Mirja Puolakkainen

Chlamydia pneumoniae Induces Tissue Factor Expression in Mouse Macrophages via Activation of Egr-1 and the MEK-ERK1/2 Pathway

Abstract— Recent studies have suggested that infection with Chlamydia pneumoniae (C pneumoniae) may contribute to the instability of atherosclerotic plaques and thrombosis and is associated with acute coronary events. Tissue factor (TF), a potent prothrombotic molecule, is expressed by macrophages and other cell types within atherosclerotic lesions and plays an essential role in thrombus formation after plaque rupture. Therefore the effects of C pneumoniae on induction of TF expression in macrophages were investigated. Infection of RAW mouse macrophages with C pneumoniae induced a time-dependent increase in procoagulant activity, expression of TF protein, and TF mRNA. C pneumoniae infection stimulated increased binding of nuclear proteins to the consensus DNA sequence for Egr-1, a key response element within the TF promoter, and increased the expression of Egr-1 protein. Transient transfections of RAW cells with mutated TF promoter constructs showed that the Egr-1 binding region is an important transcriptional regulator of C pneumoniae–induced TF expression. Furthermore, C pneumoniae–stimulated phosphorylation of ERK1/2 and Elk-1 and pharmacological inhibition of mitogen-activated protein kinase activity reduced the expression of TF and Egr-1. Antibody and polymyxin B blocking of the Toll-like receptor 4 (TLR4) partially reduced the C pneumoniae–induced expression of TF and Egr-1. In conclusion, the C pneumoniae–induced increase in TF expression in macrophages is mediated in part by Egr-1, signaling through TLR4, and activation of the MEK-ERK1/2 pathway.

Chlamydia pneumoniae uses the mannose 6-phosphate/insulin-like growth factor 2 receptor for infection of endothelial cells

ABSTRACT Several mechanisms for attachment and entry of Chlamydia have been proposed. We previously determined that the major outer membrane protein of Chlamydia trachomatis is glycosylated with a high-mannose oligosaccharide, and a similar structure inhibited the attachment and infectivity of C. trachomatis in epithelial cells. Because insulin-like growth factor 2 (IGF2) was shown to enhance the infectivity of Chlamydia pneumoniae but not C. trachomatis in endothelial cells, a hapten inhibition assay was used to analyze whether the mannose 6-phosphate (M6P)/IGF2 receptor that also binds M6P could be involved in infection of endothelial cells (HMEC-1) by Chlamydia. M6P and mannose 6-phosphate-poly[N-(2-hydroxyethyl)-acrylamide] (M6P-PAA) inhibited the infectivity of C. pneumoniae AR-39, but not C. trachomatis serovar UW5 or L2, while mannan inhibited the growth of C. trachomatis, but not C. pneumoniae. Using metabolically labeled organisms incubated with cells at 4°C (organisms attach but do not enter) or at 37°C (organisms attach and are internalized), M6P-PAA was shown to inhibit attachment and internalization of C. pneumoniae in endothelial cells but did not inhibit attachment or internalization of C. trachomatis serovar E or L2. These findings indicate that C. pneumoniae can utilize the M6P/IGF2 receptor and that the use of this receptor for attachment and entry differs between C. pneumoniae and C. trachomatis.

Further Characterization of Chlamydia pneumoniae Specific Monoclonal Antibodies

Studies using monoclonal antibodies have demonstrated species‐specific reactivities with Chlamydia pneumoniae. In this study, further characterization of C. pneumoniae specific monoclonal antibodies TT‐205 and RR‐402 and description of C. pneumoniae specific antibodies prepared against other isolates are presented. TT‐205 and RR‐402 were shown to neutralize infectivity. Neutralization in cell culture was specific and enhanced by complement. Attempts to characterize the reactive antigen by immunoblotting, immunoaffinity chromatography and radioimmunoprecipitation were unsuccessful, probably due to difficulties in solubilizing the immunoreactive epitope without denaturing it. Recognition of the determinant by the monoclonal antibodies is labile to physical and chemical treatments suggesting that the reactive epitope is conformational.

