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Infection and Immunity, January 2009, p. 76-84, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00963-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Role for the Chlamydial Type III Secretion Apparatus in Host Cytokine Expression

Daniel Prantner¹ and Uma M. Nagarajan^1,2*

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,¹ Division of Pediatric Infectious Diseases, Arkansas Children's Hospital Research Institute, Little Rock, Arkansas 72202²

Received 31 July 2008/ Returned for modification 31 August 2008/ Accepted 8 October 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In many important human pathogens, such as Shigella and Salmonella spp., the bacterial type III secretion (T3S) apparatus is required to initiate inflammation via activation of caspase-1- or NF-B-dependent genes. Using an ex vivo infection model, the goal of the present study was to determine whether the chlamydial T3S apparatus also modulates the host inflammatory response. Infections of mouse peritoneal macrophages were performed with Chlamydia muridarum, and the expression of inflammatory cytokines was monitored by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. Since there is no current genetic system for Chlamydia spp., blockade of T3S was accomplished pharmacologically using a T3S inhibitor called INP0007. It has been previously shown that INP0007 also blocks chlamydial growth in vitro and that the addition of exogenous iron completely reverses this deficit. The addition of iron to INP0007-treated C. muridarum-infected macrophages not only restored chlamydial growth deficit caused by INP0007 but also led to a multi-inclusion phenotype. Overall, T3S inhibition led to decreased interleukin-6 (IL-6), IL-1β, and CXCL10, whereas the tumor necrosis factor alpha levels were unchanged. Rescue of chlamydial growth by addition of iron sulfate did not restore cytokine production, implying that the decreased expression of many cytokines during infection was dependent on T3S and not solely on growth. In addition, the observation that the greatest effects of INP0007 were seen at late time points during infection suggests that a temporally regulated T3S effector protein(s) may be triggering the host cytokine response.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chlamydia trachomatis is an important human pathogen causing over 90 million new cases of infection per year worldwide, inducing pathology at multiple anatomical sites. Ocular serovars can cause trachoma (reviewed in reference 48), the world's leading cause of preventable blindness, while genital tract infections in women can lead to ectopic pregnancy, pelvic inflammatory disease, and infertility secondary to scarring of the fallopian tubes (23). Studying the host inflammation central to these outcomes has become an intense focus of investigation. The mouse pathogen C. muridarum has been fully sequenced, and its genome shows high conservation with C. trachomatis in both sequence and gene order (35), making C. muridarum an invaluable tool for translational studies in the mouse model. In particular, it was shown that genital infection of female Toll-like receptor 2 (TLR2) knockout (KO) mice result in decreased production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-) and the neutrophil chemokine, macrophage-inflammatory protein-2 (Mip-2) compared to wild-type controls. Significantly, the TLR2 KO mice also had decreased oviduct pathology (8). In addition, caspase-1 KO mice, which are defective in their ability to process the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 (4, 14), also exhibit less oviduct pathology during a primary infection with C. muridarum (6). Similarly, blocking IL-1 signaling with a receptor antagonist prevented the tissue destruction of human fallopian tube organ cultures infected with C. trachomatis (19), strengthening the association between overactive host inflammation and pathology. However, the chlamydial factors influencing inflammation are less understood.

Chlamydia spp. possess a biphasic life cycle lasting approximately 24 to 32 h, replicating inside a plasma membrane-derived vacuole termed the inclusion. The bacteria avoid lysosomal degradation by modifying their inclusion, enabling escape into the host exocytic pathway (38). It is hypothesized that the chlamydial type III secretion (T3S) apparatus plays a central role in this process. The T3S apparatus is a large multiprotein syringelike structure that facilitates targeted secretion of bacterial effector proteins directly into the host cytosol (12). This apparatus is highly conserved among different bacteria and is common to at least 15 gram-negative human pathogens. There are many examples in the literature addressing the involvement of T3S in inflammation, including studies with Salmonella spp. (25), Yersinia spp. (40), Shigella spp. (47), Pseudomonas spp. (11, 13, 26, 46), and Burkholderia spp. (44), which have shown that a functional T3S apparatus was required for caspase-1 activation and/or IL-1β secretion. In addition, using a broader microarray approach, a large proportion of NF-B-dependent cytokines such as IL-6, Mip-2, and monocyte chemoattractant protein 1 were significantly upregulated in macrophages infected with wild-type Edwardsiella tarda compared to infections utilizing a T3S-deficient mutant (31). Transcriptional analyses of transformed HeLa cervical epithelial cells (34, 52) and the human monocytic cell line THP-1 (36) have demonstrated that inflammatory cytokines such as IL-6 and proIL-1β also get upregulated after infection with C. trachomatis, but the T3S dependence has not yet been examined. Together, these studies highlight the rationale for determining whether the chlamydial T3S apparatus modulates the host inflammatory response.

