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* Division of Pediatric Infectious Diseases, Arkansas Children’s Hospital and University of Arkansas for Medical Sciences, Little Rock, AR 72202;
Department of Microbiology/Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205;
Metroplex Pathology Associates, Austin, TX 78703; and
Université Paris 7, Institut Jacques Monod, Centre National pour Recherche Scientifique, Unité Mixte de Recherche 7592, 75251 Paris cedex 5, France
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Abstract |
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and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF- and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF- and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF- and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology. |
Introduction |
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TLRs are a family of proteins that share homology with the Toll antimicrobial proteins of Drosophila (12). These receptors are found primarily on mammalian innate immune cells, such as macrophages and dendritic cells, but are also expressed on many epithelial cells. TLRs act as pattern recognition receptors that enable cells to recognize pathogen-associated molecular patterns (PAMPs). This sheds light on the previously unappreciated specificity afforded to the innate immune system, whereby the TLRs allow for immediate recognition of conserved bacterial, viral, and fungal elements (13, 14). Engagement of these receptors can lead to activation of phagocytes and, through activation of the transcription factor NF-
B, production of inflammatory cytokines such as TNF- and IL-6 (15, 16, 17). Of the 10 currently described TLRs, TLR2 and TLR4, along with their ligands, are perhaps the best understood in terms of innate responses to bacteria. TLRs recognize a variety of PAMPs. TLR4 is the signal-transducing receptor for LPS (18); it is aided by two accessory proteins, CD14 and MD-2 (19). TLR2 is involved in the recognition of a broad range of microbial products, including peptidoglycan (PGN) from Gram-positive bacteria (20), bacterial lipoproteins (21), mycobacterial cell wall lipoarabinomannan (22), and yeast cell walls (23).
Chlamydia has several cell wall and outer membrane components that may serve as PAMPs recognized by TLRs. Chlamydial LPS (24) and chlamydial heat shock proteins (HSPs) (25, 26, 27) are ligands for the TLR4 receptor. However, recent papers describe TLR4-independent cytokine production from inflammatory cells exposed to live chlamydial elementary bodies (EBs) (24, 28, 29), and a dominant role for TLR2 vs TLR4 in the recognition process of C. pneumoniae (also known as Chlamydiophila pneumoniae) (30).
The effect of TLR2-deficiency on the macrophage response to C. trachomatis infection and the effect of a deficiency of either TLR4 or TLR2 have not been described in an in vivo model of Chlamydia infection. We examined cytokine release in vitro from peritoneal macrophages and primary fibroblasts obtained from mice deficient in the genes for TLR4 and TLR2 to determine the relative contribution of these two pattern recognition receptors in the inflammatory cell response to C. trachomatis. In addition, we determined the roles of TLR2 and TLR4 in the in vivo response to chlamydial genital tract infection by examining the course of chlamydial infection, the local inflammatory response, and chronic histopathology in infected mice deficient for either TLR2 or TLR4.
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Materials and Methods |
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C57BL/6 mice, C57BL/10 mice, and mice homozygous for Tlr4lps-del(C57BL/10ScCr) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice homozygous for Tlr2tm1Aki were kindly provided by S. Akira (Osaka, Japan) and were bred in our own animal facility. Mice homozygous for Tlr2tm1Aki were bred with C57BL/6 mice, and the F1 progeny (heterozygotes) were used as controls for TLR2 knockout (KO) experiments. The mice were between 6 and 10 wk old at the time of purchase and between 8 and 16 wk of age for different experiments. Wild-type and control groups were age matched for all experiments. Mice were given food and water ad libitum in an environmentally controlled room with a cycle of 12 h of light and 12 h of darkness. All animal experiments were preapproved by the University Institutional Animal Care and Use Committee.
Reagents and bacteria
Purified LPS of Escherichia coli serotype 011:B4 was purchased from InvivoGen (San Diego, CA). Purified PGN, isolated from the sonicated cell wall of Streptococcus pyogenes, group A, D58, was purchased from Lee Labs (Grayson, GA). The MoPn agent of C. trachomatis (Nigg), also known as Chlamydia muridarum (30), was grown in Mycoplasma-free McCoy or HeLa229 cells, and EBs were harvested from infected cells by centrifugation as previously described (31).
