Download against Oviduct Disease to Induce Immune Pathology and Protect

Plasmid-Deficient Chlamydia muridarum Fail
to Induce Immune Pathology and Protect
against Oviduct Disease
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of June 18, 2017.
Catherine M. O'Connell, Robin R. Ingalls, Charles W.
Andrews, Jr., Amy M. Scurlock and Toni Darville
J Immunol 2007; 179:4027-4034; ;
doi: 10.4049/jimmunol.179.6.4027
http://www.jimmunol.org/content/179/6/4027
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References
The Journal of Immunology
Plasmid-Deficient Chlamydia muridarum Fail to Induce
Immune Pathology and Protect against Oviduct Disease1
Catherine M. O’Connell,2* Robin R. Ingalls,† Charles W. Andrews, Jr.,‡ Amy M. Scurlock,§¶
and Toni Darville*§¶
C
hlamydia trachomatis ocular infections cause trachoma,
the world’s leading cause of preventable blindness (1). C.
trachomatis is also the leading cause of bacterial sexually
transmitted infections worldwide (2). In women, untreated infections may progress to serious reproductive tract sequelae. Observational and randomized studies yield risk estimates of 15– 80%
for developing pelvic inflammatory disease (3); 5–25% for ectopic
pregnancy (4); and 10 –20% for tubal factor infertility (5, 6) subsequent to chlamydial infection. The protective immunity that develops in response to infection is not long-lived (7), and animal
models indicate that repeated infection elicits more severe disease
(8 –12). Although antibiotic therapy eliminates chlamydial infection, it does not ameliorate established pathology. Furthermore, the
administration of antimicrobial agents may blunt the development
of natural immunity to C. trachomatis in humans (13), as has been
demonstrated in mouse models of infection (14).
Data from both human studies and animal models point to
CD4⫹ Th1 cells as essential to recovery from infection and resistance to reinfection (15–22). However, the immunopathogenesis of
chlamydial disease is not clear. Both cellular (23) and immunological hypotheses (24) have been proposed. Evidence that the
innate immune response may be the primary mediator of pathology
*Department of Microbiology and Immunology, University of Arkansas for Medical
Sciences, Little Rock, AR 72205; †Boston University School of Medicine, Boston,
MA 02118; ‡Milstead Pathology PC, Atlanta, GA 30012; §Department of Pediatrics,
University of Arkansas for Medical Sciences, Little Rock, AR 72205; and ¶Arkansas
Children’s Hospital Research Institute, Little Rock, AR 72202
Received for publication April 24, 2007. Accepted for publication June 25, 2007.
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 Horace C. Cabe Foundation, Bates-Wheeler Foundation, Arkansas Children’s Hospital Research Institute, and University of Arkansas
Medical Sciences. R.R.I. is supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases AI064749.
2
Address correspondence and reprint requests to Dr. Catherine M. O’Connell,
Department of Pediatrics (Infectious Diseases Section), Children’s Hospital of
Pittsburgh of University of Pittsburgh Medical Center, 3705 Fifth Avenue, Pittsburgh, PA 15213. E-mail address: Catherine.O’[email protected]
www.jimmunol.org
comes from the mouse model of genital tract infection, in which
the presence of neutrophils that release and activate proteolytic
molecules during acute infection correlates directly with development of fibrotic occlusion of the oviduct (25, 26).
Pattern recognition receptors (PRRs)3 found on innate immune
cells enable recognition of signature structures of pathogens, called
pathogen-associated molecular patterns. Engagement of PRRs,
which include TLRs, by pathogen-associated molecular patterns
leads to activation of innate immune cells that stimulate and direct
the activity of pathogen-specific T cells (27). We have shown that
TLR2-deficient mice do not develop oviduct pathology after chlamydial infection, demonstrating that TLR2 signaling is directly
involved in disease development (28). Because chlamydiae are
present as both extra- and intracellular/intravacuolar forms, they
have the potential to stimulate multiple PRR pathways. In this
study, we report novel Chlamydia muridarum mutants that retain
the ability to infect the murine genital tract, but fail to cause disease. These mutants fail to stimulate TLR2-dependent signaling in
cell culture and in vivo. Mice previously infected with the mutants
were protected against disease when challenged with virulent C.
muridarum, evidence that a protective adaptive immune response
generated in the absence of TLR2 signaling is sufficient to protect
from pathology caused by the innate responses induced upon
infection.
