. Circulating monocytes from patients undergoing treatment with experimental azithromycin for coronary artery disease were examined for C pneumoniae infection by cell culture. Antibiotics did not inhibit chlamydial growth within monocytes. Electron microscopy showed development of chlamydial inclusion bodies. Reverse transcription–polymerase chain reaction demonstrated continuous synthesis of chlamydial mRNA for 10 days without lysis of the monocytes. The in vivo presence of viable pathogen not eliminated by azithromycin was shown by cultural recovery of C pneumoniae from the circulating monocytes of 2 patients with coronary artery disease. ConclusionsC pneumoniae uses monocytes as a transport system for systemic dissemination and enters a persistent state not covered by an otherwise effective antichlamydial treatment. Prevention of vascular infection by antichlamydial treatment may be problematic: circulating monocytes carrying a pathogen with reduced antimicrobial susceptibility might initiate reinfection or promote atherosclerosis by the release of proinflammatory mediators.

Chlamydia pneumoniae Infection of Vascular Smooth Muscle and Endothelial Cells Activates NF-κB and Induces Tissue Factor and PAI-1 Expression A Potential Link to Accelerated Arteriosclerosis

BACKGROUND Recent reports link C. pneumoniae infection of arteriosclerotic lesions to the precipitation of acute coronary syndromes, which also feature tissue factor and plasminogen activator inhibitor 1 (PAI-1) overexpression. We investigated whether or not C. pneumoniae can induce thrombogenicity by upregulation of procoagulant proteins. METHODS AND RESULTS Human vascular endothelial and smooth muscle cells were infected with a strain of C. pneumoniae isolated from an arteriosclerotic coronary artery. Tissue factor, PAI-1, and interleukin-6 expression was increased in infected cells. Concomitantly, NF-kappaB was activated and IkappaBalpha degraded. p50/p65 heterodimers were identified as the components responsible for the NF-kappaB activity. CONCLUSIONS These data provide evidence that C. pneumoniae infection can induce procoagulant protein and proinflammatory cytokine expression. This cellular response is accompanied by activation of NF-kappaB. Our results demonstrate how C. pneumoniae infection may initiate acute coronary syndromes.

Nod1-Mediated Endothelial Cell Activation by Chlamydophila pneumoniae

Seroepidemiological and animal studies, as well as demonstration of viable bacteria in atherosclerotic plaques, have linked Chlamydophila pneumoniae infection to development of chronic vascular lesions and coronary heart disease. Inflammation and immune responses are dependent on host recognition of invading pathogens. The recently identified cytosolic Nod proteins are candidates for intracellular recognition of bacteria, such as the obligate intracellular chlamydia. In the present study, mechanisms of endothelial cell activation by C. pneumoniae via Nod proteins were examined. Viable, but not heat-inactivated, chlamydia activated human endothelial cells, suggesting that invasion of these cells is necessary for their profound activation. Endothelial cells express Nod1. Nod1 gene silencing by small interfering RNA reduced C pneumoniae–induced IL-8 release markedly. Moreover, in HEK293 cells, overexpressed Nod1 or Nod2 amplified the capacity of C pneumoniae to induce nuclear factor &kgr;B (NF-&kgr;B) activation. Interestingly, heat-inactivated bacteria were still able to induced a NF-&kgr;B reporter gene activity via Nod proteins when transfected intracellularly, but not when provided from the extracellular side. In contrast, TLR2 sensed extracellular heat-inactivated chlamydia. In conclusion, we demonstrated that C pneumoniae induced a Nod1-mediated and Nod2-mediated NF-&kgr;B activation in HEK293 cells. In endothelial cells, Nod1 played a dominant role in triggering a chlamydia-mediated inflammatory process.

Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens

ABSTRACT The reported rate of detection of Chlamydia pneumoniaeDNA within atherosclerotic lesions by PCR varies between 0 and 100%. In this study, identical sets of coded experimental atheroma samples (n = 15) and spiked controls (n = 5) were analyzed by 16 test methods in nine centers by means of PCR. The positive controls were correctly identified to levels of 1, 0.1, and 0.01 inclusion bodies of C. pneumoniae/ml of tissue homogenate by 16 (100%), 11 (69%), and 3 (19%) of the test methods, respectively. Three out of 16 negative controls (19%) were rated positive. Positivity rates for atheroma samples varied between 0 and 60% for the different test methods, with the maximum concordant result for positivity being only 25% for one carotid artery sample. There was no consistent pattern of positive results among the various laboratories, and there was no correlation between the detection rates and the sensitivity of the assay used.

Hydroxymethylglutaryl Coenzyme A Reductase Inhibitors Modify the Inflammatory Response of Human Macrophages and Endothelial Cells Infected With Chlamydia pneumoniae

BACKGROUND In patients with atherosclerosis, hepatic hydroxymethylglutaryl coenzyme A reductase (CSE) inhibitors may reduce the activation of inflammation. Because Chlamydia pneumoniae infection has been linked to coronary artery disease through the induction of plaque inflammation, we investigated whether cerivastatin affects the infection rate of human macrophages and endothelial cells (ECs) and their proinflammatory activation after chlamydial infection. METHODS AND RESULTS Macrophages were collected from the alveolar compartment of 6 volunteers and 10 patients with chronic bronchitis. ECs were obtained from 10 umbilical cords. The C. pneumoniae strain CWL was incubated with macrophages or ECs in the presence and absence of the CSE inhibitor cerivastatin. The infection rate was determined by immunofluorescence microscopy. The release of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and tumor necrosis factor (TNF)-alpha was quantified by ELISA. The release of oxygen radicals was determined by ferricytochrome assay. Infection rates were tendentially lower after the preincubation of macrophages with CSE inhibitors (17.2% versus 9. 3% and 18.2% versus 10.4%, respectively; P=NS). The secretion of MCP-1, IL-8, and TNF-alpha by infected macrophages from volunteers increased. Coincubation with cerivastatin resulted in significantly lower MCP-1 and IL-8 production, whereas the release of TNF-alpha remained unaffected. Similar effects regarding chemokine release were observed in ECs. CONCLUSIONS CSE inhibitors modify the inflammatory response of human immune cells to C. pneumoniae. This finding could be relevant for the therapeutic potential of CSE statins in patients with atherosclerosis and C. pneumoniae infection.