Currently, there is no system for genetic manipulation of Chlamydia spp., and no known T3S mutant has been isolated through other means. However, the speculation that T3S antagonists could be used as next-generation antibiotics, so-called "virulence blockers" (21), led to mass screening using a high-throughput reporter system searching for small organic compounds that inhibit T3S in Yersinia pseudotuberculosis (20). One specific agent called either compound 1 or INP0007 was identified and subsequently shown to inhibit secretion of T3S effectors in both Yersinia spp. (29) and Salmonella spp. (18). INP0007 and other structurally related salicylidene acylhydrazide compounds were also tested for efficacy against Chlamydia spp. and were shown to inhibit growth and development of both C. trachomatis and C. pneumoniae in vitro (2, 27, 41, 51), highlighting the potential necessity of chlamydial T3S for intracellular survival. However, this growth restriction can be overcome by the addition of exogenous iron (41), leading to speculation that T3S effectors may also play a crucial role in iron acquisition in vivo (21). Based on the involvement of T3S in the inflammatory response for other pathogenic bacteria, it is predicted that optimal host cytokine production during chlamydial infection will require functional T3S. The goal of the present study was to examine how T3S blockade effects the induction of inflammatory cytokine responses during in vitro C. muridarum infections.

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Chlamydial stocks and cell lines. C. muridarum Nigg strain was propagated in Mycoplasma-free McCoy cells grown in Dulbecco modified Eagle medium supplemented with 100 µM nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 10% fetal bovine serum, 50 mg of gentamicin sulfate/ml, and 0.5 mg of cycloheximide/ml. Infectious elementary bodies were isolated from McCoy cells by sonication, washed in phosphate-buffered saline, resuspended in SPG buffer (250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid [pH 7.2]), and stored at –80°C.

Chemicals. The T3S inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide or INP0007 (also known as compound 1) (51) was purchased from ChemBridge corporation and dissolved in dimethyl sulfoxide (Sigma) at 10 mM, divided into aliquots, and stored at –20°C until use. The antibiotic chloramphenicol was dissolved in ethanol at 10 mg/ml and stored at –20°C, whereas rifampin (Fisher) was dissolved in dimethyl sulfoxide at 35 mg/ml and stored at 4°C. Iron sulfate was dissolved in Millipore water at 10 mM and stored at 4°C. The TLR3 ligand poly(I-C) (InvivoGen) was reconstituted according to manufacturer's instructions and frozen in aliquots at –20°C at a concentration of 2.5 mg/ml.

Mouse macrophages and in vitro infections. Ten- to twelve-week-old female C57BL/6J mice (Jackson Laboratories) were injected in the peritoneum with 1 ml of 3% thioglycolate. Three days later, the peritoneal cavities were rinsed three times with 3 ml of complete medium (RPMI medium supplemented with 10% fetal bovine serum, 100 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 µM nonessential amino acids, 100 U of penicillin/ml, 100 µg of streptomycin/ml, 50 mM β-mercaptoethanol) to isolate macrophages. Cells were washed, resuspended in complete medium, and plated at 10⁶ cells per well in 24-well tissue culture dishes (BD Falcon, San Jose, CA) in an incubator set to 37°C and 5% CO₂. The medium was aspirated 2 h after plating and replaced with fresh medium to remove nonadherent cells. All infections were performed in antibiotic-free complete media on cells that were allowed to rest in culture for at least 48 h after isolation from the mouse. Immediately prior to infection, the T3S inhibitor and iron sulfate were added to the appropriate wells. C. muridarum was introduced at a multiplicity of infection of 1 unless otherwise noted, and the cells were centrifuged at 1,690 x g at 37°C for 1 h. Next, the medium was aspirated, and respective wells received fresh medium containing FeSO₄, INP0007, or both FeSO₄ and INP0007. At the indicated time points, supernatants were collected and stored at –80°C until further analysis. To confirm that cells were infected, macrophages infected in parallel, in wells containing glass coverslips, were fixed with methanol for 30 min at room temperature at 24 h postinfection and then stained with the fluorescein isothiocyanate-conjugated antichlamydial monoclonal antibody (Pathfinder; Bio-Rad, Hercules, CA) or processed for enumeration of inclusion-forming units (IFU) on a fresh McCoy monolayer as described earlier (3).

