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Toll-Like Receptor-2, but Not Toll-Like
Receptor-4, Is Essential for Development of
Oviduct Pathology in Chlamydial Genital
Tract Infection
Toni Darville, Joshua M. O'Neill, Charles W. Andrews, Jr.,
Uma M. Nagarajan, Lynn Stahl and David M. Ojcius
J Immunol 2003; 171:6187-6197; ;
doi: 10.4049/jimmunol.171.11.6187
http://www.jimmunol.org/content/171/11/6187
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
The Journal of Immunology
Toll-Like Receptor-2, but Not Toll-Like Receptor-4, Is
Essential for Development of Oviduct Pathology in Chlamydial
Genital Tract Infection1
Toni Darville,2*† Joshua M. O’Neill,* Charles W. Andrews, Jr.,‡ Uma M. Nagarajan,†
Lynn Stahl,§ and David M. Ojcius§
I
n the realm of infectious diseases, it has often been observed
that an overly aggressive inflammatory host response can be
more problematic than the infection that initiated it. This is
certainly true in the case of genital tract infection with Chlamydia
trachomatis, where the pathology that leads to fallopian tube inflammation, scarring, and infertility is the result of a robust host
inflammatory response. C. trachomatis is a Gram-negative obligate intracellular bacterium that can initiate a variety of immune
system responses from infected hosts. In mice, infection with the
mouse pneumonitis (MoPn)3 biovar of this organism is ultimately
resolved by the host via a CD4⫹ Th1 response (1– 4). However,
attention has been paid recently to the role of the innate immune
system in regulating the early response to infection by C. tracho-
*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
Received for publication July 18, 2003. Accepted for publication September 29, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Public Health Service Grant AI054624 from the National Institutes of Health and by the Horace C. Cabe Foundation and the BatesWheeler Foundation, Arkansas Children’s Hospital Research Institute and University
of Arkansas Medical Sciences, and the Fondation pour la Recherche Médicale.
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: [email protected]
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.
Copyright © 2003 by The American Association of Immunologists, Inc.
matis as well as its role in inflammation and the resulting pathological sequelae that occur (5–10). The relatively new discovery of
the involvement of Toll-like receptors (TLRs) (11) in the innate
host response has prompted investigations of these receptors as
potential regulators of the response to chlamydiae.
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 (15–17). 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 Grampositive 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) (25–27) 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
0022-1767/03/$02.00
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The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis
have not been elucidated. We examined production of TNF-␣ 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. The Journal of Immunology, 2003, 171: 6187– 6197.
6188
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.
Materials and Methods
Animals
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 ⫻ 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
␮g/ml gentamicin, 100 ␮g/ml vancomycin, and 0.2 ␮M 2-ME at a concentration of 1 ⫻ 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 ⫻ 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 ␮l 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 me-
dium, 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 ␮l with Taq polymerase
(Hot GoldStar enzyme, 0.025 U/ml) in Taqman buffer (Eurogentec, Brussels, Belgium) containing 5 mM MgCl2 and 200 ␮M concentrations of
each dNTP. cDNA was amplified using 0.8 ␮M concentrations of each
specific sense primer and 0.8 ␮M antisense primers. We also used 0.4 ␮M
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.
RT-PCR for genital tract TLR2 and TLR4 mRNA from upper
and lower genital tract tissues
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-␮l
reaction mixture containing 1⫻ PCR buffer and 1.5 mM MgCl2 (Applied
Biosystems), 3.5 mM MgCl2, 1 mM (each) dNTPs, 1 ␮M oligo(dT) primer,
1 ␮M random hexamer, 0.5 ␮l of RNasin (Promega, Madison, WI), 0.5 ␮l
RNase-free DNase (Promega), and 1 ␮g 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 ␮l 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-␮l reaction mixture containing one-twentieth of the cDNA generated from reverse transcription reaction, 1⫻ PCR buffer, 1.5 mM MgCl2, 1 mM (each)
dNTPs, 1 ␮M 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 ␮l of
250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid (pH
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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.
