Jan Rupp

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.

Association Between Azithromycin Therapy and Duration of Bacterial Shedding Among Patients With Shiga Toxin–Producing Enteroaggregative Escherichia coli O104:H4

CONTEXT An outbreak of Shiga toxin-producing enteroaggregative Escherichia coli (STEC O104:H4) infection with a high incidence of hemolytic uremic syndrome (HUS) occurred in Germany in May 2011. Antibiotic treatment of STEC infection is discouraged because it might increase the risk of HUS development. However, antibiotic therapy is widely used to treat enteroaggregative E coli infection. In the German outbreak, a substantial number of patients received prophylactic azithromycin treatment as part of a therapeutic regimen with the C5 antibody eculizumab. OBJECTIVE To analyze the duration of bacterial shedding in patients with STEC infection who did and did not receive oral azithromycin therapy. DESIGN, SETTING, AND PATIENTS At a single center in Lübeck, Germany, 65 patients with STEC infection, including patients with HUS as well as STEC-infected outpatients without manifestation of HUS, were investigated between May 15 and July 26, 2011, and were monitored for a mean of 39.3 days after onset of clinical symptoms. MAIN OUTCOME MEASURE Carriage of STEC after azithromycin therapy. RESULTS Twenty-two patients received oral azithromycin and 43 patients did not receive antibiotic treatment. Among antibiotic-treated patients, long-term STEC carriage (>28 days) was observed in 1 of 22 patients (4.5%; 95% CI, 0%-13.3%) compared with 35 of 43 patients (81.4%; 95% CI, 69.8%-93.0%) who were not treated with antibiotics (P < .001). All 22 patients receiving azithromycin treatment had at least 3 STEC-negative stool specimens after the completion of treatment, and no recurrence of STEC was observed in these patients. As proof of principle, 15 patients who initially were not treated with antibiotics and were long-term STEC carriers were treated with oral azithromycin given for 3 days and subsequently had negative stool specimens. CONCLUSION Treatment with azithromycin was associated with a lower frequency of long-term STEC O104:H4 carriage.

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.

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.

Immunoproteomic Identification and Serological Responses to Novel Chlamydia pneumoniae Antigens That Are Associated with Persistent C. pneumoniae Infections

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Hydroxymethylglutaryl Coenzyme A Reductase Inhibition Reduces Chlamydia pneumoniae-Induced Cell Interaction and Activation

Background Chlamydia pneumoniae stimulates chronic inflammation in vascular cells. Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) may have an ameliorating effect. We investigated possible mechanisms. Methods and Results We infected human macrophages that in coculture spread infection to vascular smooth muscle cells (VSMCs). Cerivastatin (250 nmol/L) reduced VSMC infection by 33%. Western blotting made it apparent that VSMC infection resulted in increased cell membrane‐associated RhoA and Racl, implying increased prenylation of these proteins. This effect was blocked by statin but circumvented by mevalonate. Cytochrome C assays showed that infected VSMCs produced increased reactive oxygen species that was blocked by statin. Infection increased nuclear transcription factor‐&kgr;B expression in VSMCs that was dose‐dependently suppressed by statin. Infected VSMCs produced and released RANTES and MCP‐1. Statin dose‐dependently blocked this production both at the mRNA and protein levels. Mevalonate and M geranylgeranylpyrophosphate circumvented these effects. Conclusions C pneumoniae can be transmitted from macrophages to VSMCs. VSMCs showed an activation profile typical of atherosclerosis, namely Racl and RhoA prenylation, nuclear transcription factor‐&kgr;B activation, reactive oxygen species production, and chemokine production. Statin reduces macrophage‐mediated C pneumoniae‐induced signaling and transmission. (Circulation. 2003;108:261‐265.)

Modulation of the Inflammatory Response to Streptococcus pneumoniae in a Model of Acute Lung Tissue Infection

Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia and is a main cause of infectious deaths. However, little is known about host-pathogen interaction in human lung tissue. We tested the hypothesis that human alveolar macrophages (AMs) and alveolar epithelial cells (AECs) are important for initiating the host response against S. pneumoniae, and we evaluated the role of Toll-like receptor (TLR) 2, TLR4, and p38 mitogen-activated protein kinase (MAPK) signaling in the inflammatory response after pneumococcal infection. We established a novel model of acute S. pneumoniae infection using vital human lung specimens. In situ hybridization analysis showed that S. pneumoniae DNA was detected in 80 to 90% of AMs and 15 to 30% of AECs after in vitro infection accompanied by increased expression of inflammatory cytokines. Enhanced phosphorylation of p38 MAPK and increased TLR2 and 4 mRNA expression were observed in infected lung tissue. Thirty to fifty percent of AMs and 10 to 20% of AECs showed evidence of apoptosis 24 hours after pneumococcal infection. After macrophage deactivation with Clodronate/liposomes, infected lung tissue exhibited a significantly decreased release of inflammatory mediators. Inhibition of p38 MAPK signaling markedly reduced inflammatory cytokine release from human lungs, whereas TLR2 blockade revealed only minor effects. AMs are central resident immune cells during S. pneumoniae infection and are the main source of early proinflammatory cytokine release. p38 MAPK holds a major role in pathogen-induced pulmonary cytokine release and is a potential molecular target to modulate overwhelming lung inflammation.

CD14 promoter polymorphism -159C>T is associated with susceptibility to chronic Chlamydia pneumoniae infection in peripheral blood monocytes.

Chlamydia pneumoniae uses peripheral blood monocytes (PBMC) for systemic dissemination and has been linked to atherogenesis by inflammation mediated via TLR2/4 and CD14. We found 12.8% of 610 coronary artery disease (CAD) patients of Central European background to be chronically infected with C. pneumoniae based on the repeated detection of chlamydial DNA in PBMC. Among those the −159C>T CD14 promoter polymorphism was more frequent (OR 1.7, 95% CI 1.08–2.65, P=0.0224) than among C. pneumoniae-negative subjects matched for age and gender. The Arg753Gln TLR2 and Asp299Gly TLR4 polymorphisms were not related to chlamydial infection. Susceptibility for chronic chlamydial infection of PBMC in CAD patients appears associated with the CD14-159C>T promoter polymorphism encoding for enhanced CD14 expression.