Assaying Chlamydia pneumoniae Persistence in Monocyte-Derived Macrophages Identifies Dibenzocyclooctadiene Lignans as Phenotypic Switchers.

Antibiotic-tolerant persister bacteria involve frequent treatment failures, relapsing infections and the need for extended antibiotic treatment. The virulence of an intracellular human pathogen C. pneumoniae is tightly linked to its propensity for persistence and means for its chemosensitization are urgently needed. In the current work, persistence of C. pneumoniae clinical isolate CV6 was studied in THP-1 macrophages using quantitative PCR and quantitative culture. A dibenzocyclooctadiene lignan schisandrin reverted C. pneumoniae persistence and promoted productive infection. The concomitant administration of schisandrin and azithromycin resulted in significantly improved bacterial eradication compared to sole azithromycin treatment. In addition, the closely related lignan schisandrin C was superior to azithromycin in eradicating the C. pneumoniae infection from the macrophages. The observed chemosensitization of C. pneumoniae was associated with the suppression of cellular glutathione pools by the lignans, implying to a previously unknown aspect of chlamydia-host interactions. These data indicate that schisandrin lignans induce a phenotypic switch in C. pneumoniae, promoting the productive and antibiotic-susceptible phenotype instead of persistence. By this means, these medicinal plant -derived compounds show potential as adjuvant therapies for intracellular bacteria resuscitation.

Impact of dibenzocyclooctadiene lignans from Schisandra chinensis on the redox status and activation of human innate immune system cells.

Redox signaling has been established as an essential component of inflammatory responses, and redox active compounds are of interest as potential immunomodulatory agents. Dibenzocyclooctadiene lignans isolated from Schisandra chinensis, a medicinal plant with widespread use in oriental medicine, have been implicated to possess immunomodulatory properties but their effects on the human innate immune system cells have not been described. In this contribution, data are presented on the impact of schisandrin, schisandrin B and schisandrin C on human monocytic cell redox status, as well as their impact on dendritic cell maturation and T cell activation capacity and cytokine production. In THP-1 cells, levels of intracellular reactive oxygen species (ROS) were elevated after 1 h exposure to schisandrin. Schisandrin B and schisandrin C decreased cellular glutathione pools, which is a phenotype previously reported to promote anti-inflammatory functions. Treatment of human primary monocytes with the lignans during their maturation to dendritic cells did not have any effect on the appearance of surface markers HLA-DR and CD86 but schisandrin B and schisandrin C suppressed the secretion of cytokines interleukin (IL)-6, IL-10 and IL-12 by the mature dendritic cells. Dendritic cells maturated in presence of schisandrin C were further cocultured with naïve CD4+ T cells, resulting in reduced IL-12 production. In THP-1 cells, schisandrin B and schisandrin C reduced the IL-6 and IL-12 production triggered by E. coli lipopolysaccharide and IL-12 production induced by an infection with Chlamydia pneumoniae. In conclusion, the studied lignans act as immunomodulatory agents by altering the cytokine secretion, but do not interfere with dendritic cell maturation. And the observed effects may be associated with the ability of the lignans to alter cellular redox status.

Growth of Chlamydia pneumoniae Is Enhanced in Cells with Impaired Mitochondrial Function.

Effective growth and replication of obligate intracellular pathogens depend on host cell metabolism. How this is connected to host cell mitochondrial function has not been studied so far. Recent studies suggest that growth of intracellular bacteria such as Chlamydia pneumoniae is enhanced in a low oxygen environment, arguing for a particular mechanistic role of the mitochondrial respiration in controlling intracellular progeny. Metabolic changes in C. pneumoniae infected epithelial cells were analyzed under normoxic (O2 ≈ 20%) and hypoxic conditions (O2 < 3%). We observed that infection of epithelial cells with C. pneumoniae under normoxia impaired mitochondrial function characterized by an enhanced mitochondrial membrane potential and ROS generation. Knockdown and mutation of the host cell ATP synthase resulted in an increased chlamydial replication already under normoxic conditions. As expected, mitochondrial hyperpolarization was observed in non-infected control cells cultured under hypoxic conditions, which was beneficial for C. pneumoniae growth. Taken together, functional and genetically encoded mitochondrial dysfunction strongly promotes intracellular growth of C. pneumoniae.

