Chlamydia pneumoniae and Chlamydia Trachomatis Infection Differentially Modulates Human Dendritic Cell Line (MUTZ) Differentiation and Activation

Chlamydia trachomatis and Chlamydia pneumoniae are important human pathogens that infect the urogenital/anorectal and respiratory tracts, respectively. Whilst the ability of these bacteria to infect epithelia is well defined, there is also considerable evidence of infection of leucocytes, including dendritic cells (DCs). Using a human dendritic cell line (MUTZ), we demonstrate that the infection and replication of chlamydiae inside DCs is species and serovar specific and that live infection with C. pneumoniae is required to upregulate costimulatory markers CD80, CD83 and human leucocyte antigen (HLA)‐DR on MUTZ cells, as well as induce secretion of interleukin (IL)‐2, IL‐6, IL‐8, IL‐12 (p70), interferon‐gamma and tumour necrosis factor‐alpha Conversely, C. trachomatis serovar D failed to upregulate DC costimulatory markers, but did induce secretion of high concentrations of IL‐8. Interestingly, we also observed that infection of MUTZ cells with C. pneumoniae or C. trachomatis serovar L2, whilst not replicative, remained infectious and upregulated lymph node migratory marker CCR7 mRNA. Taken together, these data confirm the findings of other groups using primary DCs and demonstrate the utility of MUTZ cells for further studies of chlamydial infection.     

Epithelial Cells Infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) Are Resistant to Apoptosis

The obligate intracellular pathogen Chlamydophila pneumoniae (Chlamydia pneumoniae) initiates infections in humans via the mucosal epithelia of the respiratory tract. Here, we report that epithelial cells infected with C. pneumoniae are resistant to apoptosis induced by treatment with drugs or by death receptor ligation. The induction of protection from apoptosis depended on the infection conditions since only cells containing large inclusions were protected. The underlying mechanism of infection-induced apoptosis resistance probably involves mitochondria, the major integrators of apoptotic signaling. In the infected cells, mitochondria did not respond to apoptotic stimuli by the release of apoptogenic factors required for the activation of caspases. Consequently, active caspase-3 was absent in infected cells. Our data suggest a direct modulation of apoptotic pathways in epithelial cells by C. pneumoniae.     

Incidence of Chlamydia pneumoniae Infection in Vertically HIV-1 Infected Children

 The rate of seroconversion for antibody to Chlamydia pneumoniae was analysed in blood samples of 26 vertically HIV-1 infected children and 14 seroreverter children (HIV-negative children born to HIV-positive mothers) during a 3-year study period. Seroconversion for Chlamydia pneumoniae was found in 13 of 26 HIV-1 infected children and in 1 of 14 in the seroreverter group (P=0.013). A lower mean CD4+ cell count and p24 antigen positivity at enrolment were significantly associated with seroconversion for Chlamydia pneumoniae. Signs and symptoms of acute respiratory infection were recorded in the 30 to 40 days preceding collection of the blood samples showing seroconversion for Chlamydia pneumoniae in 8 of 13 HIV-1 infected children and in the single seroreverter. This study confirms the potential role of Chlamydia pneumoniae in the pathogenesis of respiratory tract infections in HIV-1 infected subjects.     

Characterization of Apoptotic Activities During Chlamydia trachomatis Infection in Primary Cervical Epithelial Cells

Chlamydiae are obligate intracellular bacteria that infect human epithelial cells. It has been reported that Chlamydia trachomatis, induces apoptosis in epithelial cells, however, the molecular mechanisms responsible for host cell death especially in primary epithelial cells remained largely unknown as most of the studies are in cell line like HeLa. In this study we demonstrated that C. trachomatis induces apoptosis signaling pathway and apoptosis in primary cervical epithelial cells in a time and dose dependent manner. Live cervical epithelial cells were isolated from endocervical cells and induction was done with chlamydial EBs. Our results demonstrated that apoptosis in infected epithelial cells was associated with an increased activity of caspase 8; however, caspase 9 was activated to a lesser extent. Analysis of apoptosis pathway revealed that expression level of McL-1, Bcl-2, CASP8, and TRADD genes were found to be significantly upregulated (P?<?0.01), where as levels of Caspase 1, Caspase 10 and BRIC2 were found to be significantly downregulated (p?<?0.01). Our results showed that Chlamydia induces apoptosis and caspase activation in epithelial cells through caspase 8, with an increased expression of the McL-1, which confers a block at the mitochondrial level.     