RNA extraction and real-time PCR analysis. After removal of the supernatants, RNA was isolated from the macrophage monolayer by using the RNeasy kit from Qiagen. Approximately 1 mg from each RNA sample was DNase I (Promega) treated (30 min, 37°C; 10 min 70°C). Afterward, 200 ng of RNA was reverse transcribed with SuperScript III (Invitrogen) enzyme according to the manufacturer's instructions, using random hexamer and oligo(dT) for priming. Quantitative PCR was performed on samples by using an IQ-Sybr mix (Bio-Rad) and an iQ5 iCycler (Bio-Rad). The amount of cDNA present was determined for each gene with standard curves and normalized to the housekeeping gene β-actin for analysis. All primers were designed by using Beacon Design software (Bio-Rad) and are listed in Table 1.

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TABLE 1. Genes and corresponding primer sequences

Cytokine analysis. The protein levels of beta interferon (IFN-β), CXCL10, IL-6, TNF-, and IL-1β in 50 µl of culture supernatants (1/20 of the total volume) were determined by using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) performed according to the manufacturer's supplied protocols. The optical densities at 450 nm were measured by using a Biotek plate reader. For analysis of intracellular IL-1β, cells were detached from the plate in phosphate-buffered saline and lysed by three cycles of freezing and thawing. The lysates were then sonicated, and 50 µl (1/10 of the total lysate) was assayed for IL-1β.

IFU determination and fluorescence microscopy. For IFU determination, macrophage monolayers were harvested in 1 ml of SPG buffer at 24 h postinfection. The cell suspensions were then sonicated for 30 s and serially diluted from 10² to 10⁶ before being used to infect fresh McCoy monolayers in 96-well black plates. Inclusions were visualized with a mouse monoclonal antibody (EVI H1) recognizing chlamydial lipopolysaccharide (LPS) (7), followed by Alexa Fluor 488-conjugated anti-mouse immunoglobulin G (Southern Biotechnology) in 0.1% Evans blue solution. Inclusions were counted by using an Olympus fluorescence microscope to calculate the IFU/ml of each sample. For fluorescence microscopic images of chlamydial infected cells, cells on coverslips were stained, mounted on slides, and visualized on a Zeiss Axioskop2 microscope using the x40 objective and photographed with the AxioCam MRm camera. 14-3-3beta recruitment to the inclusion membrane was visualized by staining infected and treated cells with a TRITC (tetramethyl rhodamine isothiocyanate)-labeled anti-14-3-3beta antibody (C-20; Santa Cruz Biotechnology) at a 1:50 dilution.

Statistical analysis. Three or four independent experiments were performed. For multi-inclusion counting, five fields on a x40 objective image from each group were tabulated. Statistically significant differences (P < 0.05) were determined by using a two-tailed Student t test. For experiments with more than two treatment groups, a one-way analysis of variance (ANOVA) with pairwise multiple comparison (Holm-Sidak method) was performed. One-way ANOVA was performed either on the raw data obtained or after calculating the percent inhibition in expression after various treatments compared to the untreated group from three or four independent experiments by using SigmaStat (Systat Software, Inc.).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Inhibition of C. muridarum growth by the T3S inhibitor INP0007 is reversed by the addition of exogenous iron. To determine whether the chlamydial T3S apparatus plays a role in cytokine generation, C. muridarum that effectively grows in mouse macrophages and induces a strong cytokine response was used as a model system (28). The T3S antagonist INP0007 that has been previously shown to target T3S in multiple bacteria, including C. trachomatis (27, 51), was used as a T3S inhibitor. Macrophages infected with C. muridarum in the presence of INP0007 did not yield any infectious chlamydiae (Fig. 1A) or form detectable inclusions (Fig. 1B). Importantly, the inability to obtain infectious chlamydiae and form inclusions could be completely reversed by addition of 50 µM FeSO₄ (Fig. 1A and B). Both of these results are consistent with previously reported findings using C. trachomatis (27, 41, 51). Interestingly, the addition of FeSO₄ to INP0007-treated macrophages resulted in a multi-inclusion phenotype (Fig. 1B) with significantly more cells possessing two inclusions (P = 0.016) and three or more inclusions (P = 0.023) compared to untreated cells (Fig. 1C), illustrating a phenotypic effect exerted by INP0007 even after growth inhibition was reversed.