Materials and Methods
Propagation of Chlamydia and isolation of C. muridarum
CM3.1
All C. muridarum strains, including Nigg (29), CM972 (30), and CM3.1,
were propagated in McCoy cells (American Type Culture Collection CRL1696), as previously described (30). Bacteria were titrated by plaque assay
(30) or as inclusion-forming units (IFU) (31). UV-inactivated bacteria
were prepared, as described (28). The presence of the cryptic plasmid in
3
Abbreviations used in this paper: PRR, pattern recognition receptor; IFU, inclusionforming unit; KO, knockout; MOI, multiplicity of infection; ShEC, immortalized
ectocervical epithelial cell line.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
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Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can
cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do
not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital
disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of
chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to
infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine
production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no
evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient
strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human
C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human
disease. The Journal of Immunology, 2007, 179: 4027– 4034.
4028
ATTENUATED CHLAMYDIAE PROTECT AGAINST OVIDUCT DISEASE
C. muridarum was detected by PCR using the primers pMoPn F1 (5⬘-CG
TGCATGAACTTCTGAGGA-3⬘) and pMoPn R1 (5⬘-TGCAAGGAGG
TAAGCGTTCT-3⬘).
C. muridarum CM972 was plated on confluent McCoy cell monolayers
at a multiplicity of 1–2 and incubated for 2 h at 37°C without centrifugation
before being overlaid with 1⫻ DMEM, 10% FBS, 0.25% agarose, and 0.01
␮g ml⫺1 cycloheximide. The monolayers were incubated at 37°C, 5% CO2
for 5–7 days, to allow plaques to form. Under these culture conditions, C.
muridarum CM972 forms only tiny plaques at low frequency. However,
large plaques resembling those formed by C. muridarum Nigg were observed occurring at a frequency of 1–2 ⫻ 10⫺6 relative to the primary
inoculum applied to the monolayers. The plaques were harvested and individually purified by repeating the plaquing procedure without centrifugation three times before being expanded. Subsequent determination of the
plaquing efficiency of the isolates was performed, as previously described
(32). One isolate was selected for further study and designated C. muridarum CM3.1.
Murine infection and monitoring
Immune studies and tissue analysis
Genital tract secretions were collected and analyzed for cytokines, as described previously (28). Sera from mice were collected by retroorbital
bleeds at the time of sacrifice and stored at –20°C until analyzed by ELISA,
as previously described (31). 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. T cell
proliferation assays were performed, as described (26), with the addition of
anti-CD4 (BD Biosciences) to select cell cultures to investigate CD4⫹ T
cell specificity of the response. Histopathological analysis of fixed genital
tract tissues was performed, as described (31). Immunohistochemistry was
performed on paraffin-embedded tissues sectioned at 5 ␮m, deparaffinized,
and hydrated. Sections were incubated with anti-chlamydial LPS mAb (gift
from Y.-X. Zhang, Boston University, Boston, MA) at a dilution of 1/1000.
The immunohistochemical procedure was performed manually using the
EnVision G2 System/AP visualization kit (DakoCytomation), according to
the manufacturer’s instructions. The reaction was visualized by Permanent
Red Chromogen, and sections were mounted in Faramount aqueous mounting medium (DakoCytomation).
In vitro analysis of cellular responses
The following cell lines were examined: HEK293 cells stably expressing
TLR2 and MyD88 (33) and a human papillomavirus 16/E6E7 immortalized ectocervical epithelial cell line (ShEC) (34). In addition, in vitro infection was performed in murine bone marrow-derived dendritic cells
grown from bone marrow cultures following the procedure of Inaba et al.
