Differential signaling pathways are initiated in macrophages during infection depending on the intracellular fate of Chlamydia spp.

Chlamydia muridarum and Chlamydia caviae have equivalent growth rates in mouse epithelial cells but only C. muridarum replicates inside mouse macrophages, while C. caviae does not. Macrophages infected with C. muridarum or C. caviae were used to address the hypothesis that the early signaling pathways initiated during infection depend on the fate of chlamydiae in the host cell. Transmission electron microscopy of C. muridarum-infected macrophages showed intact chlamydial elementary bodies and reticulate bodies 2 h postinfection in compact vacuoles. Conversely, in macrophages infected with C. caviae, chlamydiae were observed in large phagocytic vacuoles. Furthermore, C. caviae infections failed to develop into inclusions or produce viable bacteria. Expression of proinflammatory cytokines TNFα, IL-1ß and MMP13 was similar in C. caviae- or C. muridarum-infected macrophages at 3 h postinfection, indicating that chlamydial survival is not required for initiation of these responses. IL-1ß secretion, dependent on inflammasome activation, occurred in C. caviae-infected macrophages despite no chlamydial growth. Conversely, IFNß mRNA was observed only in C. muridarum- but not in C. caviae-infected macrophages. These data demonstrate that differential signaling events are initiated during a productive versus nonproductive chlamydial infection in a macrophage.

Progesterone antagonizes the positive influence of estrogen on Chlamydia trachomatis serovar E in an Ishikawa/SHT-290 co-culture model.

Studies indicate that estrogen enhances Chlamydia trachomatis serovar E infection in genital epithelial cells. Hormones have direct and indirect effects on endometrial epithelial cells. Estrogen and progesterone exposure induces endometrial stromal cells to release effectors that subsequently regulate growth and maturation of uterine epithelial cells. Estrogen enhances C. trachomatis infection by aiding entry and intracellular development in endometrial epithelial cell (Ishikawa, IK)/SHT-290 stromal cell co-culture. Enhanced chlamydial infection was mediated by direct estrogen-stimulated signaling events in epithelial cells and indirectly via estrogen-induced stromal cell effectors. The current study investigates the effects of hormones on chlamydial development using culture conditions representative of the menstrual cycle. Chlamydia trachomatis-infected IK or IK/SHT-290 cultures were exposed to 10(-8) M estrogen (E2), 10(-7) M progesterone (P4) or a combination of both hormones (10(-8) M E2 followed by 10(-9) M E2/10(-7) M P4). Chlamydial infectivity and progeny production were significantly decreased (30-66%) in cultures exposed to progesterone or estrogen/progesterone combination compared to estrogen alone. Thus, progesterone antagonized the positive effects of estrogen on chlamydial infection. These data indicate the susceptibility of endometrial epithelial cells to C. trachomatis infection during the menstrual cycle is altered by phase specific actions of sex hormones in the genital tract.

The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-ß during Chlamydia trachomatis infection.

IFN-ß has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-ß expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-ß expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-ß expression. We determined that cGAS is required for IFN-ß expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-ß, coculture of cells depleted for either STING or cGAS rescued IFN-ß expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-ß expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-ß expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.

In vivo ultrastructural analysis of the intimate relationship between polymorphonuclear leukocytes and the chlamydial developmental cycle.

We utilized a recently developed model of intracervical infection with Chlamydia muridarum in the mouse to elicit a relatively synchronous infection during the initial developmental cycle in order to examine at the ultrastructural level the development of both the chlamydial inclusion and the onset of the inflammatory response. At 18 h after infection, only a few elementary bodies attached to cells were visible, as were an occasional intracellular intermediate body and reticulate body. By 24 h, inclusions had 2 to 5 reticulate bodies and were beginning to fuse. A few polymorphonuclear leukocytes (PMNs) were already present in the epithelium in the vicinity of and directly adjacent to infected cells. By 30 h, the inclusions were larger and consisted solely of reticulate bodies, but by 36 to 42 h, they contained intermediate bodies and elementary bodies as well. Many PMNs were adjacent to or actually inside infected cells. Chlamydiae appeared to exit the cell either (i) through disintegration of the inclusion membrane and rupture of the cell, (ii) by dislodgement of the cell from the epithelium by PMNs, or (iii) by direct invasion of the infected cell by the PMNs. When PMNs were depleted, the number of released elementary bodies was significantly greater as determined both visually and by culture. Interestingly, depletion of PMNs revealed the presence of inclusions containing aberrant reticulate bodies, reminiscent of effects seen in vitro when chlamydiae are incubated with gamma interferon. In vivo evidence for the contact-dependent development hypothesis, a potential mechanism for triggering the conversion of reticulate bodies to elementary bodies, and for translocation of lipid droplets into the inclusion is also presented.

The multifaceted role of oestrogen in enhancing Chlamydia trachomatis infection in polarized human endometrial epithelial cells.

The oestrogen receptor (ER) α-ß+ HEC-1B and the ERα+ß+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERß alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERß antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.