Isolation of murine peritoneal macrophages
Three days before murine peritoneal macrophages were harvested, mice were given a 1-ml i.p. injection of 3% thioglycollate medium. To harvest macrophages, mice were euthanized by cervical dislocation, and their peritoneal cavities were washed three times with 10 ml of macrophage culture medium. Peritoneal macrophages were plated into 24-well culture plates containing coverslips at a concentration of 1 x 106 macrophages/well yielding 90 to 100% confluence. Macrophage culture medium was RPMI 1640 (Cellgro, Herndon, VA) with 10% FBS, 10 mM HEPES, 10 mM sodium pyruvate, 1 mM L-glutamine, 10 mM nonessential amino acids, 50 µg/ml gentamicin, 100 µg/ml vancomycin, and 0.2 µM 2-ME at a concentration of 1 x 106 macrophages/well. Cells were washed with fresh medium after 60 min to remove nonadherent cells.
In vitro infection of macrophages
Three days after the murine macrophages were harvested, in vitro infections were performed. Varying concentrations of stock MoPn or UV-inactivated MoPn from the same culture stock were added to macrophage culture medium to allow for different multiplicities of infection (MOIs), such that an MOI of 1 represented 1 inclusion-forming unit (IFU) per macrophage. UV-inactivated EBs were prepared by exposing purified EBs to UV light in a laminar flow hood for 3 h. After UV inactivation, the EBs were checked to confirm that no infectious organisms were present. MOIs used were 0.5, 1, 2, 5, and 10. One milliliter of infection medium or, as a negative control, medium containing a lysate of uninfected McCoy cells, was placed over the macrophage monolayer in each well, and the plates were centrifuged (1800 x g, 60 min, 35°C). Cells plated with live EBs were then washed with macrophage medium to remove any excess EBs, and 1 ml of fresh macrophage medium was placed in each well. Finally, the plates were placed in an incubator at 35°C and 5% CO2. At 24 and 48 h after infection, 700 µl of supernatant were removed from the appropriate wells and frozen at -70°C until cytokine assays could be performed. At these same time points, coverslips were removed after aspiration of medium, fixed with methanol for at least 30 min, and stained with Pathfinder fluorescein-conjugated murine anti-chlamydial mAb according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). Macrophage monolayers were viewed with an Olympus microscope (Olympus, Melville, NY) to evaluate inclusion formation.
Establishment of murine lung fibroblasts and in vitro culture with MoPn
Lung fibroblasts from C57BL/6, TLR2tm1Aki, and Tlr4lps-del mice were isolated as previously described (32) and cultured for 5 to 6 days at 37°C in an atmosphere of 5% CO2 in RPMI 1640 containing supplemented with 20% heat-inactivated FBS (Life Technologies, Gaithersburg, MD) and 2 mM L-glutamine. After the cells reached confluence, culture medium was aspirated, and the cells were infected with C. trachomatis at an MOI of 0, 0.25, or 0.5 for 24 h.
Real time PCR for IL-1
, IL-6, and macrophage inflammatory protein-2 (MIP-2) mRNA from murine fibroblasts
RNA from uninfected and infected fibroblasts was isolated using an RNeasy kit (Qiagen, Chatsworth, CA) following the manufacturer’s instructions. Total RNA was converted into cDNA by standard reverse transcription with avian myeloblastosis virus reverse transcriptase in the manufacturer’s buffer (Roche Diagnostics, Meylan, France). Quantitative PCR was performed with 1/20 of the cDNA preparation in the GeneAmp 5700 (Applied Biosystems, Foster City, CA) (33) in 25 µl with Taq polymerase (Hot GoldStar enzyme, 0.025 U/ml) in Taqman buffer (Eurogentec, Brussels, Belgium) containing 5 mM MgCl2 and 200 µM concentrations of each dNTP. cDNA was amplified using 0.8 µM concentrations of each specific sense primer and 0.8 µM antisense primers. We also used 0.4 µM concentrations of the fluorogenic oligonucleotides specific for the gene segments in which a reporter fluorescent dye on 5' (FAM) and a quencher dye on 3' (TAMRA) were attached. Real time PCR was conducted at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min, and 72°C for 30 s. The specific activities of cDNA from different cytokines were compared with -actin and normalized to uninfected wild-type fibroblast responses by the comparative CT method as described by the manufacturer. Forward primers, reverse primers, and fluorescent probes for murine -actin, IL-1, MIP-2, and IL-6 are listed in Table I.