Comparative analysis of the growth and biological activity of a respiratory and atheroma isolate of Chlamydia pneumoniae reveals strain-dependent differences in inflammatory activity and innate immune evasion.

BACKGROUND: Chlamydia pneumoniae is a common human pathogen that is associated with upper and lower respiratory tract infections. It has also been suggested that C. pneumoniae infection can trigger or promote a number of chronic inflammatory conditions, including asthma and atherosclerosis. Several strains of C. pneumoniae have been isolated from humans and animals, and sequence data demonstrates marked genetic conservation, leaving unanswered the question as to why chronic inflammatory conditions may occur following some respiratory-acquired infections. METHODS: C. pneumoniae strains AR39 and AO3 were used in vitro to infect murine bone marrow derived macrophages and L929 fibroblasts, or in vivo to infect C57BL/6 mice via the intranasal route. RESULTS: We undertook a comparative study of a respiratory isolate, AR39, and an atheroma isolate, AO3, to determine if bacterial growth and host responses to infection varied between these two strains. We observed differential growth depending on the host cell type and the growth temperature; however both strains were capable of forming plaques in vitro. The host response to the respiratory isolate was found to be more inflammatory both in vitro, in terms of inflammatory cytokine induction, and in vivo, as measured by clinical response and lung inflammatory markers using a mouse model of respiratory infection. CONCLUSIONS: Our data demonstrates that a subset of C. pneumoniae strains is capable of evading host innate immune defenses during the acute respiratory infection. Further studies on the genetic basis for these differences on both the host and pathogen side could enhance our understanding how C. pneumoniae contributes to the development chronic inflammation at local and distant sites.

Mechanisms of apoptosis inhibition in Chlamydia pneumoniae-infected neutrophils.

The obligatory intracellular bacterium Chlamydia pneumoniae (C. pneumoniae) can survive and multiply in neutrophil granulocytes. Since neutrophils are short living cells, inhibition of neutrophil apoptosis appears to play a major role in the productive infection of neutrophils by C. pneumoniae. In the present study, we have investigated which survival pathways and which events of the apoptotic process are modulated in C. pneumoniae-infected neutrophils. All infection experiments were carried out using primary human neutrophils in vitro. We show that infection with C. pneumoniae activates PI3K/Akt as well as the ERK1/2 and p38 MAP kinases and present evidence that activation of the PI3K/Akt and ERK1/2 pathways are essential to initiate the apoptosis delay in C. pneumoniae-infected neutrophils. Both the PI3K/Akt and ERK1/2 pathways are involved in the maintained expression of the anti-apoptotic protein Mcl-1. In addition, we also showed that the PI3K/Akt pathway leads to the activation of NF-κB-dependent release of IL-8 by infected neutrophils. Infection with C. pneumoniae activates the PI3K/Akt and ERK1/2 MAPK survival pathways in neutrophils, induces the NF-κB dependent release of IL-8 and leads to the maintenance of Mcl-1 expression in neutrophils.

Temperature and host cell-dependent changes in virulence of Chlamydia pneumoniae CWL029 in an optimized mouse infection model.

