Differential infection outcome of Chlamydia trachomatis in human blood monocytes and monocyte-derived dendritic cells.

BACKGROUND: Chlamydia trachomatis is an intracellular bacteria which consist of three biovariants; trachoma (serovars A-C), urogenital (serovars D-K) and lymphogranuloma venereum (L1-L3), causing a wide spectrum of disease in humans. Monocytes are considered to disseminate this pathogen throughout the body while dendritic cells (DCs) play an important role in mediating immune response against bacterial infection. To determine the fate of C. trachomatis within human peripheral blood monocytes and monocyte-derived DCs, these two sets of immune cells were infected with serovars Ba, D and L2, representative of the three biovariants of C. trachomatis. RESULTS: Our study revealed that the different serovars primarily infect monocytes and DCs in a comparable fashion, however undergo differential infection outcome, serovar L2 being the only candidate to inflict active infection. Moreover, the C. trachomatis serovars Ba and D become persistent in monocytes while the serovars predominantly suffer degradation within DCs. Effects of persistence gene Indoleamine 2, 3-dioxygenase (IDO) was not clearly evident in the differential infection outcome. The heightened levels of inflammatory cytokines secreted by the chlamydial infection in DCs compared to monocytes seemed to be instrumental for this consequence. The immune genes induced in monocytes and DCs against chlamydial infection involves a different set of Toll-like receptors, indicating that distinct intracellular signalling pathways are adopted for immune response. CONCLUSION: Our results demonstrate that the host pathogen interaction in chlamydia infection is not only serovar specific but manifests cell specific features, inducing separate immune response cascade in monocytes and DCs.

Glucocorticoid Effects on Tissue Residing Immune Cells in Giant Cell Arteritis: Importance of GM-CSF.

Giant cell arteritis (GCA) is a systemic granulomatous vasculitis clinically characterized by a prompt response to glucocorticoid therapy. Dendritic cells (DCs) play a central role in the pathogenesis of the disease and are increased in temporal arteries from GCA patients. The aim of this study was to determine the effects of glucocorticoid therapy on granulomatous infiltrates and on peripheral DCs of GCA patients. Immunohistochemical staining of temporal artery specimens from 41 GCA patients revealed a rapid reduction of the number of DCs after initiation of glucocorticoid treatment. TUNEL staining was performed to quantify apoptotic S100+ DC, CD3+ T cells, and CD68+ macrophages in the granulomatous infiltrates. An increase of apoptotic cells up to 9 ± 2% after 4-5 days of glucocorticoid therapy and up to 27 ± 5% (p < 0.001, compared to earlier timepoints) after 6-10 days was detected. A decrease of CCL19 and CCL21 expression was observed after starting glucocorticoid therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression also significantly decreased under glucocorticoid therapy. No GM-CSF expression was detected in the control specimens. Glucocorticoid therapy leads to a rapid, time-dependent reduction of DCs in temporal arteries from GCA patients and reduction of mediators for cell migration. Our data suggest GM-CSF as a novel therapeutic target of GCA.

Actin re-organization induced by Chlamydia trachomatis serovar D--evidence for a critical role of the effector protein CT166 targeting Rac.

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166's participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.

Restriction of Chlamydia pneumoniae replication in human dendritic cell by activation of indoleamine 2,3-dioxygenase.

Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been associated with various chronic diseases such as asthma and atherosclerosis, possibly because the pathogen can exist in a persistent form. C. pneumoniae persistently infect DCs in a TNF-alpha dependent manner. The present study investigated whether C. pneumoniae infection can induce indoleamine 2,3-dioxygenase (IDO) activity in dendritic cells, and whether the restriction of chlamydial growth in the DCs by TNF-alpha is IDO dependent. Our data indicate that infection of DCs with C. pneumoniae resulted in the induction of IDO expression. Reporting on our use of anti-TNF-alpha antibody adalimumab and varying concentrations of TNF-alpha, we further demonstrate that IDO induction following infection of DCs with C. pneumoniae is TNF-alpha dependent. The anti-chlamydial activity induced by TNF-alpha and the expression of chlamydial 16S rRNA gene, euo, groEL1, ftsk and tal genes were correlated with induction of IDO. Addition of excess amounts of tryptophan to the DC cultures resulted in abrogation of the TNF-alpha-mediated chlamydial growth restriction. These findings suggest that infection of DCs by C. pneumoniae induces production of functional IDO, which subsequently causes depletion of tryptophan. This may represent a potential mechanism for DCs to restrict bacterial growth in chlamydial infections.