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FIG. 1. Growth of C. muridarum during T3S inhibition. (A) One million mouse macrophages were infected with C. muridarum and treated with 50 µM INP0007 and 50 µM FeSO4 where indicated. Cell suspensions were collected at 24 h postinfection and assayed for IFU as described in Materials and Methods. Error bars represent ± the standard deviation (SD) of three independent experiments. (B) Macrophages on coverslips were infected as in panel A except at a multiplicity of infection (MOI) of 5, stained for inclusions, and visualized by fluorescence microscopy at a x40 objective magnification. (I) Untreated cells; (II) cells treated with FeSO4; (III) cells treated with INP0007; (IV) cells treated with INP0007 and FeSO4. (C) Five fields in the indicated groups from panel B were used to tabulate the number of cells possessing either two or more than two inclusions. The data represent the means ± the SD of five fields, and significance was determined by Student t test. Results representative from three experiments are shown.

Expression of NF-B-regulated proinflammatory cytokines TNF- and IL-6 are differentially affected by the T3S inhibitor INP0007. The ability of FeSO₄ to restore chlamydial growth in the presence of INP0007 was used as a means to distinguish cytokine responses that were T3S dependent but independent of chlamydial growth. Because both NF-B activation and expression of a large proportion of NF-B-dependent cytokines such as IL-6, Mip-2, IL-8, and monocyte chemoattractant protein 1 have been shown to be dependent upon T3S in other bacterial models (16, 31), the effect of T3S inhibition on the expression of NF-B-dependent cytokines IL-6 and TNF- was examined first. IL-6 and TNF- are highly expressed during C. muridarum infections of macrophages, although the kinetics of their induction differs greatly. IL-6 mRNA levels peak at 24 h postinfection, and this induction is inhibited by the addition of INP0007 (Fig. 2A) (78% inhibition; P < 0.001). The addition of FeSO₄ to INP0007-treated cells restores chlamydial growth (data not shown) but not IL-6 mRNA levels (Fig. 2A) (70% inhibition; P = 0.0012). Further, the addition of FeSO₄ in the absence of INP0007 to the infected cells does not affect IL-6 mRNA levels, strongly indicating that the decrease seen when INP0007 and FeSO₄ are used in conjunction is mediated solely by INP0007. The production of IL-6 protein by macrophages infected but given no other treatment varied from 400 to 2,000 pg/ml between different experiments, but the percent inhibition in expression after INP0007 or INP0007/FeSO₄ treatment remained statistically very significant (P < 0.001) by one-way ANOVA (Fig. 2B). These data imply a requirement for T3S in IL-6 induction. Conversely, induction of TNF-, which peaks at 3 h postinfection and stays elevated until 8 h (28), was unaffected by INP0007 treatment at 8 h postinfection (Fig. 2C). Although there was a trend to decreased levels of TNF- mRNA at 24 h after INP0007 treatment, these differences were not statistically significant between experiments. Secretion of TNF- protein is also insensitive to INP0007 treatment (Fig. 2D), largely reflecting the mRNA levels from 8 h. Together, these data suggest that mRNA induction of TNF- that is induced early via NF-B activation is T3S independent, whereas IL-6 that is induced late is T3S dependent.

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FIG. 2. Expression of IL-6 and TNF- during T3S inhibition. Macrophages were infected with C. muridarum in the presence of 50 µM INP0007 and 50 µM FeSO4 where indicated. (A and C) At 8 and 24 h postinfection, RNA was isolated from cells to examine expression of IL-6 (A) and TNF- (C). Results representative of three experiments are shown, with error bars calculated from the SD of samples assayed in duplicate. (B and D) At 24 h postinfection, supernatants were assayed by ELISA for IL-6 (B) and TNF- (D) protein, expressed as pg/ml. The data for TNF- represent the mean ± the SEM of three independent experiments, and significance was determined by one-way ANOVA. The data for the IL-6 ELISA are representative of three experiments showing mean values ± the SD of samples assayed in duplicate.