The obligate intracellular bacterium Chlamydia (C.) pneumoniae causes respiratory infections and is associated with vascular diseases. To elucidate how temperature and host cells used for propagation alter chlamydial virulence, C. pneumoniae CWL0129 (Cpn) was cultured at 35 or 37°C in two different cell lines and then applied to mice. These mice infected with differentially propagated chlamydiae showed differences in clinical score, body weight and inflammatory cytokines in the lung. Our study demonstrates that Cpn cultured at 37°C in hamster fibroblast BHK-21 are able to colonize the mouse lung faster and better, and induce stronger symptoms and cytokine induction than bacteria cultured at 35°C. The temperature-triggered virulence alteration could not be observed for Cpn propagated in HeLa cells and was independent of host cell protein synthesis. Transcriptome analysis did not reveal temperature-induced effects on chlamydial gene expression, suggesting that the observed virulence changes are regulated on a different, so far unknown level. Preculture close to the central body temperature of its warm-blooded human or murine host might 'prepare' Cpn for subsequent in vivo infection. Our identification of culture-dependent virulence alteration helps to establish an optimized mouse lung infection model for Cpn and provides the basis to further unravel the molecular mechanisms underlying chlamydial pathogenicity.

AP-1 Transcription Factor Serves as a Molecular Switch between Chlamydia pneumoniae Replication and Persistence.

Chlamydia pneumoniae is a Gram-negative bacterium that causes acute or chronic respiratory infections. As obligate intracellular pathogens, chlamydiae efficiently manipulate host cell processes to ensure their intracellular development. Here we focused on the interaction of chlamydiae with the host cell transcription factor activator protein 1 (AP-1) and its consequence on chlamydial development. During Chlamydia pneumoniae infection, the expression and activity of AP-1 family proteins c-Jun, c-Fos, and ATF-2 were regulated in a time- and dose-dependent manner. We observed that the c-Jun protein and its phosphorylation level significantly increased during C. pneumoniae development. Small interfering RNA knockdown of the c-Jun protein in HEp-2 cells reduced the chlamydial load, resulting in smaller inclusions and significantly lower chlamydial recovery. Furthermore, inhibition of the c-Jun-containing AP-1 complexes using tanshinone IIA changed the replicative infection phenotype into a persistent one. Tanshinone IIA-dependent persistence was characterized by smaller, aberrant inclusions, a strong decrease in the chlamydial load, and significantly reduced chlamydial recovery, as well as by the reversibility of the reduced recovery after the removal of tanshinone IIA. Interestingly, not only was tanshinone IIA treatment accompanied by a significant decrease of ATP levels, but fluorescence live cell imaging analysis by two-photon microscopy revealed that tanshinone IIA treatment also resulted in a decreased fluorescence lifetime of protein-bound NAD(P)H inside the chlamydial inclusion, indicating that chlamydial reticulate bodies have decreased metabolic activity. In all, these data demonstrate that the AP-1 transcription factor is involved in C. pneumoniae development, with tanshinone IIA treatment resulting in persistence.

All subtypes of the Pmp adhesin family are implicated in chlamydial virulence and show species-specific function.

The bacterial pathogens Chlamydia trachomatis and C. pneumoniae are obligate intracellular parasites, cause a number of serious diseases, and can infect various cell types in humans. Chlamydial infections are probably initiated by binding of the bacterial outer membrane protein OmcB to host cell glycosaminoglycans (GAGs). Here, we show that all nine members of the polymorphic membrane protein (Pmp) family of C. trachomatis mediate adhesion to human epithelial and endothelial cells. Importantly, exposure of infectious particles to soluble recombinant Pmps blocks subsequent infection, thus implicating an important function of the entire protein family in the infection process. Analogous experiments with pairs of recombinant Pmps or a combination of Pmp and OmcB revealed that all Pmps probably act in an adhesion pathway that is distinct from the OmcB-GAG pathway. Finally, we provide evidence that the Pmps of C. trachomatis and C. pneumoniae exhibit species and tissue specificity. These findings argue for the involvement of C. trachomatis Pmps in the initial phase of infection and suggest that they may interact with a receptor other than the epidermal growth factor receptor recently identified for their counterparts in C. pneumoniae.

Increased sensitivity to tryptophan bioavailability is a positive adaptation by the human strains of Chlamydia pneumoniae.