Characterization of Chlamydia pneumoniae Antigens Using Human T Cell Clones

Chlamydia pneumoniae infection is followed by the development of antigen-specific cell-mediated immunity (CMI), which is detectable as a positive lymphocyte proliferation (LP) response to C. pneumoniae elementary body (EB) antigen, but the proteins inducing the T cell activation are not known. In the present work the authors used human T lymphocyte clones (TLC) raised against C. pneumoniae EB antigen to characterize C. pneumoniae proteins as T cell-stimulating antigens. A total of 55% of the TLC established recognized antigenic determinants only on C. pneumoniae species, while the rest of the TLC proliferated to both C. pneumoniae and C. trachomatis EB. The antigen specificity of the TLC was further analysed by stimulating with SDS-PAGE fractionated C. pneumoniae EB proteins. Chlamydia pneumoniae species-specific antigens were found in the molecular weight ranges 92–98, 51–55, 43–46 and 31.5–33 kDa and genus-specific antigens in the ranges 12, 26 and 65–70 kDa. The 46.5–49.5 and 55–61 kDa regions contained both species-specific and genus-specific antigens. Human leucocyte antigen (HLA) restriction analysis for the TLC isolated from an HLA DR4, 15(2) heterozygous person showed the majority (81.3%) to be restricted to the HLA DR4 molecule, the rest being DR15(2)-restricted. An interesting preliminary finding was that the expression of interferon-gamma (IFN-γ) mRNA by the TLC was predominantly associated with antigen recognition in the context of the HLA DR4 molecule, while interleukin-4 (IL-4) production was linked to antigen recognition in the context of the HLA DR15(2) molecule.     

Epithelial Cell Apoptosis Facilitates Entamoeba histolytica Infection in the Gut

Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite''s strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.Entamoeba histolytica is a protozoan parasite and the causative agent of amebic colitis, an invasive disease responsible for as many as 100,000 deaths per year worldwide.¹ The parasite proceeds from asymptomatic infection to invasive disease in a fraction of individuals, whereby it has been named “histolytica” for its cell-damaging properties.² The cellular mechanism of this damage has been a subject of considerable study in which investigators use several nonepithelial cell types. Most of these results support the notion that Entamoeba directly triggers host cell apoptosis on contact as indicated by characteristic morphological changes (chromatin condensation, membrane blebbing, and internucleosomal DNA fragmentation), DNA laddering, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positivity, and phosphatidylserine exposure^3–9; however, one study revealed that a necrotic pathway can predominate.¹⁰ These studies have been performed by using cell lines including Jurkat T cells, HL-60 granulocytes, follicular dendritic cell protein 1, and Chinese hamster ovary cells, using primary human neutrophils, macrophages, erythrocytes, and T lymphocytes, and in a mouse amebic liver abscess model. Under the conditions of these studies, the apoptotic mechanism that occurs has been reported to be not inhibitable by Bcl-2 overexpression (in a myeloid cell line³), to not require Fas/Fas ligand or tumor necrosis factor receptor1 signaling (in mouse liver abscess⁸), to involve caspase 3 but not require caspase 8 or 9 (in Jurkat T cells⁵), and to require activation of extracellular signal regulated kinase (ERK) 1/2 (in human neutrophils⁴).Amid this context little is known about the extent of apoptosis in the intestinal epithelial cell, even though this is the first and predominant cell that contacts the parasite in the gut. Reasons for this lack of study are technical challenges in animal models of intestinal amebiasis¹¹ and in establishing epithelial cell lines from colonic epithelium.¹² For these reasons we developed a new conditionally immortalized cecal epithelial cell line derived from the CBA mouse and tested the role of intestinal epithelial apoptosis in vivo by using our CBA mouse model of intestinal infection.     

Chlamydia pneumoniae Infection Among Healthy Children and Children Hospitalised with Pneumonia in Greece

Chlamydia pneumoniae has been recognized as a cause of respiratory tract infection in humans, and its prevalence has been shown to vary among different age groups and populations. The prevalence of Chlamydia pneumoniae antibody was determined by serological investigation in 343 healthy children and in 77 children consecutively hospitalised for pneumonia in southwestern Greece. Seventy-eight (22.7%) healthy children had IgG Chlamydia pneumoniae titers ≥1/8. The prevalence of Chlamydia pneumoniae antibody in the age groups 6 months–5 years, 6–9 years and 10–15 years was 7.9%, 11.4% and 36%, respectively. One child hospitalised for pneumonia had serological results consistent with acute Chlamydia pneumoniae infection. The results of the present study suggest a low prevalence of Chlamydia pneumoniae antibody among preschoolers in Greece, followed by a steep rise in children 10–15 years of age. Chlamydia pneumoniae is not a common etiologic agent of childhood pneumonia requiring hospitalisation. Electronic Publication     