TLR2 IN CHLAMYDIAL GENITAL INFECTION
5⬘-CAATAGTGATGACCTGGCCGT-3⬘
5⬘-GATCCACACTCTCCAGCTGCA-3⬘
5⬘-CTTGAGAGTGGCTATGACTTCTGTCT-3⬘
5⬘-TGAATTGGATGGTCTTGGTCCT-3⬘
5⬘-TCAGGAGGAGCAATGATCTTG-3⬘
5⬘-CCAGGTAGGTCTTGGTGTTCATT-3⬘
5⬘-AAGATACACCAACGGCTCTGAA-3⬘
5⬘-AGAGGGAAATCGTGCGTGAC-3⬘
5⬘-CAACCAACAAGTGATATTCTCCATG-3⬘
5⬘-TGACTTCAAGAACATCCAGAGCTT-3⬘
5⬘-TCCTACCCCAATTTCCAATGC-3⬘
5⬘-AAGACCTCTATGCCAACACAGT-3⬘
5⬘-TGCAAGTACGAACTGGACTTCT-3⬘
5⬘-TAGCCATTGCTGCCAACATCAT-3⬘
␤-Actin real-time PCR
IL-1␤
MIP-2
IL-6
␤-Actin RT-PCR
TLR2
TLR4
Reverse Primer
Gene
Forward Primer
5⬘-FAM-CACTGCCGCATCCTCTTCCTCCC-TAMRA-3⬘
5⬘-FAM-CTGTGTAATGAAAGACGGCACACCCACC-TAMRA-3⬘
5⬘-FAM-TGACGCCCCCAGGACCCCA-TAMRA-3⬘
5⬘-FAM-CAGATAAGCTGGAGTCACAGAAGGAGTGG-TAMRA-3⬘
6189
7.2) containing 1 ⫻ 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
(⫻40) 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 ⫻ 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-␮m cellulose acetate filter, incubated in 300 ␮l 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-␮m 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– 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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Table I. Forward primers, reverse primers, and fluorescent probes used for real time PCR; forward and reverse primers used for RT-PCR
Probe
The Journal of Immunology
6190
Results
TNF-␣ and IL-6 release in response to MoPn infection is
markedly diminished in TLR2-deficient murine macrophages
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, and 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.
Lower mRNA accumulation for IL-1␤, 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 TLR2deficient 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.
Macrophages release TNF-␣ 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
FIGURE 1. TLR2 is important for in vitro macrophage response to C.
trachomatis infection. TNF-␣ (A) and IL-6 (B) levels detected in supernatants from TLR2⫹/⫺ and TLR2⫺/⫺ murine peritoneal macrophages after
24 h of in vitro stimulation with McCoy cell lysates, LPS (1 ␮g/ml), LPS
(1 ng/ml), PGN (50 ␮g/ml), UV-inactivated (inact) EBs, or after 24 h of
infection with live C. trachomatis MoPn at MOI ⫽ 1, and MOI ⫽ 5. Data
are the mean ⫾ SD (n ⫽ 2) and are representative of three experiments. ⴱ,
p ⬍ 0.05 by ANOVA plus post hoc test.
FIGURE 2. Macrophages respond to chlamydial infection in vitro independently of TLR4. TNF-␣ (A) and IL-6 (B) levels detected in supernatants from TLR4⫹/⫹ and TLR4⫺/⫺ murine peritoneal macrophages after
24 h of in vitro stimulation with McCoy cell lysates, LPS (1 ␮g/ml), LPS
(1 ng/ml), PGN (50 ␮g/ml), UV-inactivated (inact) EBs, or after 24 h of
infection with live C. trachomatis MoPn at MOI ⫽ 1, and MOI ⫽ 5. Data
are the mean ⫾ SD (n ⫽ 2) and are representative of three experiments. ⴱ,
p ⬍ 0.05 by ANOVA plus post hoc test.