One of the most significant activities induced by interferon-gamma against intracellular pathogens is the induction of IDO (indoleamine 2,3-dioxygenase) expression, which subsequently results in the depletion of tryptophan. We tested the hypothesis that human strains of Chlamydia pneumoniae are more sensitive to tryptophan limitation than animal C. pneumoniae strains. The human strains were significantly more sensitive to IFN-γ than the animal strains in a lung epithelia cell model (BEAS-2B), with exposure to 1 U ml(-1) IFN-γ resulting in complete loss of infectious yield of human strains, compared to the animal strains where reductions in infectious progeny were around 3.5-4.0 log. Strikingly, the IFN-γ induced loss of ability to form infectious progeny production was completely rescued by removal of the IFN-γ and addition of exogenous tryptophan for the human strains, but not the animal strains. In fact, a human heart strain was more capable of entering a non-infectious, viable persistent stage when exposed to IFN-γ and was also more effectively rescued, compared to a human respiratory strain. Exquisite susceptibility to IFN-γ, specifically due to tryptophan availability appears to be a core adaptation of the human C. pneumoniae strains, which may reflect the chronic nature of their infections in this host.

Antimicrobial effect of natural polyphenols with or without antibiotics on Chlamydia pneumoniae infection in vitro.

Chlamydia pneumoniae is a human pathogen that causes multiple diseases worldwide. Despite appropriate therapy with antichlamydial antibiotics, chronic exacerbated diseases often occur and lead to serious sequelae. The use of the macrolide clarithromycin and the fluoroquinolone ofloxacin has improved the treatment of chlamydial infection, but therapy failure is still a major problem. In this work, we studied the pretreatment with natural polyphenols and subsequent treatment with clarithromycin or ofloxacin. The phenolic compounds resveratrol and quercetin improved the antichlamydial effect of clarithromycin and ofloxacin. In particular, resveratrol at 40 µM and quercetin at 20 µM exhibited significant growth inhibition on C. pneumoniae in presence of clarithromycin or ofloxacin compared to controls. In addition, we demonstrated that both resveratrol and quercetin decreased IL-17 and IL-23 production in a time-dependent manner in C. pneumoniae-infected cells. The results showed a particularly strong inhibition of the IL-23 levels released with combined treatment of resveratrol or quercetin and ofloxacin or clarithromycin, suggesting that the combined treatment may afford a synergistic effect in controlling Chlamydia infections.

Chlamydia pneumoniae infection in atherosclerotic lesion development through oxidative stress: a brief overview.

Chlamydia pneumoniae, an obligate intracellular pathogen, is known as a leading cause of respiratory tract infections and, in the last two decades, has been widely associated with atherosclerosis by seroepidemiological studies, and direct detection of the microorganism within atheroma. C. pneumoniae is presumed to play a role in atherosclerosis for its ability to disseminate via peripheral blood mononuclear cells, to replicate and persist within vascular cells, and for its pro-inflammatory and angiogenic effects. Once inside the vascular tissue, C. pneumoniae infection has been shown to induce the production of reactive oxygen species in all the cells involved in atherosclerotic process such as macrophages, platelets, endothelial cells, and vascular smooth muscle cells, leading to oxidative stress. The aim of this review is to summarize the data linking C. pneumoniae-induced oxidative stress to atherosclerotic lesion development.

Host metabolism promotes growth of Chlamydia pneumoniae in a low oxygen environment.