(35). The TLR2 agonist, Pam3Cys-Ser-(Lys)4 (Axxora), or human rTNF-␣
(R&D Systems) were used as positive control stimulants. Cells were plated
in 24-well tissue culture dishes at a density of 105 cells/well. Infections
were conducted by overlaying cells with a multiplicity of 0.5–5. Cells were
incubated for 18 – 40 h at 37°C, 5% CO2. Supernatant was harvested and
assayed for IL-8 using a DuoSet ELISA kit from R&D Systems, or for
IL-1␤, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70, GM-CSF, IFN-␥, and
TNF-␣ by multiplex bead cytokine arrays (BioSource International). All
data points were assayed in triplicate and reported as the mean ⫾ SD.
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 Holm-Sidak 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
FIGURE 1. Characterization of C. muridarum strain CM3.1. A, Single
plaques formed by C. muridarum strains Nigg, CM972, and CM3.1 in
McCoy cell monolayers cultured in parallel, after 5 days of incubation at
37°C, 5% CO2. The solid overlay was removed, and the cells were stained
with crystal violet before being photographed. B, C. muridarum CM3.1
does not contain the cryptic plasmid. Equal amounts of template were
added to PCR containing primers directed against either the cryptic plasmid (pMoPn F1/R1) or the chlamydial genome (F11/B11 directed against
ompA (51)). C, Loss of the chlamydial plasmid does not affect the growth
of C. muridarum in McCoy cells. Cells infected with Nigg (Œ), CM972
(E), and CM3.1 (f) were harvested and titrated by plaque assay. The data
represent the means ⫾ SD of two samples per time point assayed in
duplicate.
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).
Results
Plasmid-deficient derivatives of C. muridarum strain Nigg have
different phenotypes in cell culture
We recently reported curing C. muridarum Nigg of its resident
7.5-kb plasmid using novobiocin (30). This strain, CM972, displays altered plaque morphology (plaque diameter reduced ⬎0.5fold) (Fig. 1A) and reduced infectivity in cell culture when compared with its parent. The mechanism underlying the attachment/
uptake defect in this strain is not known, but the deficiency is
overcome when the bacteria are inoculated into cells via centrifugation. Subsequently, we noted that inoculation of confluent McCoy cell monolayers in a plaque assay with high titer preparations
of CM972 without centrifugation resulted in the recovery of
plaques with wild-type morphology (Fig. 1A) at a frequency of
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Six- to 8-wk-old female C3H/HeouJ mice were obtained from The Jackson
Laboratory, and mice homozygous for Tlr2tm1Aki were provided by S.
Akira (Osaka, Japan). Mice were injected intravaginally, as described (31),
with 30 ␮l of buffer containing 3 ⫻ 105 IFU of C. muridarum Nigg,
CM972, or CM3.1. Mice were monitored for cervicovaginal shedding, as
described (31). Bacterial burden in oviduct tissues was measured by PFU
determination on McCoy cells (30). Oviducts dissected free from the uterine horns of a single mouse were homogenized in 1 ml of protease inhibitor
buffer, and an aliquot was removed for isolation and titration of chlamydiae. All animal experiments were preapproved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee.
The Journal of Immunology
4029
The course of murine lower genital tract infection with
plasmid-deficient strains resembles that produced by the
parental Nigg strain
Three independent experiments were conducted in which groups
of five progesterone-treated C3H mice were each inoculated intravaginally with 3 ⫻ 105 IFU of C. muridarum strain Nigg, CM3.1,
or CM972. The intensity and duration of infection were determined by quantitative culture of cervical swabs taken at intervals
through day 42 (Fig. 2A). No difference in the rate of resolution
was seen among the groups. The intensity of infection with CM972
was 10-fold lower than that of CM3.1 and Nigg between days 2
and 10, but indistinguishable thereafter (Fig. 2A). Thus, mice infected with CM972, which manifests an attachment/uptake defect
in vitro, experience an infection that is slightly decreased in intensity early on, but later parallels that of CM3.1 and Nigg.
