Jens Gieffers

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.

Chlamydia pneumoniae and atherosclerosis.

Exposure to Chlamydia pneumoniae is extremely common, and respiratory infections occur repeatedly among most people. Strong associations exist between C. pneumoniae infection and atherosclerosis as demonstrated by: (i) sero‐epidemiological studies showing that patients with cardiovascular disease have higher titres of anti‐C. pneumoniae antibodies compared with control patients; (ii) detection of the organism within atherosclerotic lesions, but not in adjacent normal tissue by immunohistochemistry, polymerase chain reaction and electron microscopy and by culturing the organism from lesions; and (iii) showing that C. pneumoniae can either initiate lesion development or cause exacerbation of lesions in rabbit and mouse animal models respectively. The association of this organism with atherosclerosis has also provided sufficient impetus to conduct a variety of human secondary prevention antibiotic treatment trials. The results of these studies have been mixed and, thus far, no clear long‐lasting benefit has emerged from these types of investigations. Studies of C. pneumoniae pathogenesis have shown that the organism can infect many cell types associated with both respiratory and cardiovascular sites, including lung epithelium and resident alveolar macrophages, circulating monocytes, arterial smooth muscle cells and vascular endothelium. Infected cells have been shown to exhibit characteristics associated with the development of cardiovascular disease (e.g. secretion of proinflammatory cytokines and procoagulants by infected endothelial cells and foam cell formation by infected macrophages). More detailed analysis of C. pneumoniae pathogenesis has been aided by the availability of genomic sequence information. Genomic and proteomic analyses of C. pneumoniae infections in relevant cell types will help to define the pathogenic potential of the organism in both respiratory and cardiovascular disease.

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.

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.

Poor correlation between microimmunofluorescence serology and polymerase chain reaction for detection of vascular Chlamydia pneumoniae infection in coronary artery disease patients

Chlamydia pneumoniae has been associated to coronary artery disease by various methods including recovery of viable bacteria from plaques. The pathogenetical relevance of this is unclear but investigation of antichlamydial therapy in coronary arteriosclerosis is already in progress. The microimmunofluorescence test (MIF), the only species-specific serological assay available, might be considered useful in identifying patients with vascular chlamydial infection. However, this has never been systematically examined. We compared levels of C. pneumoniae antibodies in sera using MIF with direct detection of C. pneumoniae in coronary artery segments from 158 patients undergoing myocardial revascularization. A polymerase chain reaction (PCR) protocol, recently evaluated for use with vascular materials, detected C. pneumoniae infection in 34 patients. Correlation of serology and PCR was poor: in relation to PCR, MIF-IgG analysis had 21% sensitivity, 90% specificity, 37% positive predictive value, and 81% negative predictive value for detection of chlamydial presence. Thus, the MIF test currently appears not suitable to predict individual vascular C. pneumoniae infection.

Chlamydia pneumoniae directly interferes with HIF‐1α stabilization in human host cells

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia‐inducible factor‐1 (HIF‐1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF‐1α regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF‐1α stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen‐induced HIF‐1α degradation. HIF‐1α was targeted by the chlamydial protease‐like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF‐1α stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen–host interaction in chlamydial diseases.

First-choice antibiotics at subinhibitory concentrations induce persistence of Chlamydia pneumoniae.

ABSTRACT Persistent growth forms of Chlamydia pneumoniae have been associated with chronic infections in vivo. We investigated the effects of first-line therapeutics on the induction of persistence by monitoring recoverable organisms, gene expression of membrane proteins, and morphology. We found that all of the antibiotics tested have distinct and subinhibitory concentrations at which they induce persistence.

Presence of Chlamydia pneumoniae DNA in the cerebral spinal fluid is a common phenomenon in a variety of neurological diseases and not restricted to multiple sclerosis.

Chlamydial DNA and viable organisms have been reported in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. We investigated whether this phenomenon is specific for MS and not occurring in patients with other neurological diseases (OND) or in healthy controls and whether it is caused by infected blood monocytes having crossed the blood–brain barrier. Twelve (21%) of fifty‐eight MS patients and 20 (43%) of 47 OND patients had Chlamydia pneumoniae DNA in the CSF as determined by nested polymerase chain reaction. Viable organisms were cultured from one OND patient. We failed to detect C. pneumoniae in the CSF of 67 neurologically healthy persons. C. pneumoniae was detected in parallel in the blood monocytes of 2 of 6 CSF‐positive MS patients and in 8 of 10 CSF‐positive OND patients. Thus, chlamydial presence cannot exclusively be explained as being caused by contaminating infected monocytes that have crossed the blood–brain barrier. In peripheral blood mononuclear cell‐negative patients, chlamydia have been cleared from the circulation but persist in the central nervous system (CNS), indicating the establishment of a chronic process. In summary, the presence of C. pneumoniae in patients with neurological diseases is a common phenomenon and is not restricted to MS patients. The pathogenetic relevance of a chronic chlamydial CNS infection for neurological diseases remains unclear, but the hypothesis that susceptible patients may be impaired in their ability to clear chlamydiae from the CNS requires further examination.