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Mice in groups of three were euthanized before (day -1) and at different time points after (days 7, 14, and 21) infection with MoPn. The upper (uterine horns and oviducts) and lower (upper vaginal vault to the cervix) genital tracts were removed from each mouse and homogenized individually. The homogenates were then processed using an RNeasy kit (Qiagen) according to the manufacturer’s instructions to isolate RNA from each sample. The total RNA concentration and purity were evaluated by OD readings at 260 and 280 nm. DNase treatment was performed in a 20-µl reaction mixture containing 1x PCR buffer and 1.5 mM MgCl2 (Applied Biosystems), 3.5 mM MgCl2, 1 mM (each) dNTPs, 1 µM oligo(dT) primer, 1 µM random hexamer, 0.5 µl of RNasin (Promega, Madison, WI), 0.5 µl RNase-free DNase (Promega), and 1 µg of RNA from each sample. Samples were placed in a PCR block at 37°C for 15 min followed by 70°C for 10 min. Then, 0.5 µl of reverse transcriptase (SuperScript II; Invitrogen, Carlsbad, CA) was added to cause the reverse transcription reaction at 25°C for 10 min, 42°C for 40 min, and 95°C for 5 min. A control reaction containing all components except reverse transcriptase was conducted for each RNA sample to check for contaminating nucleic acids. cDNAs were amplified by using the Bioline PCR system (Canton, MA) in a 50-µl reaction mixture containing one-twentieth of the cDNA generated from reverse transcription reaction, 1x PCR buffer, 1.5 mM MgCl2, 1 mM (each) dNTPs, 1 µM forward and reverse primers, and 0.5 U Taq polymerase. The sequences of the primers used for murine -actin, TLR2 and TLR4 are listed in Table I. PCR conditions for all primers were as follows: 95°C for 10 s, and 60°C for 60 s for 35 cycles. PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.
Murine infection with Chlamydia and measurement of chlamydial shedding
Mice received 2.5 mg of Depo-Provera (Pfizer, New York, NY) in 0.1 ml of saline (medroxyprogesterone acetate; Upjohn, Kalamazoo, MI) s.c. 7 days before vaginal infection. The mice were infected by placing 30 µl of 250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid (pH7.2) containing 1 x 107 IFUs (2000 ID50) of McCoy cell-grown C. trachomatis MoPn into the vaginal vault). Infection was administered while the mice were anesthetized with sodium pentobarbital. Infection was monitored by swabbing the vaginal vault and cervix with a calcium alginate swab (Spectrum Medical Industries, Los Angeles, CA) at various times after infection and by enumerating IFUs by isolation on McCoy cell monolayers (34). Mice were infected in groups of five, and each experiment was repeated at least once. The number of inclusion bodies within 20 fields (x40) was counted under a fluorescent microscope, and IFUs were calculated.
Collection of genital tract secretions for cytokine analysis
Genital tract secretions were collected from mice on multiple days throughout the course of infection and analyzed by ELISA for various cytokines of interest. At intervals before and after infection, an aseptic surgical sponge (ear wicks, 2 x 5 mm) (DeRoyal, Powell, TN) was inserted into the vagina of an anesthetized mouse and retrieved 30 min later. The sponges were held at -70°C until the day of the cytokine assay. Each sponge was placed in a Spin-X microcentrifuge tube (Fisher Scientific, Pittsburgh, PA) containing a 0.2-µm cellulose acetate filter, incubated in 300 µl of sterile PBS with 0.5% BSA and 0.05% Tween 20 for 1 h on ice, and then centrifuged for 5 min. Spin-X filters were preblocked with 0.5 ml of sterile PBS with 2% BSA and 0.05% Tween 20 for 30 min at 25°C, centrifuged, and washed twice with 0.05 ml of sterile PBS. Samples were kept on ice and promptly loaded into an ELISA plate prepared for a specific cytokine assay.
