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Institutes of
*
Medical Microbiology, Immunology, and Hygiene, and
Clinical Chemistry and Pathobiochemistry, Technical University of Munich, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B was activated and translocated to the
nucleus, cytokines like IL-12p40 and TNF-
were secreted, and
expression of MHC class II molecules, CD40, CD80, and CD86 was
up-regulated. Importantly, secretion of cytokines as well as
translocation of NF-B were dependent on the presence of TLR2 and
independent from TLR4 with the exception of IL-12p40 secretion, which
was attenuated in the absence of either a functional TLR2 or 4. In
conclusion, we show here that recognition of the Gram-negative
bacterium C. pneumoniae depends largely on TLR2 and only
to a minor extent on TLR4. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100 times weaker compared with endotoxin from Salmonella
minnesota (12). Perhaps, Chlamydia
minimize the induction of innate immune responses to establish
successfully the intracellular replication cycle.
In contrast C. pneumoniae is able to trigger innate as well
as adaptive immune responses. For example, macrophages are triggered to
release a number of proinflammatory cytokines including TNF-, IL-1,
IL-6, and IL-8 (3, 13). Macrophages also restrict the
intracellular replication cycle of C. pneumoniae in that the
formation of fully developed reticular bodies is blocked and only
smaller inclusions prevail, probably because the fusion of chlamydial
vacuoles with lysosomes is not inhibited in contrast to permissive HeLa
cells (14, 15). Studies in CD4- or CD8-deficient as well
as SCID mice revealed that CD4+ and
CD8+ T cells are important for the clearance of
C. pneumoniae (16). Clearance seems to be
dependent on IFN-
and NO, because IFN- receptor as well as
inducible NO synthase (iNOS)2-deficient mice are very
susceptible to infection with C. pneumoniae
(16). These findings also imply that dendritic cells must
play an important role for the clearance of C. pneumoniae
from the host.
Mature dendritic cells, also termed professional APCs, are able to present minute amounts of Ag to responsive T cells because they express MHC molecules as well as costimulatory molecules like CD40, CD80, and CD86 with high density (17, 18, 19). In their immature stage dendritic cells express fewer of these surface molecules but are much more potent in uptake and processing of Ag. Pathogen-derived pattern ligands like endotoxin or CpG DNA are able to activate and mature efficiently immature dendritic cells (20).
Innate immune cells recognize microorganisms through pattern recognition receptors. The receptors involved are surface molecules like CD14, expressed on macrophages, as well as the mannose receptor (21, 22). The family of Toll-like receptors (TLR) with at least nine defined members plays an important role in the recognition process of pathogen-derived pattern ligands (23, 24) and are mammalian homologs of the Drosophila Toll receptor as well as 18-wheeler, which are responsible for the defense against fungi and bacteria, respectively (25, 26). It is still controversial whether TLRs bind directly to bacterial patterns (27). Pathogen-derived pattern structures include endotoxin, lipoteichoic acid, peptidoglycan, cell wall components of mycobacteria like lipoarrabinomannan, zymosan from yeast, as well as bacterial CpG DNA (23, 28, 29, 30, 31). Recognition of endotoxin appears to be mediated predominantly through CD14 and TLR4, whereas TLR2 appears to be involved in the recognition of crude Gram-positive bacterial cell walls, peptidoglycan, lipoarrabinomannan, lipoproteins, and zymosan (23, 28, 29, 30, 32). Recently, TLR9 was shown to play an important role in the activation of innate immune cells by bacterial CpG DNA (24). As expected from the results described above, the recognition of complete Gram-positive or Gram-negative bacteria is dependent on TLR2 or TLR4, respectively (33, 34). In vivo TLR4-mutant or TLR2-deficient mice display a higher susceptibility to Salmonella typhimurium or Staphylococcus aureus, respectively (35, 36). Main intracellular signal pathways of both TLR2 and TLR4 seem to merge at the level of the adaptor molecule MyD88 because MyD88-deficient mice are unable to recognize the components described above (37, 38, 39, 40).
This study was performed to analyze the response of murine dendritic cells derived from bone marrow (BMDDC) upon contact with C. pneumoniae. We explored whether BMDDC restrict chlamydial development and change the expression profile of important costimulatory molecules, and we analyzed whether proinflammatory cytokines are secreted. Using TLR4-mutant and TLR2-deficient mice, we also investigated whether the responses of BMDDC toward C. pneumoniae were dependent on these molecules known to be involved in host recognition of microbial patterns.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C57BL/6 mice were purchased from Harlan Winkelmann (Borchen, Germany), C3H/HeJ and C3H/HeN mice were obtained from Charles River (Sulzfeld, Germany). TLR2-deficient mice were donated by Tularik (South San Francisco, CA) and bred in our own animal facility. All mice were kept under specific pathogen-free conditions.
The generation and characterization of TLR2-deficient mice is described by Kirschning et al. (C. Kirschning, G. Meng, R. Schwandner, R. Dziarski, H. Wagner, and K. Pfeffer, manuscript in preparation). Briefly, the 3' end of the extracellular domain as well as the major part of the transmembrane region of TLR2 were deleted and replaced by gene targeting with a neomycin resistance cassette oriented in reverse direction. Homologous recombination was verified by Northern blot and genomic PCR analysis. Furthermore, the TLR2 protein was not detectable by Western blot analysis of cells from mice homozygous for the TLR2 knockout allele as compared with cells from TLR2-positive wild-type controls.