Chlamydia trachomatis persistence in vitro: an overview.

Chlamydiae growing in target mucosal human epithelial cells in vitro can transition from their normal developmental cycle progression, alternating between infectious but metabolically inactive elementary bodies to metabolically active but noninfectious reticulate bodies (RBs) and back to elementary bodies, into a state of persistence. Persistence in vitro is defined as viable but noncultivable chlamydiae involving morphologically enlarged, aberrant, and nondividing RBs. The condition is reversible, yielding infectious elementary bodies after removal of the inducers, including penicillin, interferon-gamma, iron or nutrient starvation, concomitant herpes infection, or maturation of the host cell into its physiologically differentiated state. All aberrant RB phenotypes are not the same, owing to differing up- or down-regulated chlamydial gene sets and subsequent host responses. Although all persistence-inducing conditions exist in vivo, key questions include (1) whether or not aberrant chlamydial RBs occur in vivo during the alternating acute-silent chronic-acute chlamydial infection scenario that exists in infected patients and animals and (2) whether such aberrant RBs can contribute to prolonged, chronic inflammation, fibrosis, and scarring.

Host chemokine and cytokine response in the endocervix within the first developmental cycle of Chlamydia muridarum.

The initial host response in a primary chlamydial infection is the onset of acute inflammation. However, we still know very little about the early temporal events in the induction of the acute inflammatory response and how these events relate to the initial chlamydial developmental cycle in an actual genital infection. Because it was critical to initiate a synchronous infection in the endocervix in the first 24 h to evaluate the sequential expression of the host response, we developed the surgical methodology of depositing Chlamydia muridarum directly on the endocervix. Cervical tissue was collected at 3, 12, and 24 h after inoculation and the expression array of chemokines, cytokines, and receptors was assessed to characterize the response during the initial developmental cycle. Polymorphonuclear leukocyte (PMN) infiltration was first observed at 12 h after inoculation, and a few PMNs could be seen in the epithelium at 24 h. Electron microscopic analysis at 24 h showed that virtually all inclusions were at the same stage of development, indicating a synchronous infection. Several chemokine and cytokine genes were expressed as early as 3 h after infection, but by 12 h, 41 genes were expressed. Thus, activation of the host response occurs both with the introduction of elementary bodies into the host and early replication of reticulate bodies. No significant response was observed when UV-inactivated organisms were inoculated into the cervix at any time interval. This model provides an ideal opportunity to investigate the mechanisms by which the early inflammatory response is induced in vivo.

Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells.

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.

Chlamydiae and polymorphonuclear leukocytes: unlikely allies in the spread of chlamydial infection.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells.

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.

Differences in Chlamydia trachomatis serovar E growth rate in polarized endometrial and endocervical epithelial cells grown in three-dimensional culture.

In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells.

The lipid A 1-phosphatase of Helicobacter pylori is required for resistance to the antimicrobial peptide polymyxin.

Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.

Ultrastructural analysis of chlamydial antigen-containing vesicles everting from the Chlamydia trachomatis inclusion.

Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.

Chlamydia trachomatis enters a viable but non-cultivable (persistent) state within herpes simplex virus type 2 (HSV-2) co-infected host cells.

Epidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo. We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in co-infected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 co-infection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL, ftsK, ftsW, dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but non-cultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo, these results also suggest that disease severity might be increased in co-infected individuals.

Characterization of estrogen-responsive epithelial cell lines and their infectivity by genital Chlamydia trachomatis.

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) >> MCF-7 (57%)>Ishikawa (51%) >> HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.

Pre-exposure of infected human endometrial epithelial cells to penicillin in vitro renders Chlamydia trachomatis refractory to azithromycin.

OBJECTIVE: The clinical significance of the potential for persistent human chlamydial infections in vivo is being actively reassessed because of the increased frequency of recurrent infection with the same serovar despite compliance with an effective antibiotic regimen. The ability to extend the length of time of in vitro cultivation of polarized human endometrial epithelial cells (HEC-1B) provided the opportunity to establish a model system to determine if a persistent form of Chlamydia trachomatis had the same susceptibility as the actively growing form to a cidal concentration of azithromycin. METHODS: Polarized HEC-1B cells cultivated on extracellular matrix were infected with C. trachomatis serovar E and exposed to penicillin at 24 h post-infection (hpi) to induce a persistent infection characterized by slowly metabolizing but non-dividing, ultrastructurally aberrant reticulate bodies within the chlamydial inclusion; at 48 hpi, infected cultures were exposed to a bactericidal concentration of azithromycin for 72 h. RESULTS: Persistent chlamydiae were phenotypically resistant to azithromycin; the number of chlamydial inclusions on subpassage of progeny from persistent chlamydiae following removal of penicillin and recovery was essentially the same as from progeny from persistent chlamydiae following removal of penicillin and azithromycin and recovery. Neutrophils were attracted in vitro to persistently infected HEC-1B cells that had been exposed to penicillin and azithromycin. CONCLUSIONS: Thus, this study provides evidence at the cellular microbiology level in vitro for mechanisms that could exist in vivo to create sustained, but perhaps clinically inapparent inflammation, which might eventually lead to conditions such as silent pelvic inflammatory disease.