Detection of cytokines
Genital tract sponge eluates and macrophage culture supernatants were assayed individually for cytokine activity by ELISA using commercial cytokine ELISA kits for TNF-, IL-6, IFN-
, and the chemokine, MIP-2 (R&D Systems, Minneapolis, MN).
Histopathology
Mice were sacrificed on days 7 and 35 after infection, and the entire genital tract was removed en bloc, fixed in 10% buffered formalin, and embedded in paraffin. Longitudinal 4-µm sections were cut, stained with H&E, and evaluated by a pathologist blinded to the experimental design. Each anatomic site (exocervix, endocervix, uterine horn, oviduct, and mesosalpinx) was assessed independently for the presence of acute inflammation (neutrophils), chronic inflammation (lymphocytes), plasma cells, and fibrosis. Luminal distention of the uterine horns and dilatation of the oviducts were graded from 1 to 4, with grade 4 representing severe hydrosalpinx. Right and left uterine horns and right and left oviducts were evaluated individually. A four-tiered semiquantitative scoring system were used to quantitate the inflammation and fibrosis: 0 = normal; 1+ = rare foci (minimal presence) of parameter; 2+ = scattered (1, 2, 3, 4) aggregates or mild diffuse increase in parameter; 3+ = numerous aggregates (>4) or moderate diffuse or confluent areas of parameter; 4+ = severe diffuse infiltration or confluence of parameter.
Evaluation of serum Ab responses
Sera from mice were collected by retro-orbital blood sampling at the time of sacrifice and stored at -20°C until analyzed by ELISA as previously described (35). Preimmune sera were used as negative controls. The titer for individual mice was determined as the highest serum dilution with an OD value greater than that of the control wells.
Statistics
The ANOVA plus post hoc test was used to analyze differences in cytokine production among various in vitro groups. Statistical comparisons between the murine strains for level of infection and cytokine production over the course of infection were made by a two-factor (days and murine strain) ANOVA with post hoc Tukey test as a multiple comparison procedure. The Wilcoxon rank sum test was used to compare the duration of infection in the respective strains over time. The Kruskal-Wallis one-way ANOVA on ranks was used to determine significant differences in the pathological data between groups. The z test for determination of significant differences in sample proportions was used to compare frequencies of pathological findings between specific groups. SigmaStat software was used (SPSS Science, Chicago, IL).
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and IL-6 release in response to MoPn infection is markedly diminished in TLR2-deficient murine macrophages
Macrophages play a key role in early innate immune responses and produce proinflammatory cytokines such as TNF- and IL-6 when activated. We determined the production of these cytokines by peritoneal macrophages from TLR2 KO and heterozygous littermates after in vitro infection with MoPn. Macrophages lacking TLR2 produced significantly decreased amounts of TNF- and IL-6 in response to MoPn infection (Fig. 1). At an MOI of 1, there was an average decrease in TNF- of 41 ± 9% at 24 h and 62 ± 4% at 48 h in TLR2-deficient macrophages; IL-6 levels were 81 ± 13% and 54 ± 22% lower at 24 and 48 h, respectively. TNF- and IL-6 levels detected in supernatants from macrophages incubated with UV-inactivated EBs were no different from that from macrophages exposed to McCoy cell lysates (Fig. 1). This provides evidence that recognition of chlamydiae by TLR2 and subsequent signaling requires active infection of the macrophage as UV-inactivated EBs bind to and are ingested by macrophages. Although decreases in TNF- were not as strong at an MOI of 5, marked decreases in IL-6 were seen at all MOIs. Also, in all experiments, most of the cytokine production occurred during the first 24 h of infection, with levels detected at 48 h being minimally increased over the earlier time point (data not shown).