Chlamydia pneumoniae infections of the respiratory tract are common and are associated with acute and chronic diseases such as community-acquired pneumonia (CAP) and chronic obstructive pulmonary disease (COPD). Recent studies have shown that reduced environmental oxygen availability promotes chlamydial growth in infected host cells. The underlying mechanisms remain unclear. We performed a targeted siRNA screen coupled with an automated high-throughput microscopic analysis to identify key host cell genes that play a role in promoting the hypoxic growth of C. pneumoniae. A total of 294 siRNAs - targeting 98 selected genes including central mediators of metabolic, trafficking and signaling pathways - were tested on chlamydial inclusion formation in C. pneumoniae infected A549 cells under normoxic (20% O2) and hypoxic (2% O2) conditions 48 h post infection. Evaluation of the different functional clusters of genes revealed that under hypoxic conditions, enhanced growth of C. pneumoniae was centrally mediated by the host cell glycolytic pathway. Inhibition of the phosphofructokinase (PFK), lactate dehydrogenase (LDH), glycerol-3-phosphate dehydrogenase (GPD2) and the forkheadbox O3 (FOXO3) gene-expression by siRNAs abrogated chlamydial progeny. The pivotal role of host cell glycolysis in chlamydial development under hypoxia was further confirmed by pharmacological inhibition of the pathway by 2-fluoro-deoxy-glucose. The results indicate that the microenvironment of the host cell determines the fate of C. pneumoniae by controlling pathogen-induced metabolic pathways.

Neutrophil gelatinase-associated lipocalin and interleukin-10 regulate intramacrophage Chlamydia pneumoniae replication by modulating intracellular iron homeostasis.

Neutrophil gelatinase-associated lipocalin (NGAL/Lipocalin-2/Lcn-2) is a 25kDa protein which is involved in host defence against certain Gram negative bacteria upon binding of iron loaded bacterial siderophores thereby limiting the availability of this essential nutrient to bacteria resulting in inhibition of their growth and pathogenicity. As iron is important for the growth of the intracellular bacterium Chlamydia pneumoniae we questioned whether Lcn-2 affects the course of this infection. We employed primary peritoneal macrophages obtained from wildtype and Lcn-2 -/- mice and RAW 264.7 cells which were infected with C. pneumoniae. In addition, we studied C. pneumoniae multiplication in vivo in mice receiving diets with varying iron contents. We analyzed C. pneumoniae numbers by immunohistochemistry and RT-PCR and studied the expression of iron metabolism and cytokine genes by RT-PCR, Western blot or ELISA. Infection with Chlamydiae ex vivo and in vivo revealed a significantly higher bacterial growth in peritoneal macrophages of Lcn-2 -/- than of wildtype mice. These differences were significantly more pronounced upon iron challenge, which stimulated bacterial growth. Accordingly, treatment with an anti-Lnc-2 antibody increased whereas addition of recombinant Lcn-2 reduced bacterial growth in infected macrophages. When investigating the underlying mechanisms we observed partly different expression of several iron metabolism genes between Lcn-2 +/+ and Lcn-2 -/- macrophages and most strikingly an increased formation of the anti-inflammatory cytokine IL-10 by Lcn-2 -/- macrophages. Upon treatment with an anti-IL10 antibody we experienced a significant increase of Chlamydial growth within Lcn-2 -/- macrophages along with a reduction of the major iron storage protein ferritin. Herein we provide first time evidence that Lcn-2 is involved in host defence against Chlamydia presumably by limiting the availability of iron to the pathogen. In the absence of Lcn-2, increased formation of IL-10 exerts protective effects by increasing the intracellular formation of ferritin, thereby reducing the access of iron for bacteria.

Host cell Golgi anti-apoptotic protein (GAAP) and growth of Chlamydia pneumoniae.

Chlamydia pneumoniae protein CPn0809 is a type three secretion system substrate, the exact function of which in infection pathogenesis has remained unknown. In this study, we identified by yeast two-hybrid screening a potential host cell interaction partner of CPn0809, Golgi anti-apoptotic protein (GAAP), a conserved protein found in eukaryotic cells. GAAP gene is expressed at relatively constant levels and its expression remained stable also after C. pneumoniae infection. The interaction between GAAP and C. pneumoniae was suggested by transfection studies. GAAP knock-down by siRNA in infected A549 cells resulted in an increased number of C. pneumoniae genomes and growth of the bacteria as judged by quantitative PCR and inclusion counts, respectively. Silencing of GAAP did not make the A549 cells more susceptible to apoptosis per se, and infection with C. pneumoniae prevented staurosporin-induced apoptosis also in transfected cultures. Taken together, the proposed interaction between C. pneumoniae and GAAP modulates bacterial growth in A549 cells.