Reagents and mAbs
The mAbs specific for CD11c (biotin-labeled: 09702D, PE-labeled: 09705B), streptavidin-CyChrome (13038A) as well as PE-labeled MHC class II (06355A), CD40 (09665B), CD80 (09605B), and CD86 (09275B) and corresponding isotype controls were purchased from BD PharMingen (Hamburg, Germany). The mAbs binding a genus-specific epitope of the chlamydial endotoxin (ACI-P500/DS-281099 and ACI-FITC/DS-250298) came from Progen Biotechnik (Heidelberg, Germany). The unlabeled chlamydia-specific Ab was conjugated with an Alexa 546 Protein labeling kit (A-10237) according to the manufacturer’s protocol (Molecular Probes, Leiden, The Netherlands). Saponin (S7900), cycloheximide (C7698), and LPS of Escherichia coli (L2637) were purchased from Sigma (Deisenhofen, Germany), and Urografin (3,5-diacetoamido-2,4,6-triisobenzoic acid) was obtained from Schering (Berlin, Germany).
Culture and multiplication of C. pneumoniae
C. pneumoniae CM-1 (VR-1360; American Type Culture Collection, Manassas, VA) were multiplied according to Maass et al. (41). Chlamydial elementary bodies were centrifuged (2000 x g, 60 min, 35°C) on confluent monolayers of HEp2 cells in the presence of cycloheximide (1 µg/ml). After 72 h of culture, the harvested cells were disrupted with glass beads, and chlamydial elementary bodies were purified in a sucrose urografin gradient (bottom layer, 50% w/v sucrose solution; top layer, 30% v/v urografin in 30 mM Tris-HCl buffer, pH 7.4) at 9000 x g and 4°C for 60 min. After one wash step with 0.2 µm filtered PBS (pH 7.4), purified elementary bodies were stored in SPG buffer (0.22 M sucrose, 8.6 mM Na2HPO4, 3.8 mM KH2PO4, 5 mM glutamic acid, 0.2 µm filtered, pH 7.4) at -70°C until use. To quantify the number of elementary bodies, HEp2 cells were infected and stained with the chlamydia-specific Ab. The number of inclusion-forming units (IFU) was counted as determined by fluorescence microscopy (Carl Zeiss Jena, Göttingen, Germany) 48 h after infection. For control, noninfected HEp2 cells were treated in the same way. Contamination with mycoplasma was excluded regularly by Mycoplasma-PCR using specific primers (MWG Biotech, Martinsried, Germany).
Isolation and differentiation of BMDDC
BMDDC were generated according to Lutz et al. (42) with slight modifications. Briefly, femora and tibiae of mice were rinsed with cell culture medium applied through a 26-gauge syringe. Bone marrow cells were cultured in bacterial petri dishes at a density of 4 x 106 cells/dish in the presence of 200 U/ml GM-CSF. The medium used was very low endotoxin RPMI 1640 (Biochrom KG, Berlin, Germany) supplemented with 10% FBS (Biochrom KG) and 2-ME (50 µM; Life Technologies, Karlsruhe, Germany). After 3 days of culture, fresh medium supplemented with GM-CSF (200 U/ml) was added. On day 6 of culture, nonadherent cells were transferred to six-well cell culture dishes at a density of 0.75 x 106 cells and 2 ml of fresh medium with GM-CSF (200 U/ml) per well was added. BMDDC were exposed to C. pneumoniae (5 IFU/cell unless otherwise indicated) on the 7th day of culture in the absence of antibiotics and cycloheximide, and without centrifugation.
Detection of cytokines
The cytokines IL-12p40 and TNF- were determined in the
culture supernatant of BMDDC by commercially available ELISAs (dual set
for IL-12p40 and TNF-; R&D Systems, Wiesbaden-Nordenstadt, Germany).
The assays were performed as described by the manufacturer. Cytokine
determinations were performed at least in duplicates.
FACS analysis
Dendritic cells were double stained (30 min, 4°C) after 48 h of exposure to C. pneumoniae with mAb directed against CD11c (2 µg/ml) and MHC class II (1 µg/ml), CD11c and CD40 (2 µg/ml), CD11c and CD80 (2 µg/ml), or CD11c and CD86 (2 µg/ml). After three wash steps the cells were fixed with 1% paraformaldehyde and analyzed with a FACSCalibur instrument (BD Biosciences, San Jose, CA).
Confocal microscopy
To analyze chlamydial development inside dendritic or HEp2 cells, the cells were cultured on glass platelets and exposed to C. pneumoniae for 48 h. The glass platelets were removed and, in the case of BMDDC, stained with a FITC-labeled CD11c-specific Ab (30 min, 4°C). After three wash steps the cells were fixed with paraformaldehyde (1% w/v, 30 min, 4°C), which was followed by another wash cycle. Thereafter, cells were permeabilized with saponin (0.5% w/v, 30 min, 4°C) and stained with the Alexa 546-labeled chlamydia-specific mAb. The cells were washed again and analyzed by confocal microscopy (LSM510; Carl Zeiss Jena).