Mice infected with CM972 or CM3.1 display minimal oviduct
pathology
Mice were sacrificed 42 days postinfection, and genital tract tissues were harvested and examined grossly and histologically for
pathology (31). Hydrosalpinx was only observed in oviducts of
mice infected with Nigg, whereas the oviducts from mice infected
with the plasmid-deficient strains appeared normal. Histologically,
inflammatory parameters scored in the ectocervix, endocervix, and
uterine horns did not differ among the groups (data not shown). In
contrast, significant differences were observed in the oviducts from
mice infected with Nigg when compared with CM3.1 and CM972
(Fig. 2B). Acute (neutrophils), chronic (lymphocytes), and plasma
cell infiltrates were absent, and oviduct dilatation was significantly
reduced in mice infected with plasmid-deficient strains (Fig. 2B)
and did not differ from a control group of mice that were mock
infected (31). Oviducts from Nigg-infected mice were dilated and
distended, and exhibited a loss of plicae and a flattened or denuded
epithelium with copious inflammatory infiltrates (Fig. 3A). In con-
FIGURE 2. Genital infection with plasmid-deficient C. muridarum fails
to induce oviduct pathology. A, Intensity and duration of lower genital tract
infection are not affected by loss of the chlamydial plasmid. Quantitative
IFU from cervical swabs obtained after infection with Nigg (Œ), CM972
(E), or CM3.1 (f). Data points are means ⫾ SD with 15 animals examined
each day. B, Chronic pathology is decreased in oviducts of mice infected
with plasmid-deficient C. muridarum compared with mice infected with
Nigg. Decreased acute (neutrophils), chronic (lymphocytes), and plasma
cell infiltrates, erosion, and dilatation were noted in oviducts from mice
infected with CM3.1 and CM972 vs Nigg. Each bar ⫽ median pathology
score calculated from 15 mice sacrificed 42 days postinfection: Nigg ⫽ f;
CM972 ⫽ 䡺; CM3.1 ⫽ u. ⴱ, p ⬍ 0.05 for CM3.1 vs Nigg; @, p ⬍ 0.05
for CM972 vs Nigg. C, The oviduct bacterial burden induced by infection
with CM3.1 is similar to that induced by Nigg (p ⫽ 0.05), but reduced in
mice infected with CM972 (p ⫽ 0.02) during the first 2 wk of infection.
Each data point (Nigg (Œ), CM972 (E), or CM3.1 (f)) represents the
chlamydial burden in the oviducts from an individual mouse; the barred
symbols indicate the mean obtained from three mice on each day for each
chlamydial strain.
trast, the oviducts from mice infected with CM972 (Fig. 3B) and
CM3.1 (Fig. 3C) appeared normal, with plicated epithelium and
open, unobstructed lumens. These observations were striking because C3H mice are particularly prone to develop pathology as a
consequence of chlamydial infection (31, 36).
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10⫺6. Subculturing of these derivatives of CM972 revealed that
they were able to infect McCoy cells at efficiencies comparable to
that of Nigg. In all other respects, these derivatives of CM972
resembled the parental strain. Plasmid DNA was undetectable by
PCR (Fig. 1B) or Southern hybridization (data not shown); the
isolates were unable to accumulate glycogen within intracytoplasmic inclusions, but could be stained with a C. muridarum-specific
mAb directed against the major outer membrane protein MOMP
(30). A single isolate, designated CM3.1, was selected for further
study.
A comparison of the infectious yield of CM972 with its parent,
Nigg, when grown in synchronous culture at a multiplicity of infection (MOI) of 0.5 (30) revealed no difference, suggesting that
once inside the cell, both strains replicated and formed infectious
progeny equally. To evaluate the growth properties of CM3.1 and
to compare chlamydial growth of all three strains throughout the
developmental cycle, we infected confluent monolayers of McCoy
cells growing in medium supplemented with cycloheximide with
either C. muridarum Nigg, CM972, or CM3.1 at a MOI of 0.5 via
centrifugation. The infected cells were harvested at 4-h intervals
over the course of a 36-h infection and titrated for chlamydiae. No
difference was observed between the strains over the course of the
chlamydial developmental cycle (Fig. 1C). Each strain displayed
eclipse phases (the period of time during infection when only noninfectious reticulate bodies are present in the infected cells) of
equal duration, and a similar yield of infectious progeny from ⬃24
h postinfection. This indicated that the phenotypic changes noted
for both CM972 and CM3.1 did not affect the strains’ abilities to
replicate in cell culture.