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and IL-6 in response to MoPn infection independently of TLR4
Identical experiments were then repeated using TLR4-deficient macrophages. Unexpectedly, when macrophages from TLR4 KO and control mice were used, significantly higher TNF- and IL-6 levels were detected from the TLR4 KO macrophages compared with C57BL/10 controls at all MOIs tested (1, 2, 5) (Fig. 2). UV-inactivated EBs elicited very low cytokine responses from both TLR4 KO and normal macrophages, again indicating that stimulation of murine macrophages by intact EBs requires active chlamydial infection. Thus, more cytokines are secreted in the absence of TLR4 than in its presence, suggesting that TLR4 ligation may down-modulate signaling through another TLR (presumably TLR2) or that expression of TLR2 and/or other TLRs may be higher in the absence of TLR4.
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, IL-6, and MIP-2 occurs after MoPn infection of lung fibroblasts from TLR2 KO mice compared with controls, but higher mRNA accumulation occurs in fibroblasts from TLR4 KO mice
A 24-h infection of primary fibroblasts with C. trachomatis at an MOI of 0.5 led to a large increase in transcription of the IL-1, MIP-2, and IL-6 genes. Significantly lower levels of transcription for the three cytokine genes were consistently found in TLR2-deficient fibroblasts infected at an MOI of 0.5 or 0.25, compared with wild-type fibroblasts infected with the same MOIs (Fig. 3). These results are in agreement with the decreased secretion of TNF- and IL-6 observed for TLR2-deficient macrophages, compared with wild-type macrophages.
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and IL-6 than infected wild-type macrophages, these results suggest that TLR4 may decrease cytokine production by cells infected with live C. trachomatis, regardless of the cell type used for infection.
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Previous studies with primary human epithelial cells derived from normal human vagina, ectocervix, and endocervix revealed the presence of mRNA for TLR2, but an absence of TLR4 message (36). Homogenized whole tissues from both the upper and lower genital tracts of mice before and after infection were analyzed by RT-PCR for mRNA for both TLR2 and TLR4 receptors. Positive cDNA bands for both TLR2 and TLR4 were found in three of three mice tested on day -1, 7, 14, and 21 of infection (Fig. 5). Thus, although TLR4 may not be present in epithelial cells of the lower genital tract, it is easily detected in whole genital tract tissue from both the upper and lower genital tracts. We cannot make any conclusion as to the relative levels of gene expression for these two TLRs in the genital tract during chlamydial infection because the methodology was purely qualitative.
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and MIP-2 levels are significantly decreased in genital tract secretions of TLR2 KO mice after infection, whereas IFN- and IL-6 are only marginally reduced and TLR4 KO mice have responses similar to infected controls
Given the presence of both TLR2 and TLR4 in the murine genital tract, we used daily genital tract sponges to examine the local production of TNF-, MIP-2, IFN-, and IL-6 for the first 10 days of infection. As seen in Fig. 6, compared with controls, TLR2 KO mice had significantly reduced levels of TNF- (Fig. 6A) and MIP-2 (Fig. 6C) detected in their genital tract secretions. However, although IFN- and IL-6 levels were also reduced, the decrease was not significant (Fig. 6, B and D).
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Fig. 8 shows that the course of infection as enumerated from vaginal swabs was essentially identical in all groups of mice regardless of the presence or absence of TLR2 (Fig. 8A) or TLR4 (Fig. 8B). Thus, despite the marked decrease in levels of TNF- and MIP-2 detected in genital tract secretions in TLR2 KO mice, they resolved the infection as efficiently as controls.
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Despite their similar course of infection, chronic oviduct pathology was markedly reduced in TLR2 KO mice compared with infected heterozygous littermates (Fig. 9). Histologically, none of the infected TLR2 KO mice developed hydrosalpinx or severe dilatation of their oviducts. Forty percent of the oviducts in the control mice were severely dilated, with moderate dilatation in another 50%. The majority (75%) of the oviducts from the TLR2 KO mice were only mildly dilated. On day 35 of infection, although inflammatory cells were noted throughout the genital tracts of both groups, acute inflammatory cells were significantly decreased in the TLR2 KO mice in the oviduct (Fig. 10A) and mesosalpinx (Fig. 10B). Specifically, TLR2 KO mice showed less oviduct dilatation, fewer acute inflammatory cells and less mesosalpingeal inflammation and fibrosis than controls.
