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Toxoplasma gondii Triggers Myeloid
Differentiation Factor 88-Dependent IL-12
and Chemokine Ligand 2 (Monocyte
Chemoattractant Protein 1) Responses Using
Distinct Parasite Molecules and Host
Receptors
Laura Del Rio, Barbara A. Butcher, Soumaya Bennouna,
Sara Hieny, Alan Sher and Eric Y. Denkers
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 172:6954-6960; ;
doi: 10.4049/jimmunol.172.11.6954
http://www.jimmunol.org/content/172/11/6954
The Journal of Immunology
Toxoplasma gondii Triggers Myeloid Differentiation Factor
88-Dependent IL-12 and Chemokine Ligand 2 (Monocyte
Chemoattractant Protein 1) Responses Using Distinct Parasite
Molecules and Host Receptors1
Laura Del Rio,* Barbara A. Butcher,* Soumaya Bennouna,* Sara Hieny,† Alan Sher,† and
Eric Y. Denkers2*
I
mmunity to the intracellular protozoan Toxoplasma gondii
consists of high level production of type 1 cytokines, and
both IL-12 and IFN-␥ are essential for resistance to this opportunistic pathogen (1–5). In recent years, innate immunity has
emerged as a key element shaping the strength and character of the
acquired immune response that is necessary to survive infection.
Toxoplasma provides a highly potent stimulus for IL-12 production, which, in turn, is required for Th1 response ignition. Innate
immune cells, such as polymorphonuclear neutrophils (PMN),3
dendritic cells (DC), and macrophages, are important sources of
IL-12 during T. gondii infection (6 –9). Molecular definition of
parasite factors and host receptors, elucidation of responses triggered by such receptor-ligand interactions, and determination of
*Department of Microbiology and Immunology, College of Veterinary Medicine,
Cornell University, Ithaca, NY 14850; and †Immunobiology Section, Laboratory of
Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD 20892
Received for publication December 19, 2003. Accepted for publication March
31, 2004.
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 National Institute of Allergy and Infectious Diseases
Grant AI47888. L.D.R. was supported by Ministerio de Educación, Cultura y Deporte, Spain, the Fulbright Scholar Program, and the Council for International Exchange of Scholars.
2
Address correspondence and reprint requests to Dr. Eric Denkers, Department of
Microbiology and Immunology, College of Veterinary Medicine, Cornell University,
Ithaca, NY 14853-6401. E-mail address: [email protected]
3
Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; BCA, bicinchoninic acid; MyD88, myeloid differentiation factor 88; C-18, cyclophilin-18; CCL,
chemokine ligand; DC, dendritic cell; GPI, glycosylphosphatidylinositol; KO, knockout; PEC, peritoneal exudate cell; STAg, soluble tachyzoite Ag; TLR, Toll-like receptor; WT, wild type.
Copyright © 2004 by The American Association of Immunologists, Inc.
how the responses are regulated remain high priority areas of
investigation.
Toll-like receptors (TLR) are a family of evolutionarily conserved transmembrane molecules that recognize specific molecular
patterns associated with microbes. There are 10 TLR family members that together recognize a diverse collection of pathogen-associated molecular patterns. Recognition by TLR initiates signaling pathways through the common adaptor molecule myeloid
differentiation factor 88 (MyD88), leading to activation of NF-␬B
transcription factors and members of the mitogen-activated protein
kinase family (10 –12). The finding that MyD88⫺/⫺ mice are
acutely susceptible to T. gondii infection implicates TLR in innate
immune recognition of this parasite (13). Furthermore, TLR2⫺/⫺
mice have recently been shown to display higher susceptibility to
T. gondii infection than wild-type (WT) animals (14). Nevertheless, increased mortality during infection of TLR2⫺/⫺ mice occurs
only at an extremely high parasite dose. In contrast, MyD88-deficient animals display extreme susceptibility to infection that is
identical with the phenotype of IL-12⫺/⫺ and IFN-␥⫺/⫺ mice (13).
These results indirectly implicate additional TLR and multiple
TLR ligands in the innate immune response to T. gondii.
Here we focus on neutrophil responses to T. gondii. These cells
produce cytokines such as IL-12 and TNF-␣, and chemokines such
as chemokine ligand 2 (CCL2), CCL3, CCL4, and CCL20 in response to Toxoplasma and other stimuli (15–18). Neutrophils also
respond to T. gondii by producing potent DC chemoattractants and
activation stimuli, suggesting a role for these cells in instructing
early immune activation (16). In addition, evidence supports a role
for PMN in Th1 generation and resistance to infection with Toxoplasma and other microbial pathogens (19 –24).
We now report that MyD88-dependent neutrophil production of
IL-12 and CCL2 (monocyte chemoattractant protein 1) is triggered
0022-1767/04/$02.00
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Toll-like receptors (TLR) that signal through the common adaptor molecule myeloid differentiation factor 88 (MyD88) are
essential in proinflammatory cytokine responses to many microbial pathogens. In this study we report that Toxoplasma gondii
triggers neutrophil IL-12 and chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1) production in strict dependence
upon functional MyD88. Nevertheless, the responses are distinct. Although we identify TLR2 as the receptor triggering CCL2
production, parasite-induced IL-12 release did not involve this TLR. The production of both IL-12 and CCL2 was increased after
neutrophil activation with IFN-␥. However, the synergistic effect of IFN-␥ on IL-12, but not CCL2, was dependent upon Stat1
signal transduction. Although IL-10 was a potent down-regulator of Toxoplasma-triggered neutrophil IL-12 release, the cytokine
had no effect on parasite-induced CCL2 production. Soluble tachyzoite Ag fractionation demonstrated that CCL2- and IL-12
inducing activities are biochemically distinct. Importantly, Toxoplasma cyclophilin-18, a molecule previously shown to induce
dendritic cell IL-12, was not involved in neutrophil IL-12 production. Our results show for the first time that T. gondii possesses
multiple molecules triggering distinct MyD88-dependent signaling cascades, that these pathways are independently regulated, and
that they lead to distinct profiles of cytokine production. The Journal of Immunology, 2004, 172: 6954 – 6960.
The Journal of Immunology
by distinct biochemical fractions derived from the parasite. In addition, the production of CCL2, but not IL-12, is dependent upon
TLR2 signaling. Importantly, neutrophil IL-12 production is not
dependent upon parasite cyclophilin-18 (C-18), a protein recently
shown to trigger DC IL-12 through CCR5 ligation (25). Our results
are the first to show that T. gondii possesses multiple molecules
that trigger MyD88-dependent signaling cascades. Moreover, our
data reveal a hitherto unknown underlying complexity to MyD88dependent signaling, in that multiple pathways leading to distinct
outcomes can be triggered dependent upon upstream receptor-ligand interactions.
6955
Cell culture
Cells were plated at 106/well in 96-well tissue culture plates, stimulated
with parasite Ag and recombinant cytokines, then incubated (37°C, in 5%
CO2) for 18 h before collecting supernatants. Recombinant mouse IL-10
and IFN-␥ were obtained from R&D Systems (Minneapolis, MN).
Cytokine measurement
IL-12 p40 was detected by cytokine ELISA as described previously (9).
CCL2 levels were determined using a murine-specific ELISA kit, according to the manufacturer’s instructions (BD PharMingen, San Diego, CA)
The ELISA detection sensitivities were 31.2 and 15.6 pg/ml for IL-12 p40
and CCL2, respectively.
Biochemical fractionation
Materials and Methods
Mice
Ag preparation
Tachyzoites of the RH strain were maintained in human fibroblast monolayers by biweekly passage, as previously described (26). To prepare soluble tachyzoite Ag (STAg), tachyzoites were sonicated in the presence of
a protease inhibitor mixture consisting of 0.2 mM PMSF (Sigma-Aldrich,
St. Louis, MO), 0.2 ␮M aprotinin (Roche, Indianapolis, IN), 1 ␮M leupeptin (Roche), and 1 mM EDTA (Sigma-Aldrich). The resulting sonicate
was dialyzed into PBS and centrifuged at 10,000 ⫻ g for 1 h, then the
supernatant was collected and filtered through a 0.22-␮m pore size membrane (Corning Costar, Cambridge, MA). The STAg concentration was
determined using a bicinchoninic acid (BCA) protein assay according to
the manufacturer’s instructions (Sigma-Aldrich). The amount of endotoxin
present in STAg was routinely ⬍0.3 endotoxin unit/ml, determined by a
modified Limulus amebocyte lysate assay (BioWhittaker, Walkersville,
MD). The filtrate was stored at ⫺70°C until use.
PMN purification
Mice were injected i.p. with 10% thioglycolate (Difco, Detroit, MI), and
18 h later peritoneal exudate cells (PEC) were obtained by lavage with
ice-cold PBS. Isolation of neutrophils was performed by depleting MHC
class II-positive cells using a method modified from a previously published
protocol (9). PEC were incubated for 30 min at 4°C with a rat anti-mouse
IA/IE mAb (clone M5.114; American Type Culture Collection, Manassas,
VA) and washed twice with PBS. Cells were then incubated with magnetic
beads coupled to goat anti-rat IgG for 30 min at 4°C with gentle mixing.
Beads with MHC class II-positive cells were removed using a magnet
(MPC-2; Dynal, Oslo, Norway). After repeating this cycle twice, the remaining cells were washed in PBS and resuspended in cDMEM, consisting
of DMEM supplemented with 10% FCS (HyClone, Logan, UT), 1 mM
sodium pyruvate, 0.1 mM nonessential amino acids, 30 mM HEPES, 100
U/ml penicillin, 0.1 mg/ml streptomycin (Life Technologies, Grand Island,
NY), and 50 ␮M 2-ME (Sigma-Aldrich). Neutrophils obtained in this manner were routinely 90 –95% pure and ⬎95% viable as determined by trypan
blue exclusion.
Bone marrow-derived macrophage preparation
Bone marrow cells were flushed from femur and tibia, then macrophages
were generated in the presence of growth factors as previously described
(16, 27). Briefly macrophages were generated over period of 4 days by
culture in 30% L929 cell supernatant as a source of M-CSF. On the day of
analysis, cells were washed and resuspended in cDMEM.
Gel electrophoresis and silver staining
Proteins were resolved by SDS-PAGE under reducing conditions as described
previously (28). For silver staining, gels were fixed in 6% formaldehyde and
26% ethanol for 1 h, and then washed with water overnight. After incubation
in DTT (5 ␮g/ml; 30 min), gels were incubated for 30 min in 0.1% AgNO3.
After briefly rinsing in H2O, gels were developed in 3% NaCO3 with
0.02% formaldehyde. The reaction was stopped with 0.1 M citric acid.
Immunoblotting
Protein fractions were separated by reducing SDS-PAGE and subsequently
electrotransferred onto nitrocellulose membrane (Schleicher & Schuell,
Keene, NH). Membranes were blocked in 5% nonfat dry milk (Nestle
USA, Solon, OH) in TBST for 60 min at room temperature. Detection of
Toxoplasma C-18 was accomplished using a rabbit polyclonal anti-C-18
antiserum, (25). After several washes with TBST, Ab binding was detected
using an HRP-conjugated anti-rabbit Ab (Cell Signaling Technology, Beverly,
MA) in TBST containing 5% nonfat dry milk. After washing in TBST, bands
were visualized using ECL detection, following the manufacturer’s instructions (LumiGLO; Upstate Biotechnology, Lake Placid, NY).
Statistical analysis
Significance of differences between groups was determined by Student’s t
test or the F test. A value of p ⬍ 0.05 was considered significant.
Results
T. gondii triggers neutrophil IL-12 and CCL2 production
IL-12 is well known as a central cytokine in resistance to T. gondii
(4). As shown previously (8, 9) and in Fig. 1A, neutrophils release
high levels of this cytokine during parasite Ag stimulation. The
chemokine CCL2 (monocyte chemoattractant protein 1) is an important mediator of macrophage recruitment (29 –31), and we now
show that neutrophils produce this chemokine in response to
T. gondii stimulation (Fig. 1B). We also found up-regulation of
IL-12p40 and CCL2 mRNA within 2– 4 h after STAg stimulation
(data not shown).
We next compared regulation of CCL2 vs IL-12 production.
Interestingly, exogenous IFN-␥ displayed different effects on
STAg-triggered IL-12 and CCL2 release. In the presence of suboptimal amounts of STAg (1 ␮g/ml), IFN-␥ increased IL-12 release in a dose-dependent manner, whereas the cytokine itself was
unable to induce IL-12 (Fig. 1C). In contrast, IFN-␥ alone was a
potent CCL2 stimulus and when added together with STAg acted
synergistically to promote CCL2 production (Fig. 1D).
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C57BL/6, C3H/HeJ, and C3H/HeOuJ strain mice were obtained from The
Jackson Laboratory (Bar Harbor, ME). Breeding pairs of MyD88⫺/⫺ and
TLR2⫺/⫺ mice on a partially backcrossed 129/Ola⫻C57BL/6 background
were provided by Dr. S. Akira (Osaka University, Osaka, Japan) via Dr. D.
Golenbock (University of Massachusetts Medical School, Worcester, MA).
Control 129/Ola⫻C57BL/6 F1 animals were obtained from The Jackson
Laboratory. Breeding pairs of 129S6/SvEv-Stattm1Rds strain (Stat1⫺/⫺)
were purchased from Taconic Farms (Germantown, NY), and a breeding
colony was established in the Transgenic Mouse Facility of Cornell University College of Veterinary Medicine. WT mice of the 129Sv/Ev strain
were purchased from Taconic Farms. Female mice between 5 and 12 wk of
age were used for experiments. Animals were housed under specific pathogen-free conditions at the Cornell University College of Veterinary Medicine animal facility, which accredited by the American Association for
Accreditation of Laboratory Animal Care.
STAg was fractionated by stepwise precipitation with increasing amounts
of (NH4)2SO4. The fractions were dialyzed into 0.025 mM Tris buffer, pH
7, and filtered through a 0.2-␮m pore size membrane, and protein content
was measured by BCA assay.
For anion exchange chromatography, a UnoQ column connected to a
Biologic Chromatography System apparatus (Bio-Rad, Hercules, CA) was
equilibrated with 0.025 mM Tris buffer (Sigma-Aldrich), pH 7.0. After
loading parasite protein extracts, the column was eluted (2 ml/min) with a
36-ml linear NaCl gradient (0 –1 M) in 0.025 mM Tris buffer, pH 7.0.
Fractions were desalted, and protein was concentrated by centrifugation
through a semipermeable Amicon membrane (10 kDa pore; Millipore, Billerica, MA) for 15 min at 3700 ⫻ g. The protein concentration was determined by BCA assay, and samples were stored at ⫺70°C until use.
6956
TOXOPLASMA TRIGGERS IL-12 AND CCL2
The effects of IL-10 on STAg-induced CCL2 and IL-12 also
differed. It was previously demonstrated that IL-10 down-regulates
LPS-induced PMN IL-12 production (32), and we also found potent down-regulatory effects on STAg-induced IL-12 that was
maintained even in IFN-␥-primed cells (Fig. 1E). In striking contrast, IL-10 was completely ineffective at down-modulating STAginduced CCL2 regardless of whether IFN-␥ was present (Fig. 1F).
These findings show that parasite Ag simultaneously triggers an
IL-10-sensitive pathway leading to IL-12 production and an IL10-insensitive pathway that culminates in CCL2 release.