IL-1β secretion but not induction of proIL-1β mRNA is inhibited by the T3S inhibitor INP0007. Another important member of the host proinflammatory response whose expression is regulated by NF-B is IL-1β. Like TNF-, the induction of proIL-1β mRNA is greatest early during infection, and this peak expression is not inhibited by treatment with INP0007 (Fig. 3A). In addition, a general decrease in proIL-1β mRNA was noted in INP0007-treated cells at 24 h, but this change was not statistically significant between experiments. Secretion of mature IL-1β protein is controlled at posttranslational level since it requires cleavage of proIL-1β by the host protease caspase-1 (4). IL-1β secretion following infection with C. muridarum is sensitive to INP0007 (P < 0.001) (Fig. 3B) and only partly restored by the addition of FeSO₄ (P = 0.03). These data suggest a role for both chlamydial growth and T3S in IL-1β secretion. However, it must be noted that IL-1β protein levels detected in the supernatants of infected cells were low, approaching the limits of detection of the assay itself. It has been demonstrated that detectable amounts of mature IL-1β are also present inside C. muridarum-infected macrophages (6). Therefore, intracellular lysates of infected cells were also assayed. INP0007 prevented optimal accumulation of intracellular IL-1β whether or not FeSO₄ was present to restore growth (Fig. 3C). The absence of secreted and intracellular IL-1β despite strong mRNA induction in the presence of INP0007 suggests a role for T3S in caspase-1 activation.

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FIG. 3. Expression of IL-1β during T3S inhibition. Macrophages were infected with C. muridarum in the presence of 50 µM INP0007 and 50 µM FeSO4 where indicated. (A) At 8 and 24 h postinfection, RNA was isolated from cells to examine the expression of IL-1β. Results representative of three experiments are shown, with error bars calculated from the SD of samples assayed in duplicate. At 24 h postinfection, supernatants (B) or intracellular lysates (C) were assayed for the presence of IL-1β (B) by ELISA. The data in panel B represent means ± the standard errors of the mean of three independent experiments, and significance was determined by one-way ANOVA. The data in panel C represent the means ± the SD of samples assayed in duplicate.

Expression of host type I IFN response gene CXCL10 is sensitive to the T3S inhibitor INP0007. Type I IFNs and IFN response genes represent another class of genes that are upregulated during infection of macrophages with C. muridarum, but the bacterial contributions to this process is unclear. The observation that the functionally analogous but evolutionary unrelated type IV secretion (T4S) apparatus is required to initiate the IFN response in two other bacterial models (37, 42) suggests that the T3S apparatus may be a crucial element in the response to C. muridarum. Based on this premise, expression of IFN-β and the interferon response gene CXCL10 were examined during C. muridarum infections with or without T3S inhibition. Treatment of macrophages with INP0007 led to a significant reduction in chlamydia-induced IFN-β mRNA (76% inhibition; P < 0.001) (Fig. 4A) and IFN-β protein (P = 0.003) (Fig. 4B) secretion whether or not FeSO₄ is present to restore growth. However, the addition of FeSO₄ in the absence of INP0007 treatment also led to a significant decrease in IFN-β induction (50% inhibition; P = 0.004) and secretion (P = 0.019). This effect of FeSO₄ on IFN-β induction was C. muridarum specific because FeSO₄ did not diminish the response to the Escherichia coli LPS or poly(I-C) (data not shown). However, due to the "nonspecific" effect of FeSO₄ on IFN-β induction, the role for chlamydial T3S in IFN-β secretion remains inconclusive. IFN response genes such as CXCL10, a T-cell chemokine, require the same transcription factors that induce IFN-β, such as IRF3, but are also further amplified by type I IFN signaling (24, 28). Initial induction of CXCL10 at 8 h postinfection appears to be independent of INP0007, but peak mRNA induction and protein secretion at 24 h was significantly decreased when INP0007 was present (81% inhibition; P < 0.001 for RNA and P = 0.03 for protein) (Fig. 4C and D). This deficit was not reversed by addition of FeSO₄ (77% inhibition; P < 0.001 for RNA and P = 0.006 for CXCL10 protein). Further, the addition of FeSO₄ to infected cells in the absence of INP0007 did not decrease CXCL10 mRNA or protein levels. It is important to note that the induction of both IFN-β and CXCL10 mRNA at 24 h postinfection is inhibited by 50 mg of chloramphenicol/ml (Fig. 5) and 150 mg of rifampin/ml (data not shown), indicating that de novo bacterial protein synthesis and bacterial transcription are essential for this response. Together, these data suggest a role for T3S in addition to growth for chlamydia-induced CXCL10 secretion.