4030
ATTENUATED CHLAMYDIAE PROTECT AGAINST OVIDUCT DISEASE
To determine whether the lack of oviduct pathology induced by
infection with the plasmid-deficient strains reflected the inability
of these strains to infect the upper reproductive tract, tissue sections were stained for chlamydiae with an anti-chlamydial LPS
mAb (gift from Y.-X. Zhang, Boston University, Boston, MA).
Inclusions were observed in the oviducts of mice regardless of the
infecting strain (Fig. 3, D–F). Although quantitative culture of
oviducts from individual mice revealed a statistically significant
difference in numbers of infectious bacteria present in the oviducts
of CM972-infected mice compared with mice infected with Nigg
( p ⫽ 0.02), no statistical difference in chlamydial load was observed between mice infected with CM3.1 or Nigg ( p ⫽ 0.05)
during the first 2 wk of infection (Fig. 2C). The level of infection produced in the oviducts by CM972 was statistically significantly lower than that induced by CM3.1 ( p ⫽ 0.01) (Fig.
2C). Thus, the absence of pathology associated with infection
by the plasmid-deficient strains was not caused by an inability
to establish infection at this site, nor by a significant decrease in
infectious burden.
The absence of oviduct pathology in mice infected with
plasmid-deficient C. muridarum is caused by a failure to
activate signal transduction via TLR2
Our prior studies have established an essential role for TLR2 in the
induction of oviduct pathology associated with C. muridarum infection (28). Although TLR2 knockout (KO) mice experienced an
unaltered infection course, they exhibited a marked reduction in
chronic oviduct pathology compared with wild-type mice. The reduced oviduct pathology was associated with significantly lower
levels of TNF-␣ and MIP-2 in genital secretions during the first
week of infection. The striking reduction in immune pathology
observed in mice infected with CM972 or CM3.1 led us to hy-
Infection of mice with either CM972 or CM3.1 results
in Th1-predominant adaptive immune responses as seen in
mice infected with the parental Nigg strain
Multiple studies of murine chlamydial infection report a preponderance of IgG2a vs IgG1, reflective of a Th1-dominant response.
Using ELISA (31), we detected C. muridarum elementary bodyspecific IgG2a in serum taken from mice infected with Nigg,
CM972, or CM3.1 on day 28 postinfection. Titers of IgG2a were
not different among the groups (mean ⫾ SD log10 IgG2a ⫽ 3.3 ⫾
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FIGURE 3. A–C, Histopathological evaluation of oviducts (arrows)
from mice 42 days postinfection with Nigg revealed marked dilatation
consistent with hydrosalpinx (A, ⫻50), but little or no oviduct dilatation in
mice infected with CM972 (B, ⫻50) or CM3.1 (C, ⫻50). D–F, Inclusions
formed by C. muridarum Nigg (D, ⫻60), CM972 (E, ⫻80), and CM3.1 (F,
⫻80) at day 7 postinfection detected by immunohistochemistry.
pothesize that the plasmid-deficient strains failed to stimulate
TLR2-dependent signaling. We compared TNF-␣ and MIP-2 levels in genital tract secretions of mice infected with the plasmiddeficient strains with those from mice infected with Nigg during
the first 10 days of infection. Significantly reduced levels of
TNF-␣ (Fig. 4A) and MIP-2 (data not shown) were observed, resembling the decreased responses described in Nigg-infected
TLR2 KO mice (28).