Serological response to Chlamydia pneumoniae in patients with sarcoidosis

The antigen-specific serological response to Chlamydia pneumoniae was studied in 24 patients with sarcoidosis and compared to that seen in acute C. pneumoniae respiratory infection. By the micro-immunofluorescence test, five sarcoidosis patients had acute antibody, 15 had chronic antibody and four had no antibody against C. pneumoniae. By enzyme immunoassay, 20 sarcoidosis patients had antibody against ReLPS but that cross-reacts with chlamydial LPS. Immunoblot analysis of sera using purified C. pneumoniae elementary bodies showed that recognition of the 40 kDa C. pneumoniae major outer membrane protein was rare (20%). Reactivities with proteins with Mw of 42 K (70%), 60 K (65%), 98 K (55%) and 52 K (50%) were often noted. To study reactivity of chlamydial HSP 60 in sarcoidosis sera, sarkosyl-soluble (contains the 60 kDa HSP) and sarkosyl-insoluble (contains the 60 kDa structural protein) fractions of C. pneumoniae elementary bodies were prepared. The 60 kDa structural protein was recognized with equal frequency by sera from patients with sarcoidosis and acute respiratory infection, while the HSP 60 was more frequently recognized by sera with acute respiratory infection than sarcoidosis. Recombinant fusion proteins expressed from pGEX-2T containing overlapping DNA fragments of the C. pneumoniae 60 kDa HSP gene were purified. Different recognition patterns were identified for sera from sarcoidosis patients and from patients with acute C. pneumoniae respiratory infection.

Pro-atherogenic lung and oral pathogens induce an inflammatory response in human and mouse mast cells

A broad variety of microbes are present in atherosclerotic plaques and chronic bacterial infection increases the risk of atherosclerosis by mechanisms that have remained vague. One possible mechanism is that bacteria or bacterial products activate plaque mast cells that are known to participate in the pathogenesis of atherosclerosis. Here, we show by real‐time PCR analysis and ELISA that Chlamydia pneumoniae (Cpn) and a periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa), both induce a time and concentration‐dependent expression and secretion of interleukin 8 (IL‐8), tumour necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1) by cultured human peripheral blood‐derived mast cells, but not anti‐inflammatory molecules, such as IL‐10 or transforming growth factor β1 (TGF‐β1). The IL‐8 and MCP‐1 responses were immediate, whereas the onset of TNF‐α secretion was delayed. The Cpn‐mediated pro‐inflammatory effect was attenuated when the bacteria were inactivated by UV‐treatment. Human monocyte‐derived macrophages that were pre‐infected with Cpn also induced a significant pro‐inflammatory response in human mast cells, both in cocultures and when preconditioned media from Cpn‐infected macrophages were used. Intranasal and intravenous administration of live Cpn and Aa, respectively induced an accumulation of activated mast cells in the aortic sinus of apolipoprotein E‐deficient mice, however, with varying responses in the systemic levels of lipopolysaccharide (LPS) and TNF‐α. Pro‐atherogenic Cpn and Aa induce a pro‐inflammatory response in cultured human connective tissue‐type mast cells and activation of mouse aortic mast cells in vivo.

Nitric oxide synthase plays a role in Chlamydia pneumoniae-induced atherosclerosis

OBJECTIVEnChlamydia pneumoniae infection has been associated with atherosclerosis, although the mechanisms by which C. pneumoniae contribute to atherogenesis remain unclear. Altered production of nitric oxide, a known bactericidal and anti-inflammatory agent, represents one possible mechanistic link. To examine this issue, a diet-induced, hyperlipidemic mouse model of early atherosclerosis was used.nnnMETHODSnA series of intranasal inoculations of C. pneumoniae strain AR-39 were administered to mice lacking endothelial or inducible nitric oxide synthase and to normal controls. After 18 weeks on an atherogenic diet, atherosclerotic lesion area in the aortic sinus was measured using computer-assisted morphometry.nnnRESULTSnIn the absence of C. pneumoniae infection, diet-fed eNOS(-/-) mice developed enlarged fatty streak lesions of borderline significance in comparison to uninfected, wild-type mice, while the lesion area in uninfected, diet-fed iNOS(-/-) mice did not differ significantly from lesion area in wild-type animals. In contrast, lesion area in infected eNOS(-/-) mice increased slightly, but not significantly in comparison to uninfected eNOS(-/-) mice. Lesion area in the infected iNOS(-/-) mice was significantly enlarged when compared to both uninfected iNOS(-/-) mice as well as to infected wild-type mice.nnnCONCLUSIONSnThese data suggest that production of nitric oxide by eNOS protects against development of fatty streak lesions in uninfected hyperlipidemic mice, but does not offer additional protection in infected hyperlipidemic mice, while iNOS may play a protective role, thus limiting chlamydial exacerbation of fatty streak lesions.

Mannose-binding lectin as a risk factor for acute coronary syndromes.