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FIG. 4. Expression of IFN-β and the IFN response gene, CXCL10, during T3S inhibition. Macrophages were infected with C. muridarum in the presence of 50 µM INP0007 and 50 µM FeSO4 where indicated. At 8 and 24 h postinfection, RNA was isolated from cells to examine expression of IFN-β (A) and CXCL10 (C). Results representative of three experiments are shown, with error bars calculated from the SD of samples assayed in duplicate. At 24 h postinfection, supernatants were assayed for the presence of IFN-β (B) or CXCL10 (D) by ELISA. The data represent the means ± the standard errors of the mean of three independent experiments, and significance was determined by one-way ANOVA.

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FIG. 5. Chlamydial growth dependence for IFN-β and IFN response genes. IFN-β (A) and CXCL-10 (B) induction in mouse macrophages that were infected with C. muridarum. Where indicated, 50 µg of chloramphenicol/ml (CAM) was added immediately prior to infection. Results representative of four experiments are shown, with error bars calculated from the SD of samples assayed in duplicate.

The T3S secretion inhibitor INP0007 does not inhibit TLR signaling in the presence or absence of FeSO₄. The actual bacterial target for INP0007 is unknown, but the drug has been shown to bind iron in vitro (41). Theoretically, this could influence events in the host cell such as TLR signaling pathways and cytokine production independent of its action on bacterial physiology. To address this possibility, macrophages were treated with the TLR ligand poly(I-C), which has been shown to cause upregulation of cytokines such as IFN-β and CXCL10 via TLR3 (1). Importantly, neither FeSO₄ nor INP0007 had a detrimental effect individually or collectively on TLR3-mediated induction of IFN-β (Fig. 6A) or CXCL-10 (Fig. 6B) when added in tandem with poly(I-C). Similar results were also obtained with TLR4-mediated induction of CXCL10 and IL-6 after E. coli LPS treatment (data not shown). This implies that the cytokine modulation seen during INP0007 treatment is mediated by its effect on the chlamydiae and not on the host cell.

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FIG. 6. Role of INP0007 and FeSO4 on TLR signaling. (A and B) IFN-β (A) and CXCL-10 (B) induction in mouse macrophages that were incubated with 10 µg of the TLR3 ligand poly(I-C)/ml for 6 h in the presence 50 µM INP0007 and 50 µM FeSO4. Results representative of two experiments are shown, with error bars calculated from the SD of samples assayed in duplicate.

Addition of iron during INP0007 treatment restores IncG secretion to the inclusion membrane independent of the multi-inclusion phenotype. In the present study a multi-inclusion phenotype was observed in C. muridarum (Fig. 1B)-infected cells treated with INP0007 and FeSO₄, suggesting that inclusion (Inc) proteins facilitating fusion might still be blocked after chlamydial growth restoration by FeSO₄. Therefore, secretion of the putative T3S effector IncG was monitored by staining cells for 14-3-3beta, a host protein that is recruited to the inclusion membrane in a IncG-dependent manner (39). Recruitment of 14-3-3beta to the inclusion membrane in infected cells leads to a ring-like staining pattern (Fig. 7B) as opposed to diffuse staining in uninfected or INP0007-treated cells (Fig. 7A and C). However, when infected cells are treated with both INP0007 and FeSO₄, 14-3-3beta recruitment is clearly restored (Fig. 7D), even though the multi-inclusion phenotype is still visible. These data suggest that FeSO₄ treatment of INP0007-treated cells restores the secretion of some putative T3S proteins such as IncG but does not restore normal inclusion phenotype and other likely T3S proteins that affect cytokine induction or secretion.