Detection of NF-B via EMSA
Nuclear protein extracts were prepared from BMDDC using a
modified version of a published protocol (43). Briefly,
BMDDC were exposed to C. pneumoniae for the time period
indicated and harvested subsequently by gentle pipetting. The
suspension (3 x 106 cells) was washed
once with 1 ml of ice-cold PBS. Cells were pelleted (2 min, 2000
x g), lysed in 50 µl of ice-cold buffer A (10 mM HEPES
(pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 5 mM DTT, 300
mM sucrose, 0.1% Nonidet P-40, 7.5 µg/ml pepstatin, antipain,
chymostatin, leupeptin, and aprotinin, and 5 mM PMSF), and left on ice
for 5 min. The nuclei were pelleted by pulse centrifugation,
resuspended in 100 µl buffer A, pulse centrifuged again, and the
supernatant was discarded. Fifty microliters of ice-cold buffer B (20
mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 100 mM NaCl, 5 mM PMSF,
7.5 µg/ml pepstatin, antipain, chymostatin, leupeptin, and aprotinin)
was added, and nuclei were sonicated for 10 s, snap-frozen, and
stored at -70°C until use. Protein concentration was determined
using the Bradford method (Bio-Rad, Munich, Germany).
The prototypic Ig -chain oligonucleotide was used as a probe and
labeled by annealing of complementary primer extension with the Klenow
fragment of DNA polymerase I (Roche Diagnostics, Mannheim, Germany) in
the presence of [-32P]dCTP (3000 Ci/mmol;
NEN, Brussels, Belgium) and deoxynucleoside triphosphates (Roche
Diagnostics). Nuclear extracts (4 µg protein) were incubated with
radiolabeled DNA probes (10 ng; 105 cpm) for
30 min at room temperature in 20 µl of binding buffer (20 mM HEPES
(pH 7.9), 50 mM KCl, 1 mM DTT, 0.5 mM EDTA, 10% glycerol, 1 mg/ml BSA,
0.2% Nonidet P-40, 50 ng/µl poly(dI-dC)). Samples were run in 0.25x
TBE buffer (22.3 mM Tris, 22.3 mM boric acid, 0.5 mM EDTA pH
8.0) on nondenaturing 4% polyacrylamide gels at 125 V. As a
control the binding Sp-1 activity was analyzed in the same nuclear
extracts by EMSA using specific consensus oligonucleotides (Promega,
Heidelberg, Germany) that were labeled with
[-32P]ATP (5000 Ci/mmol; NEN) and T4
polynucleotide kinase (Roche Diagnostics). Gels were dried and
visualized by autoradiography.
Luciferase assay
Luciferase assays on TLR2-transfected human embryonic kidney
(HEK)293 cells were performed as described (44). The
expression vector for murine TLR2 was provided by H. Heine (Research
Center, Borstel, Germany) and D. Golenbock (Evans Biomedical
Research Center, Boston, MA; Ref. 45). Briefly, 3 x
105 HEK293 cells were plated in 3.5-cm dishes and
transfected the following day with 0.1 µg of endothelial
cell-leukocyte adhesion molecule-1 luciferase reporter
construct, 1 µg of Rous sarcoma virus-
-galactosidase plasmid for
normalization, and 0.2 µg of an expression plasmid encoding
murine TLR2. Twenty-four hours after transfection, cells were
stimulated with purified C. pneumoniae. Six to 8 h
later, the cells were lysed for measurement of luciferase activity
using reagents from Promega (Madison, WI). The results were normalized
to -galactosidase activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c BMDDC, generated by a 7-day culture in the presence of
GM-CSF, were exposed to C. pneumoniae, and 2 days later
their morphology was analyzed by phase contrast microscopy. BMDDC
reacted with long cytoplasmic extensions upon exposure to C.
pneumoniae (data not shown). Fluorescence microscopy revealed that
at this point in time the chlamydial material visible within BMDDC did
not resemble inclusions generated in the permissive epithelial
carcinoma cell line HEp2 but was similar in appearance to inclusions
found in macrophages (Fig. 1
)
(4). The expression of MHC class II, CD40, CD80, and CD86
was up-regulated clearly after a 2-day exposure to C.
pneumoniae as shown by the FACS analysis depicted in Fig. 2. Similar results were obtained with
dendritic cells from C57BL/6 mice (data not shown). At the same point
in time, the ability of C. pneumoniae-exposed BALB/c
BMDDC to take up FITC-labeled dextran particles was impaired
indicating that BMDDC were matured (Fig. 3). Thus, BMDDC mice were stimulated to
mature upon contact with C. pneumoniae.
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Microbial pattern ligands like endotoxin or CpG DNA stimulate
murine BMDDC to secrete proinflammatory cytokines including TNF- and
IL-12 (20). Therefore, we tested whether C.
pneumoniae would trigger similar responses from BMDDC. Fig. 4 shows that BMDDC from BALB/c mice
secreted large amounts of TNF- with peak levels 4 h after
exposure. IL-12p40 was also detected in the culture supernatant and
peaked 24 h after exposure (Fig. 4). In contrast, IL-12p70 and
IL-10 levels were beyond the detection limits of the ELISA system (data
not shown).