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A predominance of IgG2a was detected in serum of infected controls, TLR2 KO, and TLR4 KO mice
Multiple studies of murine chlamydial infection report a preponderance of IgG2a vs IgG1, reflective of a Th1-dominant response (1, 34, 35, 37). We found a preponderance of IgG2a in serum taken on day 35 of infection in control, TLR2 KO, and TLR4 KO mice. IgG1 was undetectable in many of the mice in all three groups. Titers of IgG2a were not different among the groups (mean ± SEM log10 IgG2a for 10 mice in each group = 2.1 ± 0.2 for C57BL/10 mice, 2.7 ± 0.3 for TLR2 heterozygous littermates, 2.7 ± 0.3 for TLR2 KO mice, and 2.8 ± 0.4 for TLR4 KO mice).
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Discussion |
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The results of the in vitro experiments demonstrated an important contribution of TLR2 in macrophage production of TNF- and IL-6. In the absence of this receptor, peritoneal macrophages showed a statistically significant reduction in expression of these cytokines. However, despite significant decreases, inhibition was not complete. This finding underscores an important redundancy in the TLR system whereby inflammatory mediators can still be produced, albeit in smaller amounts, by cells lacking only one of these two receptors.
As regards chlamydial infection, infected epithelial cells have been shown to be intimately involved in the early innate response to infection (5, 6, 47). We investigated the role of TLR2 and TLR4 in Chlamydia-induced inflammatory cytokine gene transcription in primary murine fibroblasts and found a significant dependence on TLR2 for production of IL-1, MIP-2, and IL-6 in response to infection with MoPn. However, as with the phagocytic cell response, fibroblasts did not demonstrate complete dependency on TLR2 for activation of a cytokine response upon chlamydial infection.
Other groups have found a similar TLR2 dependency in dendritic cell, peripheral blood mononuclear cell, and macrophage activation after infection in vitro with C. pneumoniae (28, 29). No particular chlamydial protein has been definitively proved as a TLR2 ligand, but there are several candidates. Aside from various lipoproteins, chlamydiae may produce a glycanless cell wall polypeptide similar to the known TLR2 ligand PGN (48). Furthermore, some studies have shown that chlamydial heat shock protein 60 (cHSP60) is capable of binding TLR2 and TLR4. There is, however, disagreement in the literature as to which of these two receptors plays a more important role in binding cHSP60, depending on the cell line examined (25, 49, 50, 51). Finally, it is generally agreed that endogenous HSPs may bind cells via TLR2 and TLR4, revealing an interesting possibility that TLRs on a cell may be activated by ligands released at the death of an infected cell (26, 50, 52, 53). In this way, the infection could produce induction of inflammatory mediators in an indirect manner, via release of danger signals from the cytosol of the infected host cell (54, 55).
To our knowledge, this body of work is the first to demonstrate activation of TLR2 by C. trachomatis as a whole organism, in contrast to other studies that have examined individual components of this pathogen. More importantly, we have shown that only live, actively replicating chlamydiae are capable of inducing an inflammatory response from macrophages, because UV-inactivated EBs were ineffective in inducing cytokine release. Therefore, it can be inferred that during the intracellular transformation of an EB to a reticulate body, or during replication of reticulate bodies, a crucial ligand is presented or produced that can activate TLR2. This is plausible in that a previous study has demonstrated that TLRs are able to sample pathogens that are present in macrophage phagosomes (23). Although activation of TLRs by MoPn at the cell surface cannot be ruled out, the requirement for replication certainly suggests an intracellular localization of ligand binding.
It is interesting that chlamydial LPS associated with the UV-inactivated organism induced minimal cytokine release from macrophages compared with LPS from E. coli that induced TNF and IL-6 in large quantities. However, purified chlamydial LPS is 100-fold less potent than LPS from Salmonella minnesota R595 or the lipo-oligosaccharide from Neisseria gonorrhoeae at inducing an inflammatory cytokine response (56). We have observed by real time PCR >50,000-fold increase in mRNA for TNF in wild-type macrophages infected with live MoPn compared with a 300-fold increase in macrophages exposed to UV-inactivated EBs (our unpublished data). Thus, the decrease is seen at the level of gene transcription as well as protein production.