Nuclear factor-kappaB activation in endothelium by Chlamydia pneumoniae without active infection.

Causative molecular mechanisms accounting for the potential link between Chlamydia pneumoniae and atherosclerosis are unknown. Formalin and heat-inactivated C. pneumoniae activated the transcription factor nuclear factor (NF)-kappaB in cultured porcine endothelium and up-regulated the expression of E-selectin messenger RNA and protein. This up-regulation was abolished by an IkappaB super-repressor, an NF-kappaB-specific inhibitor. Live bacteria are not necessary for the activation of endothelial NF-kappaB, and C. pneumoniae may contribute to atherogenesis without active infection.

Primary cultures of female swine genital epithelial cells in vitro: a new approach for the study of hormonal modulation of Chlamydia infection.

Previous studies have demonstrated that female reproductive hormones influence chlamydial infection both in vivo and in vitro. Due to the reduced availability of human genital tissues for research purposes, an alternative hormone-responsive model system was sought to study chlamydial pathogenesis. Mature female swine eliminated from breeding programs were selected as the animals of choice because of the similarity of a sexually transmitted disease syndrome and sequelae in swine to a disease syndrome and sequelae found in humans, because of the near identity of a natural infectious chlamydial isolate from swine to Chlamydia trachomatis serovar D from humans, and because a pig's epithelial cell physiology and the mean length of its estrous cycle are similar to those in humans. Epithelial cells from the cervix, uterus, and horns of the uterus were isolated, cultivated in vitro in Dulbecco's minimum essential medium-Hanks' F-12 (DMEM-F-12) medium with and without exogenous hormone supplementation, and analyzed for Chlamydia suis S-45 infectivity. The distribution of chlamydial inclusions in swine epithelial cells was uneven and was influenced by the genital tract site and hormone status. This study confirmed that, like primary human endometrial epithelial cells, estrogen-dominant swine epithelial cells are more susceptible to chlamydial infection than are progesterone-dominant cells. Further, the more differentiated luminal epithelial cells were more susceptible to infection than were glandular epithelial cells. Interestingly, chlamydial growth in mature luminal epithelia was morphologically more active than in glandular epithelia, where persistent chlamydial forms predominated. Attempts to reprogram epithelial cell physiology and thereby susceptibility to chlamydial infection by reverse-stage, exogenous hormonal supplementation were unsuccessful. Freshly isolated primary pig epithelial cells frozen at -80 degrees C in DMEM-F-12 medium with 10% dimethyl sulfoxide for several weeks can, after thawing, reform characteristic polarized monolayers in 3 to 5 days. Thus, primary swine genital epithelia cultured ex vivo appear to be an excellent cell model for dissecting the hormonal modulation of several aspects of chlamydial pathogenesis and infection.

Differences in innate immune responses (in vitro) to HeLa cells infected with nondisseminating serovar E and disseminating serovar L2 of Chlamydia trachomatis.

The inflammatory response associated with Chlamydia trachomatis genital infections is thought to be initiated by the release of proinflammatory cytokines from infected epithelial cells. This study focuses on the interactions between C. trachomatis-infected HeLa cells and immune cells involved in the early stages of infection, i.e., neutrophils and macrophages. First, we showed that the expression of interleukin-11 (IL-11), an anti-inflammatory cytokine mainly active on macrophages, was upregulated at the mRNA level in the genital tracts of infected mice. Second, incubation of differentiated THP-1 (dTHP-1) cells or monocyte-derived macrophages (MdM) with basal culture supernatants from C. trachomatis serovar E- or serovar L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha (TNF-alpha) and upregulation of indoleamine 2,3-deoxygenase (IDO) but not of Toll-like receptor 2 and 4 mRNA expression. Third, coculture of infected HeLa cells with dTHP-1 cells resulted in a reduction in chlamydial growth, which was more dramatic for serovar E than for L2 and which was partially reversed by the addition of anti-TNF-alpha antibodies for serovar E or exogenous tryptophan for both serovars but was not reversed by the addition of superoxide dismutase or anti-IL-8 or anti-IL-1beta antibodies. A gamma interferon-independent IDO mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Lastly, with a two-stage coculture system, we found that (i) supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal and induced MdM to express antichlamydial activity and (ii) although polymorphonuclear leukocytes released more proinflammatory cytokines in response to serovar E- than in response to L2-infected cells, MdM were strongly activated by serovar L2 infection, indicating that the early inflammatory response generated with a nondisseminating or a disseminating strain is different.

Surface accessibility of the 70-kilodalton Chlamydia trachomatis heat shock protein following reduction of outer membrane protein disulfide bonds.

Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is the Chlamydia trachomatis Hsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.