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FIG. 7. 14-3-3β staining on infected cells treated with INP0007 with or without Iron. Macrophages on coverslips were infected with C. muridarum, treated with INP0007 with or without FeSO4 and stained using TRITC-conjugated 14-3-3beta. (A) Uninfected cells; (B) infected cells; (C) infected cells treated with INP0007; (D) infected cells treated with INP0007 and FeSO4.

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Small organic molecules targeting the T3S apparatus of many human pathogens have been proposed as a new class of antimicrobial agents called virulence blockers (21). Consistent with prior studies using C. trachomatis and further supporting the possible use of these compounds as antibiotics, the addition of the T3S inhibitor INP0007 prevented the growth of C. muridarum in a tissue culture infection model. Interestingly, INP0007 treatment also led to decreased secretion of many host cytokines, including IL-1β, IL-6, and CXCL10, suggesting a role for chlamydial growth or T3S secretion in the expression of these cytokines. Interestingly, restoration of chlamydial growth by the addition of FeSO₄ to INP0007-treated cells does not fully restore the expression of IL-6, IL-1β, or CXCL10. This indicates for the first time a correlation between the chlamydial T3S apparatus and the expression of a number of the host cytokines that have been shown to play an important role in the development of oviduct pathology during genital chlamydial infection (6, 8). Interestingly, a recent study showed a decreased inflammatory response in a bovine ileal loop model of Salmonella enterica infection when the bacteria were pretreated with INP0007 (18), raising the distinct possibility that in vitro findings concerning inflammation may extend to in vivo infections.

INP0007 has been shown to inhibit secretion of the T3S effector proteins SipA (18) and YopH (29) in vitro by S. enterica and Y. pseudotuberculosis, respectively. However, the exact bacterial targets for INP0007 or how it inhibits T3S has not been elucidated. The fact that it works against such a broad spectrum of pathogens indicates that its target must be a highly conserved structure such as the T3S apparatus. The specific chlamydial effectors that are affected by T3S inhibitors, in the presence or absence of iron have not yet been defined, partially due to the lack of T3S expression/reporter systems for Chlamydia spp. In particular, although it was demonstrated previously that iron rescues the growth defect mediated by INP compounds (41), the potential ability of iron to also restore secretion of putative chlamydial T3S effectors such as Inc proteins (43) was not determined. In the present study a multi-inclusion phenotype was observed in C. muridarum infected cells treated with INP0007 and FeSO₄ (Fig. 1B), hinting that Inc protein secretion may still be prevented. However, IncG-dependent recruitment of 14-3-3beta (39) to the inclusion membrane was restored in cells treated with INP0007 and FeSO₄, although the multi-inclusion phenotype was still apparent (Fig. 7). A possible interpretation from these results would be that INP0007 targets the secretion of only a subset of the chlamydial T3S effectors (33). This implies that those blocked are required for induction of cytokines and fusion of multiple inclusions but dispensable for growth as long as sufficient iron is available. Overall, this hypothesis leads to the possibility that one or more chlamydial T3S effectors are responsible either directly or indirectly for propagating the host cytokine response during infection. The most likely prospect that could influence IL-1β secretion would seem to be the T3S translocator protein CopB (10) because the homologous T3S translocator proteins Shigella IpaB (5, 17, 49) and Salmonella SipB (9, 15) have been shown to colocalize with caspase-1 and are necessary and sufficient for its activation. Conversely, activation of caspase-1 by Yersinia pestis KIM was shown to be dependent on the T3S effector YopJ (22), illustrating that translocators are not the only possible candidates. Also, it is important to note that the effectors that are recognized by host receptors to induce the expression of these cytokines need not be typical T3S effectors, as evidenced by the T3S-dependent secretion of monomeric flagellin into host cytosol by Salmonella enterica (45). However, as long as the bacterial target for INP0007 is unresolved, it can be argued that the phenotypes exerted by this compound are independent of T3S.