Differential Effects of Gamma Interferon on Chlamydia trachomatis Growth in Polarized and Nonpolarized Human Epithelial Cells in Culture

The effects of gamma interferon (IFN-γ) on Chlamydia trachomatis growth in polarized epithelial cells were examined. The range of IFN-γ concentrations causing aberrant chlamydial growth was wider in polarized than in nonpolarized cultures. Results indicate that chlamydial growth modulation in polarized cells readily leads to persistence and better reflects in vivo conditions.     

Localization of Chlamydia trachomatis Heat Shock Proteins 60 and 70 during Infection of a Human Endometrial Epithelial Cell Line In Vitro

Unlike chlamydial lipopolysaccharide, which is released from the developing inclusion to the surface of infected genital epithelial cells, both Chlamydia trachomatis heat shock protein (hsp) 60 and 70 antigens remained confined within the inclusion during the course of the chlamydial developmental cycle. Exposure of the infected cells to penicillin to induce a persistent infection or to a lipophilic microbicide did not potentiate secretion or exocytosis of the chlamydial hsp.     

Chlamydia pneumoniae Exacerbates Aortic Inflammatory Foci Caused by Murine Cytomegalovirus Infection in Normocholesterolemic Mice

Inflammatory foci induced by murine cytomegalovirus infection in normocholesterolemic mice were present temporarily in the aortic wall, but some of these foci developed into advanced lesions that persisted late after infection. The early foci induced by virus infection were significantly exacerbated following a single inoculation with Chlamydia pneumoniae.     

No Correlation between Giant Cell Arteritis and Chlamydia pneumoniae Infection: Investigation of 189 Patients by Standard and Improved PCR Methods