Induction of VEGF and MMP-9 expression by toll-like receptor 2/4 in human endothelial cells infected with Chlamydia pneumoniae.

Matrix metalloproteinases (MMP) are a family of host-derived enzymes involved in the turnover of extracellular matrix (ECM) molecules, and, in particular, it is demonstrated that the 92 KDa gelatinase MMP-9 is often expressed in atherosclerotic plaques by macrophages and smooth muscle cells. Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerosis lesions. In this study, we analyzed the TLR2/TLR4 expression in HUVEC infected with C. pneumoniae and correlated it to the production of VEGF and MMP-9. The results obtained showed an increased VEGF and MMP-9 production correlated with a time-dependent increase in cellular proliferation in HUVEC infected with C. pneumoniae at a multiplicity of infection (MOI) of 2 IFU/cell. HUVEC preincubated with VEGF antibody did not release MMP-9, as detected by zymography assessment and ELISA assay. In addition, we demonstrated that TLR2/TLR4 are expressed in HUVEC infected with viable microorganisms (25% and 17%, respectively), while UV-inactivated microorganisms induced a lesser expression (20% and 11%, respectively) compared to control cells and HUVEC exposed to heat-killed bacteria showed a percentage of TLR-expressing cells similar to the control cells. In addition, the cells preincubated for 60 min with TLR2/TLR4 neutralizing antibodies showed a decrease in C. pneumonia-induced VEGF and MMP-9 production.

Flotillin-1 (Reggie-2) contributes to Chlamydia pneumoniae growth and is associated with bacterial inclusion.

Chlamydiae are obligate intracellular pathogens replicating only inside the eukaryotic host. Here, we studied the effect of human flotillin-1 protein on Chlamydia pneumoniae growth in human line (HL) and A549 epithelial cell lines. RNA interference was applied to disrupt flotillin-1-mediated endocytosis. Host-associated bacteria were detected by quantitative PCR, and C. pneumoniae growth was evaluated by inclusion counts. C. pneumoniae attachment to host cells was unaffected, but bacterial intracellular growth was attenuated in the flotillin-1-silenced cells. By using confocal microscopy, we detected flotillin-1 colocalized with the inclusion membrane protein A (IncA) in the C. pneumoniae inclusion membranes. In addition, flotillin-1 was associated with IncA in detergent-resistant membrane microdomains (DRMs) in biochemical fractioning. These results suggest that flotillin-1 localizes to the C. pneumoniae inclusion membrane and plays an important role for intracellular growth of C. pneumoniae.

Nifedipine affects the course of Salmonella enterica serovar Typhimurium infection by modulating macrophage iron homeostasis.

BACKGROUND: Iron overload can adversely influence the course of infection by increasing microbial replication and suppressing antimicrobial immune effector pathways. Recently, we have shown that the calcium channel blocker nifedipine can mobilize tissue iron in mouse models of iron overload. We therefore investigated whether nifedipine treatment affects the course of infection with intracellular bacteria via modulation of iron homeostasis. METHODS: The effect of nifedipine on intramacrophage replication of bacteria and modulation of cellular iron homeostasis was investigated in the murine macrophage cell line RAW264.7, and the impact of nifedipine treatment on the course of systemic infection was investigated in C57BL/6 mice in vivo. RESULTS: In RAW264.7 cells, nifedipine treatment significantly reduced intracellular bacterial survival of Salmonella enterica serovar Typhimurium and Chlamydophila pneumoniae. This could be attributed to the induction of the iron exporter ferroportin 1, which limited the availability of iron for intracellular Salmonella. When C57BL/6 mice were infected intraperitoneally with Salmonella and subsequently injected with nifedipine for 3 consecutive days, bacterial counts in livers and spleens were significantly reduced and survival of the mice significantly was prolonged compared with solvent-treated littermates. Nifedipine treatment increased expression of ferroportin 1 in the spleen, whereas splenic levels of the iron storage protein ferritin and serum iron concentrations were reduced. CONCLUSIONS: Our data provide evidence for a novel mechanism whereby nifedipine enhances host resistance to intracellular pathogens via limitation of iron availability.