Effects of IFN-␥ on IL-12 production are Stat1 dependent, but
IFN-␥-driven CCL2 release does not require Stat1
Signaling by IFN-␥ proceeds largely, but not completely, through
intracellular signaling intermediate Stat1 (33, 34). Nevertheless,
the involvement of Stat1 signaling in neutrophil cytokine responses has not previously been evaluated. In this study we show
that IFN-␥ fails to promote STAg-induced IL-12 production in
Stat1⫺/⫺ neutrophils (Fig. 2A). The result is consistent with a role
for this signaling intermediate in mediating the synergistic effects
of IFN-␥ on PMN IL-12 release. In contrast, exogenous IFN-␥
signaled CCL2 release even in the absence of a functional Stat1
molecule (Fig. 2B). Furthermore, the ability of IFN-␥ to promote
STAg-induced CCL2 release also did not require Stat1, although in
this study Stat1 was needed for optimal CCL2 production. These
results show that IFN-␥ up-regulates STAg-induced IL-12 through
a Stat1-dependent pathway, but IFN-␥ can up-regulate CCL2 production in the absence of a functional Stat1 molecule.
IL-12 and CCL2 inducing activities in STAg are biochemically
distinct
The lack of coregulation in T. gondii triggered IL-12 and CCL2
production suggested that distinct parasite molecules might trigger
each cytokine. Therefore, we undertook biochemical fractionation
experiments in which STAg was subjected to sequential precipitation with increasing amounts of ammonium sulfate. As predicted, IL-12 and CCL2-inducing activities did not cofractionate.
Thus, most IL-12-inducing activity was present in the fraction precipitating under high salt concentration (Fig. 3A; 60 – 80% saturation). In contrast, CCL2-inducing activity precipitated under low
salt conditions (Fig. 3B; 0 –25% saturation). Titration of the fractions demonstrated that IL-12-inducing activity was enriched in
the 60 – 80% fraction relative to STAg ( p ⬍ 0.05) and in the
0 –25% ( p ⬍ 0.01) fraction over an extended range of protein
concentrations (Fig. 3C). Conversely, CCL2 activity was clearly
enriched in the 0 –25% fraction over a wide range of Ag doses
(Fig. 3D; p ⬍ 0.06). We confirmed that these fractions contained
distinct sets of proteins by SDS-PAGE, followed by silver staining
of proteins within the gel (Fig. 3E).
Distinct TLR/MyD88 signaling pathways are involved in
T. gondii-triggered IL-12 and CCL2 production
TLR are vitally important transmembrane molecules for recognition
and signaling in the innate immune system (10). We previously found
that absence of the common adapter for TLR signaling, MyD88,
resulted in defective IL-12 production in STAg-stimulated, bone
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FIGURE 1. STAg-triggered neutrophil IL-12 and
CCL-2 release is regulated by distinct mechanisms.
Highly purified thioglycolate-elicited neutrophils were
cultured for 18 h with STAg, and supernatants were harvested for detection of IL-12 (A) or CCL2 (B). C and D,
Purified PMN were cultured with STAg (1 ␮g/ml) and
increasing doses of rIFN-␥, then supernatants were
tested for IL-12 (C) and CCL2 (D). E and F, Purified
PMN were cultured with STAg (1 ␮g/ml) in the presence of IFN-␥ (100 ng/ml) and IL-10 (100 ng/ml) as
indicated, and supernatants were assayed for IL-12 (E)
and CCL2 (F). ⴱ, p ⬍ 0.01 relative to STAg alone.
These experiments were repeated three times with the
same result.
The Journal of Immunology
marrow-derived neutrophils (13). In this study we show that thioglycolate-elicited peritoneal PMN from MyD88⫺/⫺ mice are also
completely defective in IL-12 (Fig. 4A) and CCL2 (Fig. 4B) production during STAg stimulation.
A recent report indicating partial susceptibility of TLR2 knockout (KO) mice to high dose T. gondii infection (14) prompted us
to examine neutrophil cytokine responses in the absence of TLR2.
Fig. 3A shows that TLR2⫺/⫺ neutrophils are indistinguishable
from WT counterparts in terms of IL-12 production (Fig. 4A). In
dramatic contrast, production of CCL2 displayed an absolute requirement for a functional TLR2 molecule (Fig. 4B). To confirm
the specificity of the CCL2 defect, TLR2 KO neutrophils were
stimulated with exogenous IFN-␥. In this situation, PMN produced
CCL2, and consistent with lack of STAg signaling, no synergistic
effect was found when parasite Ag was included (Fig. 4C).
We showed previously that splenic DC from TLR2⫺/⫺ mice
produce normal levels of IL-12 during T. gondii infection (13).
Therefore, we also examined involvement of TLR2 in macrophage
IL-12 and CCL2 production. Interestingly, TLR2 was required for
both IL-12 and CCL2 production in these cells (Fig. 4, D and E).
T. gondii-triggered PMN IL-12 production does not involve
parasite cyclophilin
Recently, it was found that a Toxoplasma cyclophilin termed C-18
possesses IL-12-inducing properties that are dependent upon
CCR5 binding in splenic DCs (25). Therefore, it was possible that
C-18 was responsible for parasite-induced neutrophil IL-12 production. Indeed, C-18 was contained within our STAg preparations
(Fig. 5, A and B). Nevertheless, C-18 appeared to be present in
greater amount in the ammonium sulfate 0 – 60% saturation frac-
FIGURE 3. IL-12- and CCL2-inducing activities in STAg separate into
distinct biochemical fractions. STAg was subjected to sequential ammonium sulfate precipitation, fractions were resuspended in PBS and dialyzed, and 5 ␮g of each was tested for IL-12-inducing (A) and CCL2inducing (B) activities on purified neutrophils. Numbers indicate the range
of percent ammonium sulfate saturation over which precipitating proteins
were collected. The 0 –25 and 60 – 80% fractions as well as STAg were
subsequently titrated, and neutrophil IL-12 (C) and CCL2 (D) production
was determined. E, Silver staining of 0 –25 and 60 – 80% fractions (enriched for CCL-2- and IL-12-inducing activity, respectively) confirms their
unique biochemical compositions. These experiments were repeated three
times with essentially identical results.
tion relative to the 60 – 80% fraction (Fig. 5, A and B), even though
the latter was enriched for IL-12-inducing activity (Fig. 3).
To definitively exclude C-18 as the IL-12-inducing parasite
molecule, the 60 – 80% fraction was subjected to anion exchange
chromatography. The fractions were subsequently tested for C-18and IL-12-inducing activity on neutrophils. As clearly shown in
Fig. 5, B and C, fraction b, which contained C-18, was totally
devoid of neutrophil IL-12-inducing activity. In dramatic contrast,
fraction d possessed potent neutrophil IL-12-inducing activity, yet
this fraction contained no detectable T. gondii cyclophilin. These
results unequivocally demonstrate that T. gondii possesses a factor
in addition to C-18 that induces neutrophil IL-12, and that, in this
case, triggering proceeds in an MyD88-dependent manner.
Discussion
The TLR family in conjunction with the common adaptor MyD88
are key to innate immune recognition of bacterial ligands and are
also emerging as critical components in response to protozoan
pathogens. This study demonstrates that distinct T. gondii-derived
molecules trigger MyD88-dependent IL-12 and CCL2 production
using independent surface receptors. Downstream signaling pathways leading to IL-12 and CCL2 production are different, and each
is subject to distinct cytokine regulation (Fig. 6).
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FIGURE 2. The effect of IFN-␥ on CCL2, but not IL-12, is Stat1 independent. Purified thioglycolate-elicited neutrophils from WT mice (f) and
Stat1 KO mice (䡺) were cultured with STAg (1 ␮g/ml) in the presence or
the absence of IFN-␥ (100 ng/ml). Supernatants were harvested after 18 h
for detection of IL-12 (A) or CCL2 (B). In Stat1 KO cells: ⴱ, p ⬍ 0.01 vs
medium alone; ⴱⴱ, p ⬍ 0.05 vs STAg alone. In WT cells: ⴱ, p ⬍ 0.01 vs
medium alone; ⴱⴱ, p ⬍ 0.01 vs STAg alone. These experiments were
repeated three times with similar results.
6957
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TOXOPLASMA TRIGGERS IL-12 AND CCL2
FIGURE 4. STAg-triggered CCL2, but not
IL-12, in PMN is TLR2 dependent, but both require
MyD88. A and B, Neutrophils from WT, TLR2⫺/⫺,
and MyD88⫺/⫺ mice were stimulated with STAg,
and supernatant was collected for detection of IL-12
(A) and CCL2 (B). C, TLR2⫺/⫺ neutrophils were
stimulated with IFN-␥ in the presence or the absence
of STAg (1 ␮g/ml), and CCL2 production was determined by ELISA. In addition, bone marrow-derived macrophages from WT and TLR2⫺/⫺ were
subjected to STAg stimulation (10 ␮g/ml), and
IL-12 (D) and CCL2 (E) release was determined by
ELISA. These experiments were repeated twice with
the same results.
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Although neutrophils produce both IL-12 and CCL2 in response
to soluble parasite extracts, addition of exogenous IFN-␥ and
IL-10 to PMN cultures had drastically different effects on the production of these cytokines. IFN-␥ displayed synergistic activity on
IL-12 and CCL2 production when added with STAg. However,
although IFN-␥ alone failed to induce IL-12 release, the cytokine
itself was a potent stimulus of CCL2. The effects of IL-10 on
STAg-induced IL-12 and CCL2 also differed. It has been previously demonstrated that IL-10 displays a potent down-regulatory
activity on IL-12 production by LPS-stimulated PMN (32). In this
study IL-10 was also a strong down-regulator of STAg-induced neutrophil IL-12 regardless of whether IFN-␥ was present. In contrast,
CCL2 production was completely unaffected by exogenous IL-10.
The Stat1 molecule is key to signaling through the IFN-␥ receptor (34). Indeed, we found that this transducing molecule is
required for IFN-␥-mediated up-regulation of STAg-induced
IL-12 production. In contrast, IFN-␥ signaled CCL2 production
even in the absence of the Stat1 molecule. The latter results are in
accord with a recent microarray analysis of IFN-␥-activated bone
marrow-derived macrophages that showed Stat1-independent
IFN-␥ regulation of CCL2 gene expression (33). The different requirement of Stat1 signaling for IL-12 and CCL2 production by
neutrophils supports the idea that the pathways triggering these
soluble mediators are regulated in a nonidentical fashion. This hypothesis was confirmed by biochemical separation of Toxoplasma
proteins, because IL-12- and CCL2-inducing activities fractionated
independently.
Signaling through MyD88 is crucial for resistance during Toxoplasma infection, as shown in MyD88⫺/⫺ mice that display an
acute susceptibility indistinguishable from that of IL-12⫺/⫺ or
IFN-␥⫺/⫺ mice (13, 14, 35). The susceptibility of MyD88-deficient mice is associated with severely impaired IL-12 production
in Toxoplasma-activated DC, bone marrow-derived macrophages,
and bone marrow-derived PMN, leading, in turn, to reduced IFN-␥
production (13). In this study we show that CCL2 production is
also impaired in the absence of MyD88. Importantly, our data
demonstrate that T. gondii triggers neutrophil CCL2 production
through TLR2. In dramatic contrast, MyD88-dependent IL-12 production was independent of this TLR.
It was recently shown that TLR2⫺/⫺ animals display increased
susceptibility to very high dose T. gondii infection (14). Nevertheless, the animals were significantly more resistant than
FIGURE 5. Toxoplasma C-18 is not responsible for neutrophil IL-12
production. A, Proteins from STAg, recombinant C-18, 0 – 60 and 60 – 80%
ammonium sulfate STAg-precipitated fractions, and 60 – 80% fractions
eluted after anion exchange chromatographic separation (a– e) were resolved by SDS-PAGE and analyzed by silver staining. B, The same fractions were electrotransferred onto a nitrocellulose membrane and subjected
to Western blot analysis using a rabbit anti-C-18 antiserum. C, Neutrophils
from C57BL/6 mice were stimulated with STAg and anion exchange fractions (a– e; 10 ␮g/ml each), and IL-12 release was detected by ELISA.
These experiments were repeated three times with similar results.
The Journal of Immunology
MyD88⫺/⫺ or IFN-␥⫺/⫺ mice, which cannot survive low dose
infection (13, 35). The basis for the partial susceptibility of the
TLR2⫺/⫺ strain was unclear, as both MyD88⫺/⫺ and TLR2⫺/⫺
animals were defective in production of macrophage proinflammatory mediators.
In this study we clearly show that TLR2 KO PMN display normal IL-12 responses to Toxoplasma, although production of the
cytokine was defective in macrophages. In addition, we showed
that DC production of IL-12 in response to Toxoplasma is not
defective in the absence of TLR2 (13). Thus, the animals may
produce sufficient IL-12 to mediate partial protection that is nonetheless not enough to provide complete resistance to high dose
infection. Alternatively, defective CCL2 release could confer partial susceptibility to infection. CCL2 possesses potent chemotactic
activity toward monocytes, DC, lymphocytes, and NK cells (30,
31, 36, 37). Together with its receptor (CCR2), it has been found
to play a role in Th1 development against Cryptococcus neoformans, and its expression is increased during Leishmania infections
(31, 38). The role of CCL2 during in vivo Toxoplasma infection is
currently under investigation in our laboratory.
Toxoplasma ligands that trigger the production of cytokines are
not yet completely defined, but recent studies have identified some
parasite-derived molecules that possess this activity. A recent report implicates Toxoplasma glycosylphosphatidylinositols (GPI)
as a stimulus for TNF-␣ release in the RAW 264.7 macrophage
cell line (39). In this paper, highly purified tachyzoite-derived GPI
as well as their core glycans induced NF-␬B activation and TNF-␣
production when added to macrophages in vitro. GPI are highly
abundant protein anchors in the membranes of tachyzoites (40),
and GPI derived from other protozoan parasites, such as Plasmo-
dium falciparum or Trypanosoma brucei, are known to exhibit
some immunostimulatory activities on macrophages, as measured
by production of TNF-␣, IL-1, or NO, and the expression of inducible NO synthase (41– 43). The involvement of parasite GPI
in induction of macrophage cytokines has also been reported in
Trypanosoma cruzi, and interestingly, GPI-induced IL-12, TNF-␣,
and NO are dependent upon TLR2 (44). We have not yet identified
the Toxoplasma-derived TLR2 ligand that triggers neutrophil
CCL2 production, but the present study shows that it is chemically
distinct from an additional MyD88-dependent Toxoplasma ligand
triggering DC and neutrophil IL-12 production.
Toxoplasma tachyzoites express a secreted 18-kDa cyclophilin
(C-18) that triggers splenic DC IL-12 production through binding
to CCR5 (25, 45). Past and present results from our laboratories
implicate another pathway leading to DC IL-12 production involving MyD88 signaling (13). In neutrophils, our results unequivocally demonstrate that C-18 is not involved in STAg-induced
IL-12 production, and indeed, MyD88 is required for parasite-induced PMN IL-12 secretion. Further study is required to identify
the TLR and parasite ligand involved in this pathway.
Our data show that neutrophil IL-12 and CCL2 production is
triggered by distinct molecules derived from the parasite, and that
the signaling pathways involved in the production of these mediators are distinct (summarized in Fig. 6). Both responses are dependent on MyD88 signaling, but, importantly, they use distinct
receptors and trigger different signaling pathways subject to their
own mechanisms of regulation. These data dramatically demonstrate that distinct TLR work in concert to provide optimal resistance against infection with a single microbial pathogen. Defining
the molecular components of the different TLR pathways and determining how they are activated by microbial Ag will provide
crucial insight into innate immune response initiation and may
ultimately prove useful for the treatment of disease caused by T.
gondii and other microbial pathogens.
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