A total of 189 temporal artery biopsy samples from giant cell arteritis (GCA) patients were investigated using sensitive PCR targeting Chlamydia pneumoniae. Chlamydial DNA was detected in 17 samples, 11 of which were positive for chlamydial antigens. Our data did not reveal strong evidence that C. pneumoniae plays an important role in the pathogenesis of GCA.Giant cell arteritis (GCA) is a vasculitis disease affecting medium- to large-sized arteries. Some studies have presented evidence of a coincidence of cyclic fluctuations of Chlamydia pneumoniae epidemics and cases of GCA (3, 13). Conflicting data exist regarding the role of C. pneumoniae in the pathogenesis of GCA. Some authors found a strong correlation between GCA and detection of C. pneumoniae (12, 15), whereas others failed to detect the pathogen (6, 7, 11). The discrepancies among the published data may derive from variations in either the quality or quantity of the DNA used or the specificity and sensitivity of the PCR system employed. To address these and other potential impediments to screening success, the DNA purification method and the PCR screening system used here were optimized for investigation of paraffin-embedded temporal artery biopsy samples. Temporal artery biopsy samples from 224 GCA patients who underwent consecutive biopsy procedures at the University Eye Hospital (Freiburg, Germany) between 1993 and 2006 were screened. Classification criteria for GCA according to the 1990 guidelines of the American College of Rheumatology (ACR) were applied to select the cases. In this manner we identified 115 potential case subjects. A total of 109 patients whose temporal artery biopsy specimens from at least one side gave negative results and whose postbiopsy clinical patterns were not consistent with GCA were used as controls.For isolation of genomic DNA, a QiAmp DNA Micro kit (Qiagen, Hilden, Germany) was used according to the manufacturer''s instructions. Alternatively, a hot phenol-chloroform DNA isolation method was used as described previously (15). The two methods yielded equal amounts of genomic DNA. With the exception of primers p90n and p91n (5′-ACACTCGCAAGGGTGAAACT-3′ and 5′-AAGTCCAGGTAAGGTCCTTCG-3′, respectively), which were designed with Primer3 software (http://primer3.sourceforge.net), the other primers were as previously described (4, 8-10). A PCR assay targeting the β-globin gene was used to compare the two methods. By the use of primers GH20 and PC04, detection of human genomic DNA in minimal amounts of paraffin-embedded tissue was possible.Serial dilutions of DNA were used, and β-globin amplification was found to be possible at a dilution of 100 pg of DNA. After successful detection of human β-globin DNA, C. pneumoniae outer PCR (i.e., PCR targeting a larger fragment which can be unambiguously amplified and which contains the smaller intended fragment) was performed. Samples were analyzed using standard PCR targeting the 16S rRNA gene (primers CpnA and CpnB) and Touchdown enzyme time release PCR (TETR-PCR) targeting the 16S-to-23S spacer region (primers CPN-90 and CPN-91) (5, 10, 11, 15) of C. pneumoniae to determine the most sensitive PCR. TETR-PCR allowed amplification of C. pneumoniae fragments (197 bp) smaller than those amplified by the standard PCR method (463 bp) initially used in previous studies (5, 15). Both PCR techniques are recommended by the U.S. Centers for Disease Control and Prevention and the Laboratory Center for Disease Control (Canada) (2). Comparison of the two PCR methods revealed sensitivities of 1 inclusion-forming unit (IFU) (Fig. ​(Fig.1B,1B, lane 5) and 10² IFU (Fig. ​(Fig.1A,1A, lane 3), respectively.Open in a separate windowFIG. 1.Outer PCR detection of C. pneumoniae in paraffin-embedded biopsy samples. (A) Standard PCR amplification products of the target 16S rRNA gene generated using primers CpnA and CpnB; (B) TETR-PCR amplification products of the target 16S-23S spacer rRNA gene generated using primers CPN-90 and CPN-91. Lanes M, DNA molecular weight marker VIII (Roche). Lanes 1 to 9 of both panels show the results obtained with C. pneumoniae DNA template containing 10⁴ to 10⁻⁴ IFUs. Lane 10, positive control; lane 11, negative control (without template). Data are representative of the results of three experiments.To further increase sensitivity, a nested PCR was applied for the two PCR methods using 10% of the PCR products from the outer PCR with primers pTW50 and pTW51 to amplify the CpnA-CpnB fragment or with primers p90n and p91n to amplify the CPN-90-CPN-95 fragment. As shown in Fig. ​Fig.2A,2A, lane 4, the sensitivity of the standard PCR increased to a limit of 1 IFU whereas the TETR-PCR was able to detect 0.1 IFU (Fig. ​(Fig.2B,2B, lane 5).Open in a separate windowFIG. 2.Nested PCR detection of C. pneumoniae in paraffin-embedded biopsy samples. (A) Standard PCR amplification products of the target 16S rRNA gene generated using primers pTW50 and pTW51; (B) TETR-PCR amplification products of the target 16S-23S spacer rRNA gene generated using primers p90n and p91n. CpnA-CpnB and CPN-90-CPN-91 amplified PCR products (10%) were used as a template for primer pair pTW50 and pTW51 and primer pair p90n and p91n, respectively. Lanes M, DNA molecular weight marker VIII (Roche). Lanes 1 to 7 of both panels contain C. pneumoniae DNA outer products obtained with 10³ to 10⁻³ IFUs. Lane 8, negative control (without template). Data are representative of the results of three experiments.The optimized methods were employed to screen samples from 115 and 109 GCA and control patients, respectively. Human genomic DNA was detected in 102 of 115 GCA specimens and in 87 of 109 control specimens by the use of β-globin PCR. It has been shown in other studies that it is difficult to amplify long fragments from formalin-fixed and paraffin-embedded tissue (1, 14). This might be one reason underlying the failure to detect chlamydial DNA in paraffin-embedded tissue samples of GCA patients in previous studies (6, 7, 11). Therefore, by using β-globin PCR we were able to identify and exclude 35 of 224 samples from further investigations, preventing false-negative results (15).Due to its ability to process a shorter fragment length and its higher sensitivity, we used the TETR-PCR. A total of 3 of 102 GCA specimens and 2 of 87 control specimens were positive for C. pneumoniae DNA by outer PCR. When the nested PCR was applied, an additional nine and three samples from GCA and control patients, respectively, were shown to be positive for C. pneumoniae DNA. Sequencing of all 17 nested PCR amplicons identified the obtained fragments as specific for C. pneumoniae. These findings contradict another report in the literature: Regan and coworkers reported in 2002 that none of the investigated temporal arteries from 90 GCA patients were positive for C. pneumoniae (11). The improvement in DNA purification and the increased sensitivity of the PCR method probably explain in large part the results with respect to C. pneumoniae-positive patients in our study. However, the relatively low level of chlamydial positivity in the GCA specimen results may have been due to the disease onset being caused by antigens other than those corresponding to C. pneumoniae. As determined by Fisher''s exact test, the difference between patient groups with regard to positive C. pneumoniae PCR results was not statistically significant (P = 0.45). As shown in Table ​Table1,1, none of the clinical parameters for C. pneumoniae DNA were statistically different between the GCA and control patients. In addition, no statistical differences were detected when we compared the ACR-positive and the ACR-negative patients with respect to C. pneumoniae detection (10.9% versus 8.3%; P = 1.0).