Chlamydophila pneumoniae attachment and infection in low proteoglycan expressing human lymphoid Jurkat cells.

This study investigated the proteoglycan (PG)-dependent mechanism of Chlamydophila pneumoniae attachment to lymphocytic cells. Lymphoid Jurkat cells and epithelial HEp-2 cells were statically infected with C. pneumoniae (TW183). Transmission electron microscopy and assessment of inclusion-forming units indicated that the bacteria grew normally in Jurkat cells and were capable of producing secondary infection; however, they grew at a slower rate than in HEp-2 cells. RT-PCR analysis indicated that HEp-2 cells strongly expressed PG-core protein encoding genes, thereby sustaining glycosaminoglycans (GAGs), such as heparin, on the cellular surface. Similar gene expression levels were not observed in Jurkat cells, with the exception of glypican-1. Immunofluorescence analysis also supported strong heparin expression in HEp-2 cells and minimal expression in Jurkat cells, although heparan sulfate pretreatment significantly inhibited bacterial attachment to both cell types. Immunofluorescent co-staining with antibodies against chlamydial LPS and heparin did not identify bacterial and heparin co-localization on Jurkat cells. We also confirmed that when C. pneumoniae was statically infected to human CD4(+) peripheral blood lymphocytes known not expressing detectable level of heparin, the bacteria attached to and formed inclusion bodies in the cells. Thus, the attachment mechanism of C. pneumoniae to Jurkat cells with low PG expression is unique when compared with HEp-2 cells and potentially independent of GAGs such as heparin.

Analysis of Chlamydia pneumoniae infection in mononuclear cells by reverse transcription-PCR targeted to chlamydial gene transcripts.

Chlamydia pneumoniae (C. pneumoniae) is an important etiological agent of respiratory infections including pneumonia. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that monocytes can assist the spread of infection to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells, we have established an in vitro model in the human Mono Mac 6 cell line. In the present study, we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real-time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells. Furthermore, our study shows that genes related to secretion are transcribed, and secreted bacterial proteins are also translated during infection of monocytes, creating novel opportunities for the management of chlamydial infection of monocytes.

Increased inflammation and impaired resistance to Chlamydophila pneumoniae infection in Dusp1(-/-) mice: critical role of IL-6.

The MAPK phosphatase DUSP1 is an essential negative regulator of TLR-triggered innate immune activation. Here, we have investigated the impact of DUSP1 on inflammatory and antimicrobial host responses to the intracellular pathogen Chlamydophila pneumoniae. Following nasal infection, DUSP1-deficient mice mounted an enhanced pulmonary cytokine (IL-1beta, IL-6) and chemokine response (CCL3, CCL4, CXCL1, CXCL2), leading to increased leukocyte infiltration. Of interest, the increased inflammatory response, in the absence of DUSP1, was associated with higher bacterial numbers in the lungs, although the expression of IFN-gamma and critical antichlamydial effector molecules, such as iNOS, was intact. Blockade of IL-6 trans-signaling by injection of a soluble gp130-Fc fusion protein corrected the overshooting chemokine production as well as the increased chlamydial load in Dusp1(-/-) mice. Furthermore, IL-6 enhanced the replication of C. pneumoniae in embryonic fibroblasts in vitro. These data show that DUSP1 is required to achieve a balanced response to chlamydial infection and identify IL-6 as critical for amplifying inflammation and benefiting chlamydial growth through direct effects on infected cells.