The transcript profile of persistent Chlamydophila (Chlamydia) pneumoniae in vitro depends on the means by which persistence is induced.

Expression of specific bacterial genes is differentially regulated during persistent, vs. active, chlamydial infection. Transcript patterns were examined using real-time reverse transcriptase-PCR in four in vitro models of persistence for Chlamydia pneumoniae strain CWL 029, using HeLa cells and normal human monocytes as host. Differential expression of genes encoding cell division proteins was variable when persistence was induced by interferon-gamma, penicillin G, or deferoxamine mesylate treatment, and in the monocyte model of persistence. Expression of genes encoding hsp60s and those specifying sigma-factors also was variable among models. These in vitro observations indicate that chlamydial persistence is not characterizable by a single transcript profile under all circumstances, supporting the idea that persistent infection in vivo is a complex, flexible strategy that promotes long-term survival of these organisms. Each model system studied here can provide information regarding the molecular characteristics of persistent C. pneumoniae infection. However, we do not know which aspect(s) of which model correspond to in vivo disease or other contexts.

Identification of high- and low-virulent strains of Chlamydia pneumoniae by their characterization in a mouse pneumonia model.

Contradicting reports exist about the pathogenicity of Chlamydia pneumoniae and the severity of the respiratory disease they cause. This study aimed to clarify, in mice, our hypothesis that marked differences in virulence of well-defined C. pneumoniae strains might exist for lung infections. C57BL/6J mice were intranasally infected with equal amounts of five different, identically prepared laboratory strains of C. pneumoniae. Based on the clinical score, weight, histopathological score, the granulocyte marker-enzyme myeloperoxidase, and the amount of Chlamydiae in the lung tissue, the C. pneumoniae isolates exhibited clear differences in overall growth characteristics or clearance, and pathological potential. Thus, we could identify chlamydial strains (Kajaani-K6 and CWL-029), where mice became seriously ill, as well as a relatively low-virulent isolate (TWAR-183). Cytokine profiles also varied drastically between the five strains in extent and kinetic. Our results indicate that C. pneumoniae isolates differ markedly with regard to their interaction with the host and their pathological potential. This might also be true for the infection in humans. Because the genomic diversity of C. pneumoniae is rather small, more subtle genomic deviations account most likely for the apparent functional differences. Our results will be useful to identify additional virulence factors in the future.

Host cell responses to Chlamydia pneumoniae in gamma interferon-induced persistence overlap those of productive infection and are linked to genes involved in apoptosis, cell cycle, and metabolism.

The respiratory pathogen Chlamydia (Chlamydophila) pneumoniae is associated with chronic diseases, including atherosclerosis and giant-cell arteritis, which are accompanied by the occurrence of these obligate intracellular bacteria in blood vessels. There, C. pneumoniae seems to be present in a persistent state. Persistence is characterized by modified bacterial metabolism and morphology, as well as a reversible arrest of chlamydial development. In cell culture, this persistent state can be induced by gamma interferon (IFN-gamma). To elucidate this long-term interaction between chlamydiae and their host cells, microarray screening on epithelial HeLa cells was performed. Transcription of persistently (and productively) infected cells was compared with that of mock-infected cells. Sixty-six host cell genes were regulated at 24 h and/or 96 h of IFN-gamma-induced persistence. Subsequently, a set of 17 human host cell genes related to apoptosis, cell cycle, or metabolism was identified as permanently up- or down-regulated by real-time PCR. Some of these chlamydia-dependent host cell responses were diminished or even absent in the presence of rifampin. However, other expression patterns were not altered by the inhibition of bacterial RNA polymerase, suggesting two different modes of host cell activation. Thus, in the IFN-gamma model, the persisting bacteria cause long-lasting changes in the expression of genes coding for functionally important proteins. They might be potential drug targets for the treatment of persistent C. pneumoniae infections.

Ligand specificity of the anaphylatoxin C5L2 receptor and its regulation on myeloid and epithelial cell lines.

During complement activation the pro-inflammatory anaphylatoxins C3a and C5a are generated, which interact with the C3a receptor and C5a receptor (CD88), respectively. C5a and its degradation product C5a-des-Arg(74) also bind to the C5a receptor-like 2 (C5L2). C3a and C3a-des-Arg(77), also called acylation-stimulating protein, augment triglyceride synthesis and glucose uptake in adipocytes and skin fibroblasts. Based on data obtained using transfected HEK293 and RBL cells, C5L2 is additionally proposed as a functional receptor for C3a and C3a-des-Arg(77). Here we use (125)I-ligand binding assays and flow cytometry with fluorescently labeled ligands to demonstrate that neither C3a nor C3a-des-Arg(77) binds to C5L2. C5L2 expression and its regulation are investigated on various cell lines by a novel C5L2-restricted binding assay and quantitative real time PCR. Dibutyryl cAMP and interferon-gamma induce up-regulation of this receptor on myeloblastic cell lines (U937 and HL-60), whereas tumor necrosis factor-alpha (TNF-alpha) has no effect. In contrast, epithelial HeLa cells are found to constitutively express C5L2 but not the C5a receptor. In HeLa cells, interferon-gamma and TNF-alpha drastically reduce C5L2 expression. No C5a-dependent Ca(2+) signaling is observed even in these cells endogenously expressing C5L2. Taken together, C5L2 is not a receptor for C3a or C3a-des-Arg(77). Thus, this receptor is unlikely to be directly involved in lipid metabolism. Instead, the identification of stimuli modifying C5L2 expression indicates that C5L2 is a highly regulated scavenger receptor for C5a and C5a-des-Arg(74).

The transcription factors AP-1 and Ets are regulators of C3a receptor expression.

The anaphylatoxin C3a is a proinflammatory mediator generated during complement activation. The tight control of C3a receptor (C3aR) expression is crucial for the regulation of anaphylatoxin-mediated effects. Key factors regulating constitutive expression of the C3aR in the mast cell line HMC-1 and receptor induction by dibutyryl-cAMP in monomyeloblastic U937 cells were determined by functional characterization of the C3aR promoter. Nucleotides -18 to -285 upstream of the translational start site proved to be critical for promoter activity in HMC-1 cells. Binding sites for the transcription factors AP-1 and Ets could be located. Overexpressed c-Jun/c-Fos (AP-1) and Ets-1 led synergistically to increased promoter activity that was substantially reduced by site-directed mutagenesis of the corresponding elements within the C3aR promoter. In HMC-1 cells, Ets interacted directly with the predicted binding motif of the C3aR promoter as determined by electromobility shift assays. AP-1 binding to the C3aR promoter was augmented during C3aR induction in U937 cells. A retroviral gene transfer system was used to express a dominant negative mutant of Ets-1 in these cells. The resulting cells failed to up-regulate the C3aR after stimulation with dibutyryl-cAMP and showed decreased AP-1 binding, suggesting that Ets acts here indirectly. Thus, it was established that Ets and the AP-1 element mediates dibutyryl-cAMP induction of C3aR promoter activity, hence providing a mechanistic explanation of dibutyryl-cAMP-dependent up-regulation of C3aR expression. In conclusion, this study demonstrates an important role of AP-1 and a member of the Ets family in the transcriptional regulation of C3aR expression, a prerequisite for the ability of C3a to participate in immunomodulation and inflammation.

Silencing or permanent activation: host-cell responses in models of persistent Chlamydia pneumoniae infection.

Chlamydia pneumoniae causes respiratory infections. In chronic diseases associated with Chlamydia, such as arteriosclerosis, C. pneumoniae is present in a persistent form, which might participate in pathogenesis of chronic inflammatory disease. To elucidate how these intracellular bacteria modulate host-cells during persistence, we compared the expression pattern of a range of host genes after short (24 h) and long (up to 7 days) times of chlamydia infection in HeLa-cells. One day post infection, in three cell-culture models of persistence, namely treatment with penicillin or IFN-gamma, or iron-depletion, infection induced the genes of CTGF, IL-6, IL-8, IL-11, LIF, EGR-1 and ETV4 in a similar fashion. However, after a longer time, two modes of host-cell reaction emerged that were dependent on the persistence model used. After IFN-gamma and penicillin treatment chlamydia-induced host-cell gene expression was inhibited, while it stayed upregulated in iron-depletion. Human monocytes/macrophages, in which persistence naturally occurs, were additionally investigated: for several genes, UV-inactivated and viable chlamydia caused long-lasting upregulation. Thus, this study reveals (i) the ability of C. pneumoniae to participate in two putative pathomechanisms of persistence, silencing and permanent activation, which might represent different in vivo situations and (ii) a strong dependence on the mode of persistence induction.