TABLE 1.

Comparison of demographic data, specific symptoms, and nonspecific laboratory parameters for patients whose temporal artery biopsy samples were C. pneumoniae DNA positive versus those whose samples were C. pneumoniae DNA negative

Diagnosis of Chlamydia pneumoniae

This paper considers the use of diagnostic techniques for Chlamydia pneumoniae in both acute and chronic infections, together with the detection of upper respiratory tract carriage. It is concluded that such diagnosis is still unsatisfactory, although techniques for acute infection are more reliable than those for chronic conditions. Reliable and inexpensive methods, particularly for the diagnosis of chronic C. pneumoniae infection, are urgently needed, if only because of the recent demonstration of the beneficial effects of antibiotic treatment in patients with atherosclerosis carrying markers for the presence of this organism.     

Chlamydia pneumoniae infection in human monocytes

Chlamydia pneumoniae infection has been associated with cardiovascular diseases in seroepidemiological studies and by demonstration of the pathogen in atherosclerotic lesions. It has the capacity to infect several cell types, including monocyte-derived macrophages, which play an essential role in the development of atherosclerosis. However, the persistence of C. pneumoniae in mononuclear cells is poorly understood. To study the morphology and biological characteristics of the infection, human peripheral blood monocytes were infected with C. pneumoniae. Freshly isolated monocytes resisted the development of infectious progeny, and confocal and transmission electron microscopy showed that the morphology of the inclusions and chlamydial particles was abnormal. Addition of tryptophan or antibodies against gamma interferon did not diminish the inhibition of C. pneumoniae, suggesting that other factors are involved in the chlamydiostatic activity of the monocytes. Chlamydial mRNA was expressed at least 3 days after infection, however, and a capability for infected monocytes to induce a positive lymphocyte proliferative response was detected for up to 7 days, indicating that C. pneumoniae remains metabolically active in the monocytes in vitro. These results are in accordance with the hypothesis that C. pneumoniae may participate in the maintenance of local immunological response and inflammation via infected monocytes and thus enhance atherosclerosis.     

A Chlamydia pneumoniae Component That Induces Macrophage Foam Cell Formation Is Chlamydial Lipopolysaccharide

Chlamydia pneumoniae infection is associated with atherosclerotic heart and vessel disease, but a causal relationship between this pathogen and the disease process has not been established. Recently, it was reported that C. pneumoniae induces human macrophage foam cell formation, a key event in early atheroma development, suggesting a role for the organism in atherogenesis. This study further examines C. pneumoniae-induced foam cell formation in the murine macrophage cell line RAW-264.7. Infected RAW cells accumulated cholesteryl esters when cultured in the presence of low-density lipoprotein in a manner similar to that described for human macrophages. Exposure of C. pneumoniae elementary bodies to periodate, but not elevated temperatures, inhibited cholesteryl ester accumulation, suggesting a role for chlamydial lipopolysaccharide (cLPS) in macrophage foam cell formation. Purified cLPS was found to be sufficient to induce cholesteryl ester accumulation and foam cell formation. Furthermore, the LPS antagonist lipid X inhibited C. pneumoniae and cLPS-induced lipid uptake. These data indicate that cLPS is a C. pneumoniae component that induces macrophage foam cell formation and suggest that infected macrophages chronically exposed to cLPS may accumulate excess cholesterol to contribute to atheroma development.     

Chlamydia pneumoniae (TWAR).

Chlamydia pneumoniae (TWAR) is a recently recognized third species of the genus Chlamydia that causes acute respiratory disease. It is distinct from the other two chlamydial species that infect humans, C. trachomatis and C. psittaci, in elementary body morphology and shares less than 10% of the DNA homology with those species. The organism has a global distribution, with infection most common among children between the ages of 5 and 14 years. In children, TWAR infection is usually mild or asymptomatic, but it may be more severe in adults. Pneumonia and bronchitis are the most common clinical manifestations of infection, and TWAR is responsible for approximately 10% of cases of pneumonia and 5% of cases of bronchitis in the United States. The microimmunofluorescence serologic assay is specific for TWAR and can distinguish between recent and past infections. The organism can be isolated in cell culture; however, PCR techniques have recently facilitated its detection in tissues and clinical specimens.     

Anthrax Toxin Entry into Polarized Epithelial Cells

We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl- secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl- secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease.