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* Institut de Recherche pour le Développement, Montpellier, France;
Center d’Immunologie Pierre-Fabre, Saint-Julien en Genevois, France; and
Hopital Necker, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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activates human macrophages. In vivo, active immunization with
Tc52 relieves the immunosuppression associated to acute infection and
elicits a specific immune response. As dendritic cells (DC) have a
central role in the initiation of immune responses, we investigated
whether Tc52 may modulate DC activity. We show that Tc52 induces human
DC maturation. Tc52-treated immature DC acquire CD83 and CD86
expression, produce inflammatory chemokines (IL-8, monocyte
chemoattractant protein-1, and macrophage-inflammatory protein-1
),
and present potent costimulatory properties. Tc52 binds to DC by a
mechanism with the characteristics of a saturable receptor system and
signals via Toll-like receptor 2. While Tc52-mediated signaling
involves its reduced glutathione-binding site, another portion of the
molecule is involved in Tc52 binding to DC. Finally, we report that
immunization with Tc52 protects mice in vivo against lethal infection
with T. cruzi. Together these data evidence complex
molecular interactions between the T. cruzi-derived
molecule, Tc52, and DC, and suggest that Tc52 and related class of
proteins might represent a new type of pathogen-associated molecular
patterns. Moreover, the immune protection data suggest that Tc52 is
among candidate molecules that may be used to design an optimal
multicomponent vaccine to control T. cruzi
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
numerous pathogen-derived molecules (e.g., CpG-containing sequences,
OmpA, or LPS) trigger DC maturation (1, 2, 3). Like other
innate cells, immature DC discriminate between infectious agents and
self by using a restricted number of receptors that recognize
structures shared by large groups of pathogens (also called
pathogen-associated molecular patterns (PAMPs)) (4). This
recognition results in cell activation via signaling receptors such as
members of the Toll-like receptor (TLR) family (4).
Immature DC express different members of the TLR family
(5) and, among them, signaling via TLR2 and TLR4 induces
DC maturation (6, 7).
During maturation, DC express increased levels of surface Ags involved
in T cell activation such as costimulatory (e.g., CD54 and CD86) and
MHC class I and II molecules (1). They produce numerous
cytokines (e.g., IL-1
and IL-6) and chemokines (e.g., monocyte
chemoattractant protein-1 (MCP-1), macrophage-inflammatory protein
(MIP)-1, MIP-1, IL-8, and RANTES) that favor lymphocyte
recruitment and activation (8). They also down-regulate
the expression of receptors for inflammatory chemokines (e.g., CCR1 and
CCR5) and up-regulate those for constitutive chemokines (e.g., CCR7 and
CXCR4), thus allowing maturing DC to migrate from the periphery to the
lymph nodes (8). At the same time, they lose their
capacity to process Ag and neoexpress some molecules (such as CD83 on
human cells). In the T cell areas of the lymphoid organs, myeloid
migratory DC have acquired potent immunostimulatory properties and
sensitize recirculating naive Ag-specific T cells (1).
The obligate intracellular protozoan Trypanosoma cruzi is the etiological agent of Chagas disease (9). The life cycle of T. cruzi includes stages in the insect vector (epimastigotes and metacyclic trypomastigotes) and the vertebrate host (bloodstream trypomastigotes and intracellular amastigotes). Chagas disease is associated with immunological and immunopathological reactions. The acute and chronic phases of infection are characterized by extensive polyclonal B and T cell activation and, paradoxically, T cells isolated at the acute phase of infection show a marked state of unresponsiveness. Different mechanisms and T. cruzi-derived molecules seem to be involved in host immune dysfunction (10). In addition to mitogenic molecules, such as LPS-like substances (11) and a T. cruzi Tc24-released protein (12), the parasites express different T cell suppressive factors such as the neuraminidase-transsialidase shed acute phase Ag (13) and a membrane glycoprotein, AGC10 (14). Nevertheless, T. cruzi is vulnerable to immune attack by Abs, T cells (CD4+ and CD8+), NK cells, and granulocytes (9). This provides hope of developing active protection against this pathogen.
The T. cruzi-released protein Tc52 contains a tandemly
repeated structure characteristic of GSTs, notably of the 
group,
and to a set of small heat shock proteins (15), and was
found to be among factors crucial for parasite survival and virulence
(16). In fact, in trypanosomes, oxidized reduced
glutathione and glutathione reductase defense system is replaced by a
system based on trypanothione (17). Tc52 protein functions
in vitro as a thioltransferase and has been described as the missing
link between the reduced glutathione (GSH)- and trypanothione-based
metabolisms (18). Like other proteins belonging to the
thioredoxin and glutaredoxin families, Tc52 exerts immunoregulatory
functions (19). In vitro, Tc52 modulates T cell
proliferation by scavenging cysteine and GSH (20), and it
activates human macrophages: it synergizes with IFN- to increase
inducible NO synthase mRNA levels and NO production, and it modulates
IL-1, IL-12, and IL-10 mRNA expression (21). Moreover,
we have shown that Tc52 could be detected in blood circulation of
T. cruzi-infected mice (20). In fact,
circulating Tc52 levels rose from 2 ng/ml (day 2 postinfection) to 43
ng/ml (day 45 postinfection) when the challenge infection was done with
2 x 102 trypomastigotes per mouse.