Chlamydia pneumoniae Multiply in Neutrophil Granulocytes and Delay Their Spontaneous Apoptosis

The obligate intracellular bacterial pathogen Chlamydia pneumoniae (Cp) is responsible for a range of human diseases, including acute respiratory infection. Although experimental intratracheal infection with Cp results in a massive recruitment of neutrophil granulocytes (polymorphonuclear neutrophils (PMN)), the role of these cells in the defense against Cp is unclear. In this study the interactions of PMN with Cp were investigated. In vitro coincubation experiments showed that human granulocytes were able to internalize Chlamydia in an opsonin-independent manner. Importantly, phagocytosed Cp were not killed; the ingested bacteria survived and multiplied within PMN. Although uninfected granulocytes became apoptotic within 10 h, infected PMN survived up to 90 h. Coincubation with Cp significantly decreased the ratio of apoptotic PMN, as detected by morphological analysis, annexin V, and TUNEL staining. The observed antiapoptotic effect was associated with a markedly lower level of procaspase-3 processing and, consequently, reduced caspase-3 activity in infected PMN. LPS was found as a major, but not exclusive, component responsible for the observed antiapoptotic effect. Chlamydia LPS affected PMN apoptosis both by acting directly on the cells and by inducing the autocrine production of the antiapoptotic cytokine IL-8. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, Cp can extend the life span of neutrophil granulocytes, making them suitable host cells for survival and multiplication within the first hours/days after infection.

Phagocytes transmit Chlamydia pneumoniae from the lungs to the vasculature.

Chlamydia pneumoniae, a major cause of community-acquired pneumonia, primarily infects the respiratory tract. Chronic infection of nonrespiratory sites, such as the vascular wall, the brain or blood monocytes, requires evasion from the lungs and spreading via the bloodstream. The cell types involved in dissemination are insufficiently characterised. In this study, New Zealand White rabbits were infected intratracheally with C. pneumoniae, and lung manifestation and systemic dissemination were monitored by polymerase chain reaction and immunohistochemistry. Infection of the lungs was characterised by an early phase dominated by granulocytes and a late phase dominated by alveolar macrophages (AM). Granulocytes, AM and alveolar epithelial cells acted as host cells for chlamydiae, which remained detectable for up to 8 weeks. AM transported the pathogen to the peribronchiolar lymphatic tissue, and subsequently C. pneumoniae entered the spleen and the aorta via dissemination by peripheral blood monocytes. In conclusion, Chlamydia pneumoniae-infected alveolar macrophages transmigrate through the mucosal barrier, and give the pathogen access to the lymphatic system and the systemic circulation. Infected peripheral blood monocytes are the vector system within the bloodstream and transmit the infection to the vascular wall. This is the first description of granulocytes acting as a reservoir for Chlamydia pneumoniae early in infection.

Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease

The common respiratory pathogen Chlamydia pneumoniae has been implicated in the pathogenesis of coronary artery disease and acute myocardial infarction. In order to verify the endovascular presence of potentially viable chlamydia by detection of genomic DNA, we examined atherosclerotic arteries from various vascular regions using a C. pneumoniae specific nested polymerase chain reaction (PCR). The samples were obtained during surgical revascularization procedures or at autopsy. Chlamydial DNA was detected in 51/238 (21%) atherosclerotic samples. A total of 17 non-atherosclerotic control samples were PCR-negative. Chlamydial presence was detected in 36/140 (26%) vascular samples obtained at coronary revascularization procedures, in 9/61 (15%) samples from carotid artery stenosis, 3/17 (18%) samples from the aorta, and 3/20 (15%) iliac artery samples. Histomorphological discrimination of infected and non-infected arterial samples was not possible. Antichlamydial IgG and IgM response as examined by microimmunofluorescence assay did not aid identification of individual endovascular infection. C. pneumoniae is present in a significant proportion of atherosclerotic arteries. Its occurrence in atheromatous plaques is not limited to coronary arteries and may be considered indicative of an infectious component in atherosclerosis. However, it remains unclear whether chlamydia actually initiates atherosclerotic injury, facilitates its progression, or merely colonizes pre-existing atheromata.

Detection of Chlamydia pneumoniae within Peripheral Blood Monocytes of Patients with Unstable Angina or Myocardial Infarction

Because individual diagnoses of vascular infection with Chlamydia pneumoniae depend entirely on surgically removed tissues, a better assay to predict vascular infection is needed. Polymerase chain reaction detection of chlamydial DNA was applied to CD14-positive cells collected from 238 patients with angiographically identified unstable angina or acute myocardial infarction. C. pneumoniae was detected in 52 (28%) of 188 persons with unstable angina and in 13 (26%) of 50 persons with myocardial infarction. Differences between groups were not significant. C. pneumoniae is present in monocytes/macrophages of a significant proportion of persons with progressive coronary artery disease. Infarction is not accompanied by a rise in chlamydial detection rates. The potential role of chlamydiae in coronary atherosclerosis may therefore be more related to acceleration of disease or systemic effects by persistent infection than to sudden initiation of infarction by acute infection.