Background. Mannose-binding lectin (MBL) is a multifunctional protein involved in innate immunity. We tested whether MBL and elevated viral and bacterial antibodies were risk factors for acute coronary events. Design. Controlled cohort study. Methods. A total of 354 patients with unstable angina pectoris (UA) or acute myocardial infarction (AMI) were compared with 334 paired controls. Results. Enterovirus titres were associated with increased risk of UA (odds ratio 10.04, P<0.001) and AMI (odds ratio 3.18, P=0.003), but titres did not correlate with either MBL concentration or genotype. Chlamydiapneumoniae heat shock protein 60 IgG concentrations were also associated with increased risk of UA (odds ratio 1.63, P=0.049). Compared to asymptomatic controls, patients had lower complement C3 serum concentrations (P<0.001), higher MBL serum concentration, and more frequently had MBL genotypes that determined high MBL levels (P<0.001). High MBL genotypes had odds ratios of 1.16 (P=0.010) for UA and 1.12 (P=0.007) for AMI. The elevation of MBL concentrations in the acute phase correlated with MBL concentrations after recovery (r=0.85, P<0.001). Conclusions. Elevated microbial titres, indicating an on-going inflammation, were associated with cardiovascular events. MBL might have a dual role both decreasing susceptibility to infections and increasing the risk of acute coronary syndromes.

Trichomonas vaginalis and early evolving DNA and protein sequences of the CDC2/28 protein kinase family

The human sexually transmittted parasite Trichomonas vaginalis is a representative of one of the three earliest evolving eukaryotic lineages. We investigated whether T. vaginalis has DNA sequences and peptides related to cell division control molecules universal among yeasts and higher eukaryotes. A T. vaginalis ceil division control (CDC2/28) homologue was amplified by the polymerase chain reaction and sequenced. The absolute similarity with other CDC2/28 genes was 47%, with conservative replacement similarity of 67%. Western blots demonstrated a single T. vaginalis peptide reactive with antiserum to the PSTAIRE peptide, an expressed component of CDC2/28 genes in higher eukaryotes. Although eukaryotic, T. vaginalis has properties similar to those of bacteria and is the earlist evolving eukaryote reported to possess CDC2/28 DNA and peptide homologues. These observations suggest that the molecular origins of cell division control in eukaroytes preceded mitochondria, 28S ribosomes and regulated glycolysis.

Chlamydia pneumoniae Infection in Polarized Epithelial Cell Lines

ABSTRACT We set up a polarized cell culture model to study the pathogenicity of a common respiratory tract pathogen, Chlamydia pneumoniae. Immunofluorescence staining of ZO-1 (a tight junction protein) and Na+K+ ATPase (a protein pump localized at the basolateral membrane in the polarized epithelial cells), as well as TER measurements, suggested that the filter-grown Calu-3 cells, but not the A549 cells, were polarized when grown on collagen-coated membranes. Both the flat and the filter-grown cultures were infected with C. pneumoniae. Infection in the polarized Calu-3 cultures produced more C. pneumoniae genome equivalents than infection in the flat cultures. However, this progeny was not as infective as that in the flat cultures. The maximum amount of C. pneumoniae was detected at 6 days postinfection in the filter-grown A549 cells, indicating a slower developmental cycle than that observed in the flat A549 cultures. The effect of cycloheximide on the growth of C. pneumoniae in the polarized cells was negligible. Furthermore, the infection in the polarized Calu-3 cells was resistant to doxycycline, and several cytokines were released mainly on the apical side of the polarized cells in response to C. pneumoniae infection. These findings indicate that the growth of chlamydiae was altered in the filter-grown epithelial culture system. The diminished production of infective progeny of C. pneumoniae, together with the resistance to doxycycline and polarized secretion of cytokines from the infected Calu-3 cells, suggests that this model is useful for examining epithelial cell responses to C. pneumoniae infection, and it might better resemble in vivo infection in respiratory epithelial cells.

Innate immunity and vaccines in chlamydial infection with special emphasis on Chlamydia pneumoniae

Chlamydial infections are prevalent worldwide. Immunological events related to both innate and adaptive immunity during chlamydial infection can aid in recovery from the disease, but they can also cause harmful effects (immunopathology). The host genetic factors (variation in innate immunity and adaptive response-related genes) can predispose individuals to infection and its sequelae as well as determine the effects of intervention. No effective vaccine is available for human use. Modern technologies and data obtained using different omics techniques (genomics, proteomics, transcriptomics and immunomics) might help in designing novel, more efficient vaccines, hopefully also against chlamydial infections.