Purified and rested bone marrow-derived dendritic cells were
incubated with chlamydiae at a MOI of 1 or 2, as described previously for macrophages (28). After 24 h, an aliquot of supernatant
was removed and assayed for cytokines. Intracellular staining of
the infected DCs with anti-chlamydial LPS Ab 3 and 24 h postinfection revealed that all three strains of C. muridarum had been
taken up and eliminated by 24 h. No inclusions were observed,
consistent with the report of Zhang et al. (37) that C. muridarum
does not replicate in DCs. Dendritic cell supernatants were analyzed for IL-1␤, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70, GMCSF, IFN-␥, and TNF-␣. Of the cytokines assayed, incubation of
the DCs with Nigg resulted in significant increases above medium
controls for IL-6, IL-12p40/p70, TNF-␣, and GM-CSF. Dendritic
cells incubated with CM3.1 (Fig. 4B) or CM972 (data not shown)
secreted these cytokines at significantly reduced levels. Furthermore, the cytokine responses elicited by the plasmid-deficient
strains were similar to those induced by Nigg in TLR2 KO DCs
(Fig. 4B).
Cervical epithelial cells play a critical role in early immune
signaling in response to infection by sexually transmitted pathogens. We previously reported that primary and immortalized human cervical epithelial cells express a variety of TLRs, with the
exception of TLR4 and the associated protein MD-2 (38). In subsequent studies, we showed that infection of ShEC cells with C.
trachomatis resulted in a dose-dependent induction of IL-8 secretion that was entirely dependent on MyD88 expression (32). Using
HEK293 cells stably transfected with TLR2 or TLR4/MD-2, we
determined that chlamydial-induced IL-8 secretion was predominantly TLR2 dependent, with minimal TLR4-dependent activity
(32). To investigate the effect of infection by C. muridarum Nigg
and its plasmid-deficient derivatives on the secretion of IL-8 by
relevant epithelial cells, ShEC and HEK293/TLR2 cells were infected in triplicate. After 24-h incubation, the culture supernatants
were analyzed for the presence of IL-8 by ELISA. In both the
ShEC cells (Fig. 4C) and HEK293/TLR2 cells (Fig. 4D), a dosedependent increase in IL-8 secretion occurred in response to infection with Nigg. However, infection with CM972 or CM3.1 did
not result in any increase in IL-8 secretion over that seen with cells
cultured in medium alone (Fig. 4, C and D). The infectious progeny from these cell cultures were enumerated on McCoy cells, and
no differences were observed between the strains (data not shown).
Thus, although the plasmid-deficient strains productively infect
human epithelial cells at a level equivalent to Nigg, active replication of the attenuated strains failed to elicit TLR2-dependent cell
activation.
The Journal of Immunology
4031
0.2 for Nigg, 2.8 ⫾ 0.4 for CM972, and 3.2 ⫾ 0.4 for CM3.1).
IgG1 was low or absent. We also evaluated the Chlamydia-specific
T cell proliferative response in the draining iliac nodes of mice 28
days postinfection. The iliac node cells from mice infected with
Nigg or the plasmid-deficient strains exhibited robust CD4⫹ T cell
responses after stimulation in vitro with UV-inactivated elementary bodies (data not shown). The equivalent rate of resolution of lower genital tract infection (Fig. 2A), the detection of
normal titers of IgG2a Abs, and the detection of normal T cell
responses indicated an intact adaptive response in mice infected
with plasmid-deficient strains in the absence of TLR2-dependent signaling.
Prior infection with plasmid-deficient C. muridarum protects
against the development of chlamydial disease after challenge
with strain Nigg
A single experiment was conducted in which groups containing
five progesterone-treated C3H mice were each inoculated intravaginally with 3 ⫻ 105 IFU of C. muridarum strain Nigg, CM3.1,
or CM972. The intensity and duration of infection were monitored
until resolution (Fig. 4A). The courses of infection were similar
between the groups, as previously determined (Fig. 5A). Ninetyeight days postprimary infection, each group was challenged with
3 ⫻ 105 IFU of C. muridarum strain Nigg. Mice in all three groups
became infected upon challenge with the wild-type strain (Fig.
5B). The intensity and duration of challenge infection with Nigg
were reduced compared with primary infection in all three groups,
but this decrease was statistically significant ( p ⬍ 0.001) only for
mice primarily infected with Nigg; p ⫽ 0.05 for mice primarily
infected with CM972 and p ⫽ 0.18 for mice primarily infected
with CM3.1. Clearance of challenge infection with Nigg was delayed in mice primarily infected with the plasmid-deficient strains
( p ⫽ 0.02 for Nigg/Nigg vs CM3.1/Nigg; p ⫽ 0.05 for Nigg/Nigg
vs CM972/Nigg; p ⫽ 0.03 for CM3.1/Nigg vs CM972/Nigg by
two-way repeated measures ANOVA).
The mice were sacrificed 42 days after challenge, and genital
tract tissues were harvested and examined for histopathology (31).
Eight of 10 oviducts from mice primarily infected with Nigg exhibited hydrosalpinx, compared with 2 of 10 oviducts from mice
primarily infected with CM3.1. In mice primarily infected with
CM972, hydrosalpinx was not seen. Inflammatory parameters
were statistically significantly higher in mice primarily infected
with Nigg compared with mice primarily infected with either
CM972 or CM3.1 (Fig. 5C). Furthermore, 6 of 10 oviducts from
Nigg/Nigg-infected mice showed evidence of scarring fibrosis, a
pathological finding not observed for the mice primarily infected
with the plasmid-deficient strains and challenged with Nigg (Fig.
5, D and E).
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FIGURE 4. Infection with plasmid-deficient strains of C. muridarum leads to reduced levels of cytokines largely dependent on TLR2. A, Means ⫾
SD TNF-␣ in genital secretions of five C3H/HeN mice infected with Nigg (Œ), CM972 (E), or CM3.1 (f). p ⫽ 0.025 for Nigg vs CM3.1; p ⫽ 0.017
for Nigg vs CM972; p ⫽ 0.06 for CM3.1 vs CM972 (two-way repeated measures ANOVA). B, Plasmid-deficient strains induce TNF-␣ responses
in wild-type DCs similar to those induced by Nigg in TLR2 KO DCs. Supernatants from wild-type DCs (f) or TLR2 KO DCs (䡺) cultured 24 h
with MOI ⫽ 2 of Nigg or CM3.1, or the positive control TLR2-ligand, Pam3Cys, were assayed for TNF-␣ by bead array. Bars ⫽ means ⫾ SD
calculated from triplicate wells in a single representative experiment. Plasmid-deficient strains of C. muridarum fail to induce TLR2-dependent
production of IL-8 in human cervical epithelial cells (C) or HEK293/TLR2 cells (D). Supernatants 24 h postinfection with Nigg (f), CM972 (䡺),
or CM3.1 (u) at MOIs of 0.5, 1, and 5 were assayed by ELISA for IL-8. Bars ⫽ means ⫾ SD IL-8 concentration calculated from triplicate wells.
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ATTENUATED CHLAMYDIAE PROTECT AGAINST OVIDUCT DISEASE
Discussion
Despite the availability of suitable animal models for the study of
genital tract infection by C. trachomatis, understanding of the
mechanisms by which the bacterium causes disease has been limited by the lack of defined genetic mutants. In this work, we describe a pair of C. muridarum mutants that are attenuated in their
ability to cause disease, but retain the ability to infect their murine
host. The plasmid-deficient strain CM972 was previously noted to
express a marked attachment/uptake defect in cell culture (30). In
the mouse model of genital tract infection with CM972, a 10-fold
reduced infectious burden was seen during the first 2 wk of infection. This observation may explain why plasmid-deficient strains
are so rarely observed among clinical isolates of C. trachomatis.
Spontaneous loss of the plasmid during infection may lead to a
competitive disadvantage and ultimately reduced transmissibility.