Of interest are the increased cytokine responses observed at all inoculum levels in TLR4 KO macrophages and fibroblasts. That the TLR4 KO cells responded to MoPn infection by producing more cytokines than controls under the same conditions is significant because it is counterintuitive. One possibility for this discrepancy is that TLR4 KO macrophages have increased expression of other receptors, such as TLR2, and these account for the increased production of cytokines in response to infection. Another possible explanation is that in TLR4 KO cells, heterotolerance effects on NF-B signaling are removed (57). Heterotolerance to TLR2 ligands induced by TLR4 signaling would not be possible in TLR4-deficient cells, leaving responses mediated via binding to TLR2 uninhibited and accentuated. Thus, heightened TLR2-mediated signaling would overshadow any contribution of TLR4 to macrophage or fibroblast activation. Recently, it was shown that IL-4 down-regulates TLR2 expression on monocytes and dendritic cells (58). We measured IL-4 in supernatants from MoPn-infected wild-type and TLR4 KO macrophages by ELISA and found barely detectable levels of IL-4 (16 ± 4 pg/ml in wild-type and 14 ± 2 in TLR4-deficient macrophages). Thus, TLR4-induced production of IL-4 is not the mechanism for decreased cytokine release observed in wild-type cells compared with TLR4-deficient cells stimulated with E. coli LPS or infected with C. trachomatis. The absence of a heightened cytokine response in vivo in TLR4 KO mice could be explained by the increased availability of down-regulatory responses in an in vivo infection model (59, 60, 61, 62).
Our in vivo experiments in TLR2 KO mice also yielded unexpected results. Although significantly lower levels of the inflammatory cytokines TNF- and MIP-2 were found in genital tract secretions of the TLR2-deficient mice than in controls during the first 2 weeks of infection, there was no difference in the course of infection between the two groups. The cytokine response that remained in the absence of TLR2 signaling was sufficient to recruit effector cells to the genital tract, resulting in equivalent eradication of organisms even in the early days after infection. The similar time to clearance of organisms from the lower genital tract also speaks to the redundancy of the innate immune system; even in the absence of TLR2, the infection is cleared at a normal rate, indicating that other mechanisms can still initiate an effective acquired immune response.
The lack of a significant reduction in IFN- and IL-6 in TLR2 KO mice may explain their ability to effectively eradicate genital tract infection with C. trachomatis. IFN- is clearly important in host defense against chlamydiae, with direct and indirect inhibitory effects demonstrated in vitro (63, 64, 65, 66, 67, 68, 69) and in vivo (2, 3, 4, 37, 70, 71, 72). IL-6 has been shown to be partly responsible for blocking the suppressor activity of CD4+CD25+-regulatory T cells (TR), allowing for activation of pathogen-specific adaptive immune responses (73). The efficient resolution of genital tract infection observed in the TLR2 KO mice, implies that an intact adaptive CD4+ Th1 response occurred in these mice, given that these cells are essential for eradication of genital infection in the murine model (38, 74). The detection of normal titers of IgG2a Abs in the TLR2 KO mice also suggests that a normal Th1 response occurred in these mice, resulting in an infection course resembling that in mice with intact TLR2 function.
Histopathology data revealed that the presence of TLR2 contributes to chronic inflammatory sequelae found after infection has resolved. It is possible that the TLR2 KO mice cleared upper tract infection faster than control mice, resulting in a more rapid resolution of inflammation and subsequent decrease in chronic oviduct pathology. However, the absolutely comparable rate of resolution observed from cervical swabs in the TLR2 KO mice compared with control mice makes this unlikely. In murine strains with demonstrated differential susceptibilities to chlamydial genital tract infection, resolution of lower and upper tract infection are affected equally by the different genetic backgrounds (35). It is possible that in the face of significant reductions of select inflammatory mediators (TNF- and MIP-2) over the course of infection in the TLR2 KO mice, the end result is efficient eradication of the chlamydial pathogen and abrogation of later scarring of the oviduct. In humans, functional polymorphisms in the TLR2 gene are associated with lepromatous leprosy (75), the most severe form of this disease. However, macrophage TNF- responses are virtually absent upon exposure of lepromatous patients’ macrophages to mycobacteria (76). This is in contrast to our model in which cytokine responses, although significantly reduced with TLR2 deficiency, are not absent.