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BMDDC derived from C3H/HeN mice express TLR2 and TLR4 as analyzed
by RT-PCR (data not shown). Therefore, we analyzed the requirement of
these TLRs for the recognition of C. pneumoniae by BMDDC
derived from TLR4-mutant C3H/HeJ mice and TLR2-deficient mice. BMDDC
from TLR4-mutant C3H/HeJ mice as well as BMDDC from C3H/HeN control
mice up-regulated the expression of MHC class II, CD40, CD80, and CD86
upon exposure to C. pneumoniae, suggesting that a functional
TLR4 was not required (data not shown). Furthermore, the analysis of
TNF- secretion after stimulation with titrated amounts of C.
pneumoniae showed that BMDDC from TLR4-mutant C3H/HeJ mice
secreted equal amounts of TNF- as did BMDDC from normal C3H/HeN mice
(Fig. 5A). Control stimulation
with endotoxin revealed the expected result that BMDDC from
endotoxin-hyporesponsive C3H/HeJ mice failed to secrete TNF-, yet
BMDDC from C3H/HeN, TLR2-deficient, and wild-type control mice
responded well (Fig. 5A). In contrast, the release of
TNF- by TLR2- deficient BMDDC was severely impaired upon exposure to
C. pneumoniae (Fig. 5A). In addition, HEK293
cells transfected with a NF-B-luciferase reporter construct
responded to C. pneumoniae provided they were cotransfected
with a murine TLR2 expression plasmid (Fig. 6). Similarly, peritoneal washout
macrophages from TLR2-deficient or TLR4-mutant mice displayed the same
TLR dependence of TNF- secretion upon exposure to C.
pneumoniae (data not shown). Thus, this phenotype is not confined
to BMDDC. In contrast to these observations, production of IL-12p40 by
C. pneumoniae-stimulated BMDDC appeared to be dependent on
both TLR2 and TLR4. As shown in Fig. 5B, the secretion of
IL-12p40 by TLR4-mutant or TLR2-deficient dendritic cells reached only
50% of the level of the respective control BMDDC. Taken together,
these data suggest that both TLR2 and TLR4 are involved in the
recognition of C. pneumoniae by BMDDC. However, their
respective importance depends on the BMDDC response analyzed. Thus,
TLR2 is essential for TNF- secretion, whereas for optimal IL-12p40
secretion both TLR2 and TLR4 are required.
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B in BMDDC is dependent on TLR2
The transcription factor NF-B participates in the
transcriptional regulation of a variety of immune response genes like
MHC class II genes and cytokine genes (46). Because MHC
class II molecules are up-regulated (data not shown) and TNF- is
secreted by BMDDC from C3H/HeJ mice exposed to C.
pneumoniae, we expected that NF-B is also activated in BMDDC of
TLR4-mutant mice. Indeed, as shown in Fig. 7 exposure of BMDDC from C3H/HeJ and
C3H/HeN mice to C. pneumoniae for 1, 2, and 5 h or
TNF- induced nuclear translocation of NF-B. In contrast,
stimulation with endotoxin translocated NF-B, as expected, only in
dendritic cells from C3H/HeN mice (Fig. 7). In contrast, the extent of
nuclear translocation of NF-B in dendritic cells from TLR2-deficient
mice was attenuated strongly upon contact with C. pneumoniae
in comparison to dendritic cells from wild-type control mice at all
time points analyzed (Fig. 7). However, both types of BMDDC responded
equally to stimulation with endotoxin (Fig. 7). Taken together,
C. pneumoniae-derived pattern ligands are recognized via
TLR2, which in turn activates NF-B, presumably via the IL-1R/TLR
signal pathway involving MyD88 and TNFR-associated factor 6 (44, 47, 48).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B was activated, and proinflammatory cytokines were secreted. In
contrast to endotoxin-induced BMDDC stimulation, the responses depend
mainly on TLR2 and only partially on TLR4.
The chlamydial material observed in BMDDC by fluorescence microscopy
resembles structures seen in macrophages, i.e., small, not completely
developed inclusions (4). Ultrastructural analysis of
these structures in macrophages by electron microscopy revealed small
inclusions that contain only a few chlamydial particles
(4). Furthermore, chlamydia within these small inclusions
seem to persist and are metabolically active (4). Similar
aberrant forms develop in IFN--treated HeLa cells infected with
C. trachomatis (49). Upon removal of IFN-,
typical inclusions develop together with a new progeny of infectious
elementary bodies (50). The replication of C.
pneumoniae in HEp2 cells is also inhibited upon treatment of cells
with IFN-, TNF-, or lymphotoxin (51, 52), and in one
study IFN- treatment appeared to induce aberrant and persistent
forms of C. pneumoniae in HEp2 cells (53). As
shown here (Fig. 4
), BMDDC secrete rapidly large amounts of TNF-
upon contact with C. pneumoniae. We speculate that TNF-
causes restriction of chlamydial development, which may result in
persistence of C. pneumoniae. If so, BMDDC from TNFR
p55-deficient mice may not restrict chlamydial development. In vivo
mice lacking the IFN- receptor or iNOS are highly susceptible to
infection with C. pneumoniae (16). We have not
yet analyzed whether BMDDC produce NO upon exposure to C.
pneumoniae and whether this mechanism is responsible for the
restricted development in BMDDC. However, in mice infected with
Leishmania major a substantial part of L. major
foci were associated with iNOS-positive macrophages or dendritic cells,
and pharmacological inhibition of iNOS caused an increase of the
parasite burden (54).
Pathogen-derived pattern ligands like endotoxin and CpG DNA stimulate
dendritic cells to increase the expression of MHC class II, CD40, CD80,
and CD86 and to secrete cytokines like TNF- or IL-12p40
(20). It is at present unclear which component of C.
pneumoniae is responsible for the activation of BMDDC. Because
chlamydial lipid A contains only five instead of six fatty acids with a
chain length of 14–20 C atoms the stimulatory capacity of chlamydial
endotoxin has been reported as weak (10, 11, 12). To explore
whether chlamydial endotoxin participates in the activation of BMDDC we
analyzed the response of endotoxin-hyporeactive C3H/HeJ BMDDC. Here we
describe that NF-B activation and translocation, TNF-
secretion, and up-regulation of costimulatory molecules of C3H/HeJ
BMDDC was as strong as in control C3H/HeN BMDDC (Figs. 5 and 7, and
data not shown). Furthermore, polymyxin B failed to block stimulation
of dendritic cells by C. pneumoniae in terms of TNF- and
IL-12p40 secretion (data not shown). These findings suggest that
chlamydial endotoxin is unable to stimulate dendritic cells via TLR4 if
they are exposed to the intact microorganism. Whether chlamydial
endotoxin triggers BMDDC via TLR2 or whether a yet undefined C.
pneumoniae-derived pattern ligand signals via TLR2 needs to be
investigated.
In terms of the responses analyzed here, TLR2 appears more
important than TLR4 for C. pneumoniae-induced activation
of BMDDC. Translocation of NF-B as well as the release of
cytokines measured in this study were reduced substantially in BMDDC
derived from TLR2-deficient mice (Figs. 5 and 7). In addition,
activation of an NF-B luciferase construct in HEK293 cells was
dependent on the presence of TLR2 (Fig. 6). These observations do not
rule out the possibility that TLRs other than TLR2 and 4 are involved
in C. pneumoniae-induced responses of BMDDC not analyzed
here. Also, other cell types may use a different set of TLRs to
recognize C. pneumoniae. Accordingly, it was recently shown
that TLR2 and TLR6 cooperate in macrophage activation by Gram-positive
bacteria (34). Thus, a combination of TLR2 and other TLRs
could also play a role in C. pneumoniae-induced stimulation
of BMDDC. The question remains which chlamydial compound is responsible
for the TLR2-mediated activation of BMDDC. Peptidoglycans from
Gram-positive bacteria were found to trigger innate immune cells in a
TLR2-dependent fashion (23). As already mentioned, this
macromolecule could hardly be detected biochemically in
Chlamydia (7, 8, 9). In contrast,
Chlamydia are sensitive to penicillin G known to interfere
with the synthesis of peptidoglycan (55). In light of
these findings, it appears unlikely that chlamydial peptidoglycans play
a major role in the activation of cells of the innate immune system.
Because TLR2 is not involved in CpG DNA-induced cellular activation
(31) and the frequency of CpG motifs in the genome of
C. pneumoniae is low compared with other
bacteria (our unpublished observation), chlamydial
CpG DNA is also an unlikely candidate. Despite these considerations the
potency of chlamydial DNA to stimulate innate immune cells can now be
tested using TLR9-deficient mice because this TLR was shown to mediate
activation of innate immune cells by bacterial CpG DNA
(24). In search of the stimulatory chlamydial component,
we consider one of the chlamydial heat shock proteins (hsp) as a
possible candidate. In particular, chlamydial hsp60 was described to
activate macrophages to secrete cytokines like TNF- and to regulate
expression of matrix metalloproteinases (56). Indeed,
using purified hsp60 from C. pneumoniae we are now able to
show that this protein stimulates potently innate immune cells (C.
Prazeres da Costa, C. Kirschning, D. Busch, S. Prebeck, S.
Dürr, H. Wagner, and T. Miethke, manuscript in
preparation).
The dominant role of TLR2 vs TLR4 in the recognition process of
C. pneumoniae by BMDDC was surprising because we expected
initially that for recognition of a Gram-negative bacterium like
C. pneumoniae TLR4 would be more relevant. TLR2 was
demonstrated to be involved in the recognition of components of
the Gram-positive cell wall (23, 33) as well as whole
bacteria like S. aureus, Listeria monocytogenes,
Borrelia burgdorferi, and Mycobacterium avium
(33, 57). Furthermore, a dominant-negative mutant of TLR2
inhibited TNF- secretion in RAW-TT10 macrophages upon stimulation
with S. aureus but not with S. minnesota
(30). In contrast, TLR4-mutant and -deficient mice show
convincingly that TLR4 is involved in the recognition of endotoxin and
thus Gram-negative bacteria (23, 28, 58, 59). However, it
has been shown that for recognition of a number of Gram-negative
endotoxins, TLRs other than TLR4 may be involved additionally
(30, 40). Also, both TLR2 and TLR4 appear to be
responsible for recognition of Mycobacterium tuberculosis
(60). Thus for recognition of diverse components of whole
bacteria several TLRs may be needed, and different cellular responses
may rely on different TLRs. To our knowledge, this is the first example
that for recognition of a whole Gram-negative bacterium by innate
immune cells TLR2 appears to be central whereas TLR4 plays only a minor
role. Obviously, to study the relative importance of a given TLR for
the recognition process of bacteria by immune cells a number of
cellular responses have to be analyzed. Thus, one important aspect of
this study is that different cellular responses induced by bacteria, as
exemplified here with TNF- and IL-12p40 secretion, may require
different TLRs, which may be stimulated by different
pathogen-associated pattern ligands.
Does TLR2 play an important role for recognition of C. pneumoniae in vivo? This important question is presently our focus of interest. Until now the best evidence that this is the case came from i.p. injection of C. pneumoniae and restimulation of peritoneal macrophages with endotoxin in vitro. In wild-type mice, stimulation of peritoneal macrophages with C. pneumoniae in vivo induced a hyporesponsiveness of macrophages toward a stimulation with endotoxin in vitro. This phenomenon could not be observed in TLR2-deficient mice (data not shown), indicating that macrophages recognized C. pneumoniae in vivo via TLR2.
Another issue raised by our findings is the function of C.
pneumoniae-stimulated BMDDC. Initially, we expected that C.
pneumoniae might decrease the expression of MHC class II molecules
on the cell surface of BMDDC because this would impede presentation of
chlamydial proteins to Ag-specific T cells and would thus generate an
immune escape mechanism. Indeed, it has been shown that infection of
epithelial cells with C. trachomatis prevents the
IFN--induced expression of MHC class II molecules (61).
In contrast to our expectation, BMDDC increased the expression of MHC
class II molecules as well as costimulatory molecules upon exposure to
C. pneumoniae. Therefore, one would predict that C.
pneumoniae-specific T cells are activated effectively by BMDDC to
limit the spread of C. pneumoniae in vivo. Indeed, dendritic
cells pulsed with killed C. trachomatis and injected into
mice provide protection against subsequent infection (62).
In contrast C. pneumoniae-exposed and -stimulated dendritic
cells could also initiate autoimmune responses due to presentation of
cross-reactive epitopes. As shown by Penninger et al. (63)
injection of chlamydial peptides derived from the 60-kDa cysteine-rich
outer membrane protein induced myocarditis due to a cross-reactive
epitope with the myosin heavy chain molecule. Because in that study
only peptides were injected it is unclear whether a systemic infection
of experimental animals with C. pneumoniae or C.
trachomatis would result in autoimmune disease. Clearly, further
experimentation is needed to answer these questions.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: iNOS, inducible NO synthase; TLR, Toll-like receptor; BMDDC, murine dendritic cells derived from bone marrow; IFU, inclusion-forming units; hsp, heat shock protein; HEK, human embryonic kidney.
Received for publication September 6, 2000. Accepted for publication July 10, 2001.
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  Y. Bulut, K. Shimada, M. H. Wong, S. Chen, P. Gray, R. Alsabeh, T. M. Doherty, T. R. Crother, and M. Arditi Chlamydial Heat Shock Protein 60 Induces Acute Pulmonary Inflammation in Mice via the Toll-Like Receptor 4- and MyD88-Dependent Pathway Infect. Immun., July 1, 2009; 77(7): 2683 - 2690. [Abstract] [Full Text] [PDF]
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 N. Rodriguez, R. Lang, N. Wantia, C. Cirl, T. Ertl, S. Durr, H. Wagner, and T. Miethke Induction of iNOS by Chlamydophila pneumoniae requires MyD88-dependent activation of JNK J. Leukoc. Biol., December 1, 2008; 84(6): 1585 - 1593. [Abstract] [Full Text] [PDF]
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 Y. Naiki, R. Sorrentino, M. H. Wong, K. S. Michelsen, K. Shimada, S. Chen, A. Yilmaz, A. Slepenkin, N. W. J. Schroder, T. R. Crother, et al. TLR/MyD88 and Liver X Receptor {alpha} Signaling Pathways Reciprocally Control Chlamydia pneumoniae-Induced Acceleration of Atherosclerosis J. Immunol., November 15, 2008; 181(10): 7176 - 7185. [Abstract] [Full Text] [PDF]
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S. Chen, R. Sorrentino, K. Shimada, Y. Bulut, T. M. Doherty, T. R. Crother, and M. Arditi Chlamydia pneumoniae-Induced Foam Cell Formation Requires MyD88-Dependent and -Independent Signaling and Is Reciprocally Modulated by Liver X Receptor Activation J. Immunol., November 15, 2008; 181(10): 7186 - 7193. [Abstract] [Full Text] [PDF]
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 B. Schmeck, W. Beermann, P. D. N'Guessan, A. C. Hocke, B. Opitz, J. Eitel, Q. T. Dinh, M. Witzenrath, M. Krull, N. Suttorp, et al. Simvastatin Reduces Chlamydophila pneumoniae-Mediated Histone Modifications and Gene Expression in Cultured Human Endothelial Cells Circ. Res., April 25, 2008; 102(8): 888 - 895. [Abstract] [Full Text] [PDF]
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S.-J. Jiang, C.-C. Kuo, M. W. Berry, A. W. Lee, and L. A. Campbell Identification and Characterization of Chlamydia pneumoniae-Specific Proteins That Activate Tumor Necrosis Factor Alpha Production in RAW 264.7 Murine Macrophages Infect. Immun., April 1, 2008; 76(4): 1558 - 1564. [Abstract] [Full Text] [PDF]
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G. E. Kaiko, S. Phipps, D. K. Hickey, C. E. Lam, P. M. Hansbro, P. S. Foster, and K. W. Beagley Chlamydia muridarum Infection Subverts Dendritic Cell Function to Promote Th2 Immunity and Airways Hyperreactivity J. Immunol., February 15, 2008; 180(4): 2225 - 2232. [Abstract] [Full Text] [PDF]
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S. Bas, L. Neff, M. Vuillet, U. Spenato, T. Seya, M. Matsumoto, and C. Gabay The Proinflammatory Cytokine Response to Chlamydia trachomatis Elementary Bodies in Human Macrophages Is Partly Mediated by a Lipoprotein, the Macrophage Infectivity Potentiator, through TLR2/TLR1/TLR6 and CD14 J. Immunol., January 15, 2008; 180(2): 1158 - 1168. [Abstract] [Full Text] [PDF]
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 C. Hermann Review: Variability of host pathogen interaction Innate Immunity, August 1, 2007; 13(4): 199 - 218. [Abstract] [PDF]
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 D. Droemann, J. Rupp, T. Goldmann, U. Uhlig, D. Branscheid, E. Vollmer, P. Kujath, P. Zabel, and K. Dalhoff Disparate Innate Immune Responses to Persistent and Acute Chlamydia pneumoniae Infection in Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., April 15, 2007; 175(8): 791 - 797. [Abstract] [Full Text] [PDF]
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F. Cao, A. Castrillo, P. Tontonoz, F. Re, and G. I. Byrne Chlamydia pneumoniae-Induced Macrophage Foam Cell Formation Is Mediated by Toll-Like Receptor 2 Infect. Immun., February 1, 2007; 75(2): 753 - 759. [Abstract] [Full Text] [PDF]
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K. A. Shirey, J.-Y. Jung, and J. M. Carlin Up-Regulation of Gamma Interferon Receptor Expression Due to Chlamydia-Toll-Like Receptor Interaction Does Not Enhance Signal Transducer and Activator of Transcription 1 Signaling Infect. Immun., December 1, 2006; 74(12): 6877 - 6884. [Abstract] [Full Text] [PDF]
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O. Equils, D. Lu, M. Gatter, S. S. Witkin, C. Bertolotto, M. Arditi, J. A. McGregor, C. F. Simmons, and C. J. Hobel Chlamydia Heat Shock Protein 60 Induces Trophoblast Apoptosis through TLR4 J. Immunol., July 15, 2006; 177(2): 1257 - 1263. [Abstract] [Full Text] [PDF]
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J. Katz, P. Zhang, M. Martin, S. N. Vogel, and S. M. Michalek Toll-Like Receptor 2 Is Required for Inflammatory Responses to Francisella tularensis LVS. Infect. Immun., May 1, 2006; 74(5): 2809 - 2816. [Abstract] [Full Text] [PDF]
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K. A. Shirey and J. M. Carlin Chlamydiae Modulate Gamma Interferon, Interleukin-1{beta}, and Tumor Necrosis Factor Alpha Receptor Expression in HeLa Cells Infect. Immun., April 1, 2006; 74(4): 2482 - 2486. [Abstract] [Full Text] [PDF]
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 C. M. O'Connell, I. A. Ionova, A. J. Quayle, A. Visintin, and R. R. Ingalls Localization of TLR2 and MyD88 to Chlamydia trachomatis Inclusions: EVIDENCE FOR SIGNALING BY INTRACELLULAR TLR2 DURING INFECTION WITH AN OBLIGATE INTRACELLULAR PATHOGEN J. Biol. Chem., January 20, 2006; 281(3): 1652 - 1659. [Abstract] [Full Text] [PDF]
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 X. Yang, D. Coriolan, K. Schultz, D. T. Golenbock, and D. Beasley Toll-Like Receptor 2 Mediates Persistent Chemokine Release by Chlamydia pneumoniae-Infected Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, November 1, 2005; 25(11): 2308 - 2314. [Abstract] [Full Text] [PDF]
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 X. Yang, D. Coriolan, V. Murthy, K. Schultz, D. T. Golenbock, and D. Beasley Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1069 - H1076. [Abstract] [Full Text] [PDF]
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Y. Naiki, K. S. Michelsen, N. W. J. Schroder, R. Alsabeh, A. Slepenkin, W. Zhang, S. Chen, B. Wei, Y. Bulut, M. H. Wong, et al. MyD88 Is Pivotal for the Early Inflammatory Response and Subsequent Bacterial Clearance and Survival in a Mouse Model of Chlamydia pneumoniae Pneumonia J. Biol. Chem., August 12, 2005; 280(32): 29242 - 29249. [Abstract] [Full Text] [PDF]
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K. Yaraei, L. A. Campbell, X. Zhu, W. C. Liles, C.-c. Kuo, and M. E. Rosenfeld Effect of Chlamydia pneumoniae on Cellular ATP Content in Mouse Macrophages: Role of Toll-Like Receptor 2 Infect. Immun., July 1, 2005; 73(7): 4323 - 4326. [Abstract] [Full Text] [PDF]
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U. M. Nagarajan, D. M. Ojcius, L. Stahl, R. G. Rank, and T. Darville Chlamydia trachomatis Induces Expression of IFN-{gamma}-Inducible Protein 10 and IFN-{beta} Independent of TLR2 and TLR4, but Largely Dependent on MyD88 J. Immunol., July 1, 2005; 175(1): 450 - 460. [Abstract] [Full Text] [PDF]
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 J.-P. Carralot, C. Dumrese, R. Wessel, R. Riessen, I. Autenrieth, S. Walter, O. Schoor, S. Stevanovic, H.-G. Rammensee, and S. Pascolo CD8+ T cells specific for a potential HLA-A*0201 epitope from Chlamydophila pneumoniae are present in the PBMCs from infected patients Int. Immunol., May 1, 2005; 17(5): 591 - 597. [Abstract] [Full Text] [PDF]
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N. Rodriguez, F. Fend, L. Jennen, M. Schiemann, N. Wantia, C. U. P. da Costa, S. Durr, U. Heinzmann, H. Wagner, and T. Miethke Polymorphonuclear Neutrophils Improve Replication of Chlamydia pneumoniae In Vivo upon MyD88-Dependent Attraction J. Immunol., April 15, 2005; 174(8): 4836 - 4844. [Abstract] [Full Text] [PDF]
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J. Rey-Ladino, K. M. Koochesfahani, M. L. Zaharik, C. Shen, and R. C. Brunham A Live and Inactivated Chlamydia trachomatis Mouse Pneumonitis Strain Induces the Maturation of Dendritic Cells That Are Phenotypically and Immunologically Distinct Infect. Immun., March 1, 2005; 73(3): 1568 - 1577. [Abstract] [Full Text] [PDF]
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B. Opitz, S. Forster, A. C. Hocke, M. Maass, B. Schmeck, S. Hippenstiel, N. Suttorp, and M. Krull Nod1-Mediated Endothelial Cell Activation by Chlamydophila pneumoniae Circ. Res., February 18, 2005; 96(3): 319 - 326. [Abstract] [Full Text] [PDF]
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 I. H Haralambieva, I. D Iankov, P. V Ivanova, V. Mitev, and I. G Mitov Chlamydophila pneumoniae induces p44/p42 mitogen-activated protein kinase activation in human fibroblasts through Toll-like receptor 4 J. Med. Microbiol., December 1, 2004; 53(12): 1187 - 1193. [Abstract] [Full Text] [PDF]
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M. G. Netea, B. J. Kullberg, L. E. H. Jacobs, T. J. G. Verver-Jansen, J. van der Ven-Jongekrijg, J. M. D. Galama, A. F. H. Stalenhoef, C. A. Dinarello, and J. W. M. Van der Meer Chlamydia pneumoniae Stimulates IFN-{gamma} Synthesis through MyD88-Dependent, TLR2- and TLR4-Independent Induction of IL-18 Release J. Immunol., July 15, 2004; 173(2): 1477 - 1482. [Abstract] [Full Text] [PDF]
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N. W. Brattig, C. Bazzocchi, C. J. Kirschning, N. Reiling, D. W. Buttner, F. Ceciliani, F. Geisinger, H. Hochrein, M. Ernst, H. Wagner, et al. The Major Surface Protein of Wolbachia Endosymbionts in Filarial Nematodes Elicits Immune Responses through TLR2 and TLR4 J. Immunol., July 1, 2004; 173(1): 437 - 445. [Abstract] [Full Text] [PDF]
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A. G. Rothfuchs, C. Trumstedt, H. Wigzell, and M. E. Rottenberg Intracellular Bacterial Infection-Induced IFN-{gamma} Is Critically but Not Solely Dependent on Toll-Like Receptor 4-Myeloid Differentiation Factor 88-IFN-{alpha}{beta}-STAT1 Signaling J. Immunol., May 15, 2004; 172(10): 6345 - 6353. [Abstract] [Full Text] [PDF]
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V. Redecke, H. Hacker, S. K. Datta, A. Fermin, P. M. Pitha, D. H. Broide, and E. Raz Cutting Edge: Activation of Toll-Like Receptor 2 Induces a Th2 Immune Response and Promotes Experimental Asthma J. Immunol., March 1, 2004; 172(5): 2739 - 2743. [Abstract] [Full Text] [PDF]
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T. Darville, J. M. O'Neill, C. W. Andrews Jr., U. M. Nagarajan, L. Stahl, and D. M. Ojcius Toll-Like Receptor-2, but Not Toll-Like Receptor-4, Is Essential for Development of Oviduct Pathology in Chlamydial Genital Tract Infection J. Immunol., December 1, 2003; 171(11): 6187 - 6197. [Abstract] [Full Text] [PDF]
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C. L. Doxsee, T. R. Riter, M. J. Reiter, S. J. Gibson, J. P. Vasilakos, and R. M. Kedl The Immune Response Modifier and Toll-Like Receptor 7 Agonist S-27609 Selectively Induces IL-12 and TNF-{alpha} Production in CD11c+CD11b+CD8- Dendritic Cells J. Immunol., August 1, 2003; 171(3): 1156 - 1163. [Abstract] [Full Text] [PDF]
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F. Bea, M. H. Puolakkainen, T. McMillen, F. N. Hudson, N. Mackman, C. C. Kuo, L. A. Campbell, and M. E. Rosenfeld Chlamydia pneumoniae Induces Tissue Factor Expression in Mouse Macrophages via Activation of Egr-1 and the MEK-ERK1/2 Pathway Circ. Res., March 7, 2003; 92(4): 394 - 401. [Abstract] [Full Text] [PDF]
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S. Dessus-Babus, T. L. Darville, F. P. Cuozzo, K. Ferguson, and P. B. Wyrick Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis Infect. Immun., June 1, 2002; 70(6): 3234 - 3248. [Abstract] [Full Text] [PDF]
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Y. Bulut, E. Faure, L. Thomas, H. Karahashi, K. S. Michelsen, O. Equils, S. G. Morrison, R. P. Morrison, and M. Arditi Chlamydial Heat Shock Protein 60 Activates Macrophages and Endothelial Cells Through Toll-Like Receptor 4 and MD2 in a MyD88-Dependent Pathway J. Immunol., February 1, 2002; 168(3): 1435 - 1440. [Abstract] [Full Text] [PDF]
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) and
C3H/HeN mice (
); lower graphs in A and
B, response of TLR2-deficient (