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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).
TLR2 IN CHLAMYDIAL GENITAL INFECTION
The Journal of Immunology
Similarly, higher levels of cytokine gene transcription were
found in TLR4-deficient fibroblasts after a 24-h infection with C.
trachomatis compared with wild-type fibroblasts infected with the
same MOIs (Fig. 4). Given that infected TLR4-deficient macrophages also secrete higher concentrations of TNF-␣ 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.
mRNAs for both TLR2 and TLR4 are present in the upper and
lower genital tract before and during infection
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
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FIGURE 3. TLR2 is important for in vitro fibroblast response to C.
trachomatis infection. Fold increase in amount of mRNA for IL-1␤ (A),
IL-6 (B), and MIP-2 (C) quantitated by real time PCR from TLR2⫹/⫹ and
TLR2⫺/⫺ fibroblasts after incubation in medium alone or MoPn C. trachomatis, MOI ⫽ 0.25 and MOI ⫽ 0.5, for 24 h. The values were standardized against ␤-actin and cytokine values from uninfected TLR2⫹/⫹
fibroblasts. Data are representative of three separate experiments.
6191
FIGURE 4. TLR4 is dispensable for in vitro fibroblast response to C.
trachomatis infection. Fold increase in amount of mRNA for IL-1 (A), IL-6
(B), and MIP-2 (C) quantitated by real-time PCR from TLR4⫹/⫹ and
TLR4⫺/⫺ fibroblasts after incubation in medium alone or MoPn C. trachomatis, MOI ⫽ 0.25 and MOI ⫽ 0.5 for 24 h. The values were standardized against ␤-actin and cytokine values from uninfected TLR4⫹/⫹
fibroblasts. Data are representative of three separate experiments.
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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TLR2 IN CHLAMYDIAL GENITAL INFECTION
although IFN-␥ and IL-6 levels were also reduced, the decrease
was not significant (Fig. 6, B and D).
TLR4-deficient mice produced similar amounts of inflammatory
mediators to controls early during infection (Fig. 7). Thus, despite
the increased cytokine levels detected in vitro from Chlamydiainfected TLR4-deficient macrophages and fibroblasts, increased
levels were not seen with in vivo infection in TLR4 KO mice.
Course of MoPn infection is equivalent in TLR2 KO, TLR4 KO,
and wild-type mice
FIGURE 5. mRNA for TLR2 and TLR4 are present in upper (U) and
lower (L) genital tract tissues from uninfected and infected mice. Data
represent ethidium bromide-stained gel of cDNA bands for TLR2 and
␤-actin from the same mouse samples, and for TLR4 and ␤-actin from the
same mouse samples. Bands are from a single representative mouse at time
before infection, day ⫺1 (D-1), day 7 of infection (D7); day 14 of infection
(D14), and day 21 of infection (D21). The lower band in the gel for TLR2
depicts primer-dimers. Three mice were analyzed at each time point, and
all gave similar results.
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,
Infected TLR2 KO mice display less pathology than wild-type
mice
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
FIGURE 6. TLR2 is important for TNF-␣ and MIP-2 responses with in vivo Chlamydia infection, but IFN-␥ and IL-6 responses are minimally affected
by TLR2 deficiency. Mean (⫾ SEM) TNF-␣ (A), IFN-␥ (B), MIP-2 (C), and IL-6 (D) levels in genital tract secretions of TLR2⫹/⫺ and TLR2⫺/⫺ mice
during the first 10 days of infection with MoPn of C. trachomatis. Data represent the combined results of two separate experiments (n ⫽ 5 per strain per
experiment). Each sample was run in duplicate. p ⬍ 0.005 by two-factor ANOVA for TNF-␣ (A) and MIP-2 (C) between TLR2⫹/⫺ vs TLR2⫺/⫺ groups.
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TNF-␣ 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
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.
The Journal of Immunology
6193
acute inflammatory cells and less mesosalpingeal inflammation
and fibrosis than controls.