However, the attachment and uptake defect associated with
CM972 were not responsible for the failure of this strain to induce
oviduct pathology because strain CM3.1, a spontaneous mutant
derived from CM972, does not induce oviduct pathology, whereas
it infects normally in cell culture and in vivo. Thus, loss of the
plasmid from C. trachomatis is pleiotropic, impacting two virulence-associated phenotypes in addition to the ability of the chlamydiae to accumulate glycogen within their inclusion (30, 39).
An explanation for the attenuation of CM972 and CM3.1 was
derived from our discovery that neither strain is capable of stimulating a TLR2-dependent response. Our previous work has associated TLR2-dependent cytokine production with the induction of
oviduct pathology by chlamydiae (28). Evaluation of the ability of
the plasmid-deficient strains to elicit TLR2-dependent cytokines
during in vivo infection revealed a deficiency in the extent of their
response. The signaling defect was consistently observed in sentinel cells such as DCs and cervical epithelium, and was confirmed
in reconstituted TLR2-expressing epithelial cells. The possibility
that the plasmid-deficient strains were limited in their ability to
infect oviduct epithelium was ruled out as a contributor to reduced
pathology because inclusions could be observed via immunohistochemical staining and infectious bacteria were recovered from
dissected oviducts of infected mice.
Several explanations can be proposed for how the plasmid regulates TLR2-dependent signaling, as follows: directly, by failing to
express a plasmid-encoded TLR2 ligand, or indirectly, by regulation of an enzyme or pathway responsible for expression or modification of the ligand. Using confocal microscopy, we have determined that TLR2 colocalizes to inclusions produced by in vitro
infection of HEK293/TLR2 cells with CM972 and CM3.1 (data
not shown), as observed previously for Nigg (32). Thus, trafficking
of TLR2 to the inclusion is not affected by plasmid loss. However,
we have not ruled out the potential for plasmid control of chlamydial products that influence trafficking of coadaptor molecules
required for TLR2 signaling. Additional work is needed to identify
the TLR2 ligand, but the DC cytokine cellular response that we
have described will serve as a bioassay for the evaluation of candidate molecules.
Multiple studies have revealed that genetic polymorphisms of
PRRs (40, 41) and their adaptor molecules (42) are associated with
susceptibility to disease. However, the effects of TLR polymorphisms are not necessarily detrimental, considering that specific
polymorphisms in TLR4 have been linked to resistance to Legionnaires’ disease and decreased atherosclerosis (43, 44). Our data
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FIGURE 5. Prior infection with plasmid-deficient C. muridarum does not protect against infection, but protects against disease after challenge with strain
Nigg. Quantitative IFU from cervical swabs obtained after A, primary infection with Nigg (Œ), CM972 (E), or CM3.1 (f), and B, challenge infection with
Nigg after resolution of a primary infection with either strain Nigg (Œ), CM972 (E), or CM3.1 (f). Numbering in B reflects days following primary
infection. Data points are means ⫾ SD of mice positive for infection on each day with n ⫽ 5 per group. C, The oviducts of mice primarily infected with
plasmid-deficient C. muridarum and challenged with Nigg exhibit minimal pathology. Acute, chronic, plasma cell infiltrates and oviduct dilatation were
significantly decreased in mice primarily infected with CM3.1 or CM972 and challenged with Nigg vs mice primarily infected with Nigg and challenged
with Nigg. Each bar ⫽ median pathology score calculated from 10 oviducts. Nigg ⫽ f; CM972 ⫽ 䡺; CM3.1 ⫽ u. ⴱ, p ⬍ 0.001 for CM3.1/Nigg vs
Nigg/Nigg; @, p ⬍ 0.001 for CM972/Nigg vs Nigg/Nigg. D and E, Fibrotic oviduct occlusion occurs as a consequence of Nigg/Nigg infection. Significant
tubal dilatation and fibrosing obliteration of the oviduct lumen (arrowhead) were observed in mice primarily infected with Nigg and challenged with Nigg
(D, ⫻10; E, ⫻50).
The Journal of Immunology
Acknowledgments
We acknowledge the superb technical assistance of James D. Sikes and the
assistance of Phaedra Yount in the preparation of the manuscript.
Disclosures
The authors have no financial conflict of interest.
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