It has been previously published in studies with C. trachomatis that the addition of exogenous iron or holotransferrin as iron donors during T3S-mediated inhibition completely restored growth (41). Further, using the E-lux expression system in Y. pseudotuberculosis, the same authors claimed no effect of excess iron on the expression of any T3S genes examined or of YopH secretion when used in conjunction with T3S inhibitors (41), although this was not tested for chlamydiae. Taken together, it was suggested that the iron-binding ability of the T3S drugs was independent of its effect on T3S. The observation that the addition of iron did not affect the INP0007 dependence of any of the host cytokines genes examined above suggests that T3S inhibition by these compounds is separate from their role in iron sequestration. Conversely, it has also been proposed that the chlamydial T3S system may be involved in iron acquisition and thereby necessary for chlamydial growth (21). The fact that iron restores the growth defect caused by T3S inhibition would then indicate that C. muridarum has T3S-independent means to thrive in cells in vitro as long as iron is not limiting. Interestingly, iron depletion has been shown to induce persistence in C. pneumoniae (50), and a recent study shows that iron depletion limits intracellular chlamydial growth in macrophages (32). This leads to another possibility that INP0007 could function as a siderophore and deplete iron besides its T3S inhibition function. Determining the link between iron acquisition by chlamydiae and the T3S apparatus is clearly necessary to resolve this conundrum.

An independent question to address is which host receptors are involved in triggering the T3S-induced cytokine response. This is particularly important because the observed difference in cytokine expression in INP0007-treated cells could be due to a direct effect of the drug on host receptors or their signaling pathways. Cytokine expression by TLR3 (Fig. 6) and TLR4 (data not shown) pathways were not affected by INP0007 or iron treatment, illustrating that the effects of these additives were specific to chlamydial infection and not due to a eukaryotic target. The TLR2-MyD88 pathway plays a role in TNF- and IL-1β induction during chlamydial infection (8), although the receptors involved in the induction of IL-6, IFN-β, and CXCL10 are still unclear. All of the cytokines that were examined are NF-B dependent, although individual gene promoters have additional regulatory elements. Interestingly, the induction of TNF- and IL-1β RNA were not T3S dependent (Fig. 3B). Both of these genes are induced and peak early (3 h) postinfection (28), unlike IL-6, IFN-β, and CXCL10, which peak much later at 24 h postinfection (Fig. 3A). It has been suggested that the earliest chlamydial T3S effectors, such as TARP, may not be completely blocked by T3S inhibitor treatment (27), so genes such as TNF- could be triggered following recognition of these early effectors. Alternatively, NF-B activation following a direct interaction of chlamydiae with TLR2 may play a dominant role for TNF- expression (30) but not for IL-6. As for IL-1β protein secretion, transcriptional upregulation is not sufficient. A second independent signal is required to activate the host protease caspase-1 in order to cleave proIL-1β to its active form. The fact that mRNA induction of IL-1β is INP0007 independent implies that the INP0007-mediated decrease in IL-1β secretion is due to a block in caspase-1 activation. This observation is consistent with the finding by Wolf et al. that the addition of INP0007 prevented the activation of caspase-1 in HeLa cells infected with C. trachomatis L2 (51). During infection of macrophages in vitro with C. muridarum, detection of active caspase-1 fragments by Western blotting occurs at 12 to 24 h postinfection (6). In general, INP0007 seems to have its greatest effects on cytokines expressed or enzymatically cleaved at later points of a primary infection. This observation raises the possibility that the effector inducing this response could be temporally regulated.

Overall, in the present study a well-established pharmacological inhibitor of T3S was used to illustrate the important role for chlamydial T3S in the establishment of the host cytokine response. In order to understand this phenomenon better, it will be crucial to determine how INP0007 impairs T3S inhibition, how iron reverses this chlamydial growth defect, and finally which specific chlamydial effector molecule(s) initiate the cytokine response.

 
This study was supported by Public Health Service grant AI067678 (U.M.N.) from the National Institutes of Health and in part by the Arkansas Bioscience Institute, Arkansas Children Hospital Research Institute, to U.M.N.

We thank Patrick Bavoil and Roger Rank for critical reading of the manuscript. A T3S inhibitor INP0400 structurally similar to INP0007 was provided by Pia Keyser (Innate Pharmaceuticals, Umea, Sweden).

 
* Corresponding author. Mailing address: Division of Pediatric Infectious Diseases, Arkansas Children's Hospital Research Institute, 1120 Marshall Street, Rm. 2052, Little Rock, AR 72202. Phone: (501) 364-2479. Fax: (501) 364-2403. E-mail: nagarajanuma{at}uams.edu

Published ahead of print on 13 October 2008.

Editor: A. J. Bäumler

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, January 2009, p. 76-84, Vol. 77, No. 1
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