In contrast to the improvement in pathology in TLR2 KO mice, infection in the TLR4 KO mice resulted in chronic tissue damage equal to infected controls. Thus, equivalent in vivo cytokine responses observed in the TLR4 KO mice and controls are reflected in similar outcomes of infection. These data argue against a direct role for cHSP60-induced TLR4-mediated tissue damage in genital tract infection but does not rule out a role for HSP60 in the pathological process. Interestingly, a recent report by Morre et al. (77) found no association of the functional polymorphism in the gene encoding TLR4 function (Asp299Gly) with tubal infertility in C. trachomatis IgG-positive Dutch women.
In conclusion, this study revealed important information regarding the interaction of C. trachomatis with TLRs. Although parts of this organism (chlamydial LPS and cHSP60) are recognized by TLR4, the intact organism stimulates innate immune cells and fibroblasts independently of TLR4. TLR2 plays a significant role as a pattern recognition receptor for this organism, and stimulation of TLR2 requires active infection with Chlamydia. The normal clearance of organisms from the genital tracts of TLR2 KO mice and the nonsignificant reduction in key cytokines in these mice point to the redundancy of the immune response and the number of collateral pathways available for innate responses to infections. Importantly, the in vivo data indicate a protective role for TLR2 deficiency in genital tract infection sequelae due to C. trachomatis, given that mice deficient in TLR2 exhibited significantly decreased oviduct pathology. The remarkably improved outcome in these mice suggests that an improved balance in protective vs pathological inflammatory responses occurs with TLR2 deficiency with respect to Chlamydia infection of the female genital tract.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Toni Darville, Department of Microbiology/Immunology, B506C, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205. E-mail address: darvilletonil{at}uams.edu
3 Abbreviations used in this paper: MoPn, mouse pneumonitis agent of Chlamydia trachomatis; EB, elementary body; HSP, heat shock protein; cHSP60, chlamydial heat shock protein 60; KO, knockout; MIP-2, macrophage-inflammatory protein-2; MOI, multiplicity of infection; PGN, peptidoglycan; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; IFU, inclusion-forming unit.
Received for publication July 18, 2003. Accepted for publication September 29, 2003.
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References |
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interferon on resolution of murine chlamydial genital infection. Infect. Immun. 60:4427.-dependent and -independent pathways. J. Immunol. 158:3344.[Abstract]
interferon-secreting T cells in the genital tract produces optimal protection against Chlamydia trachomatis infection in mice. Infect. Immun. 66:4030.B signaling pathway components. J. Immunol. 170:508., and TNF-. J. Immunol. 157:798.[Abstract]
and TGF- responses regulate the occurrence of mucosal inflammation. Immunol. Today 18:61.[Medline]
interferon-mediated inhibition of intracellular Chlamydia psittaci replication in T24 cells. Infect. Immun. 53:347. interferon-mediated cytotoxicity to Chlamydia-infected fibroblasts. Infect. Immun. 57:870. interferon-mediated cytotoxicity related to murine Chlamydia trachomatis infection. Infect. Immun. 56:2023. interferon on Chlamydia-infected host cells: microbicidal activity follows microbistasis. Infect. Immun. 57:1318.-mediated inhibition. J. Immunol. 162:3541. interferon. Infect. Immun. 68:6038. interferon gene knockout mice. Infect. Immun. 65:2145.[Abstract]
knockout mice show Th2-associated delayed-type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection. Eur. J. Immunol. 29:3782.[Medline]
Interferon production by cytotoxic T lymphocytes is required for resolution of Chlamydia trachomatis infection. Infect. Immun. 66:5457.This article has been cited by other articles:
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