The genital tract tissues from TLR4 KO mice exhibited pathology equal to control mice. Inflammatory cell infiltration and degree
of oviduct dilatation were no different from infected wild-type
mice sacrificed on the same day (data not shown).
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).
Discussion
Resolution of a primary chlamydial genital tract infection in the
mouse ultimately relies on a Th1 cell-mediated immune response
(1– 4, 38 – 40). This adaptive immunity in turn is initiated by the
innate immune system. It is likely that C. trachomatis initially
invades genital tract epithelial cells as well as resident tissue macrophages and dendritic cells. Infected cells respond by releasing
inflammatory mediators and chemokines that induce an influx of
NK cells and neutrophils into the area, activate nearby phagocytes
and APCs, and ultimately dictate the ensuing acquired response.
We demonstrated the presence of both TLR2 and TLR4 in the
upper and lower genital tract tissues of noninfected and infected
mice in our initial experiments, which allowed us to proceed with
an examination of their relative importance, if any, in chlamydial
infection. Evidence has previously been presented showing that
TLRs are key in translating microbial recognition into expression
of specific subsets of chemokines and cytokines from macrophages
and dendritic cells (13, 21, 29, 41, 42). Furthermore, TLR2 and
TLR4 have been shown to induce different sets of chemokines and
cytokines, implying that the innate immune response can be customized for different pathogens (43– 46). It is therefore vital to
elucidate the role of these two receptors in the expression of inflammatory mediators by infected cells in response to C.
trachomatis.
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.
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FIGURE 7. TLR4 is not important for in vivo cytokine responses elicited by Chlamydia infection. Mean (⫾ SEM) TNF-␣ (A), IFN-␥ (B), MIP-2 (C),
and IL-6 (D) levels in genital tract secretions of TLR4⫹/⫹ and TLR4⫺/⫺ mice during the first 10 days of infection with MoPn of C. trachomatis. Data
represent the combined results of two separate experiments (n ⫽ 5 per strain per experiment). Each sample was run in duplicate.
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FIGURE 9. Oviduct pathology is significantly decreased in TLR2 KO mice. Histopathological evaluation of oviducts from TLR2⫹/⫺ and TLR2⫺/⫺ mice on
day 35 of infection reveals minimal oviduct dilatation
in the TLR2 ⫺/⫺ mice (A, ⫻4; B, ⫻10; C, ⫻20), but
marked dilatation consistent with hydrosalpinx in the
wild-type mice (D, ⫻4; E, ⫻10; F, ⫻20). Arrows,
Oviducts.
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 –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
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FIGURE 8. Intensity and duration of in vivo genital tract infection are
not affected by TLR2 or TLR4 deficiency. Quantitative isolations from
lower genital tract swabs obtained after infection with MoPn C. trachomatis in TLR2⫹/⫺ and TLR2⫺/⫺ mice (A) and in TLR4⫹/⫹ and TLR4⫺/⫺
mice (B). Data points represent means ⫾ SEM of duplicate determinations
with 10 animals examined on each day.
TLR2 IN CHLAMYDIAL GENITAL INFECTION
The Journal of Immunology
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 UVinactivated 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 100fold 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 – 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– 69) and in vivo (2– 4, 37, 70 –
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
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FIGURE 10. Inflammation and chronic pathology are decreased in upper genital tract tissues of infected TLR2 KO mice compared with controls.
Decreased levels of acute inflammation were seen in the oviducts (A) and
mesosalpingeal tissues (B) from TLR2⫺/⫺ mice. Dilatation of oviducts (A)
and fibrosis of mesosalpinx (B) were also less in the TLR2⫺/⫺ mice. Bars,
Median pathology scores calculated from 10 mice of each group sacrificed
on day 35 of infection. ⴱ, p ⬍ 0.05 by ANOVA on ranks.
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6196
Acknowledgments
We acknowledge the superb technical assistance of Jim D. Sikes and
Thomas Jungas.
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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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