Increasing the parasite inoculum led to a significant increase of Tc52
levels in the blood (our unpublished observations). Finally,
active immunization with Tc52 abrogates immunosuppression observed
during the acute phase of the infection and stimulates specific B and T
cell responses (21).
Although they have a central role in innate and adaptive immunity, few recent investigations have explored the T. cruzi-DC relationship (22, 23). We tested in this study whether Tc52 may modulate human DC function. We report that Tc52 binds to and induces human and murine DC maturation and protects mice against lethal infection with T. cruzi.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tc52 was purified from parasite as described elsewhere (24). Briefly, epimastigote (T. cruzi TcY7 clone) soluble extracts obtained by sonication and centrifugation were prepared and adjusted to 5 mg/ml proteins in buffer (20 mM HEPES (pH 7.25), 1 mM EDTA, 0.15 M KCl supplemented with 0.5 mM final concentration of PMSF), and passed through S-hexyl glutathione affinity matrix (Sigma-Aldrich, St. Louis, MO). The column was then washed with 30 ml of the same buffer, and bound material was eluted using 20 ml of buffer containing 2.5 mM S-hexyl glutathione. All the eluates were dialyzed against PBS (10 mM sodium phosphate (pH 7.2), 0.15 M NaCl) and analyzed by SDS-PAGE. The proteins were visualized using the silver staining method.
To remove contaminating endotoxin, Tc52 was passed through a column containing polymyxin B coupled to resin (Pierce, Rockford, IL). The endotoxin level, determined by the Limulus assay (Charles River Breeding Laboratories, Charleston, SC) was <0.25 EU/mg Tc52.
Human cells
T cells were purified from PBMC by rosetting with SRBCs. Purity assessed by FACS analysis using a FITC-labeled anti-CD3 mAb (BD Biosciences, Erembodegem, Belgium) was >96%. Monocytes were purified from PBMC by positive selection using a magnetic cell separator (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Purity assessed by FACS analysis using a FITC-labeled anti-CD13 mAb (Cymbus, Hants, U.K.) was >95%. Monocytes were cultured in complete medium (CM) consisting of RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 mM HEPES, and 0.1 mM nonessential amino acids (all from Life Technologies, Cergy Pontoise, France) at 5 x 106 cells/5 ml/well in six-well tissue culture plates (Costar, Cambridge, MA) with 20 ng/ml IL-4 and 20 ng/ml GM-CSF (R&D Systems, Abingdon, U.K.). At day 6, immature DC were recultured at 105 cells/200 µl/well in 96-well flat-bottom tissue culture plates (Costar) in cytokine-containing CM, and were exposed or not to different concentrations of Tc52, 10 ng/ml LPS (from Escherichia coli isotype 0111:B4, Sigma-Aldrich), or casein (Sigma-Aldrich) used as a control protein. In some experiments, 5 µg/ml polymyxin B sulfate or 20 mM GSH, oxidized GSH, or L-methionine (all from Sigma-Aldrich) were also added. In others, Tc52 was added to immature DC in culture medium consisting of gluthatione- and cysteine-free RPMI 1640 medium (Life Technologies) supplemented with cytokines. Macrophages were generated by culturing monocytes for 6 days with 10 ng/ml GM-CSF.
Murine cells
C57BL/6 (H-2b), C3H/HeJ, and C3H/HeN (H-2d) mice were from Harlan (Gannat, France). C57BL/6 TLR2 knockout (TLR2-/-) mice were generated as described (25). Murine DC were generated by culturing bone marrow cells in CM supplemented with 50 µM 2-ME and containing 3 ng/ml murine GM-CSF (R&D Systems). At day 5, nonadherent immature DC expressing intermediate levels of I-A were cultured at 106 cells/ml in 96-well tissues culture plates (Costar). Cells were either exposed or nonexposed to Tc52 (5 µg/ml) or LPS (10 ng/ml) and IL-6 was quantified in the 24-h cell-free supernatants by ELISA (sensitivity of 3 pg/ml; R&D Systems). Peritoneal macrophages from C3H/HeN mice were elicited by thioglycolate.
Flow cytometric analysis
FACS analysis was performed using a FACSVantage cytofluorometer (BD Biosciences) with the following mAbs: FITC-labeled anti-CD86 (BD PharMingen, San Diego, CA), anti-CD54, and anti-HLA-DR (both from BD Biosciences) mAbs. The binding of the anti-CD83 mAb (BD Biosciences) was revealed by FITC-labeled anti-mouse IgG Ab (Silenus, Hauworth, Australia). Control isotype mAbs were from BD Biosciences. Results are expressed as mean fluorescence intensity (MFI) values after subtraction of the MFI obtained with the control mAb or as a percentage of positive cells.
NF-
B translocation
NF-B translocation was measured by FACS (26).
Briefly, immature DC at 2 x 105 cells/well
in 96-bottom wells plate were incubated with 0.4–5 µg/ml Tc52 or 10
ng/ml LPS for 30 min at 37°C. After washing, nuclei were prepared by
incubating the cells with 200 µl PIPES-Triton buffer (10 mM PIPES,
0.1 M NaCl, 2 mM MgCl2 (all from Sigma-Aldrich),
and 0.1% Triton X-100 (Boehringer Mannheim, Mannheim, Germany) in
H2O for 30 min at 4°C. Nuclei were washed and
stained with an anti-NF-B p65 mAb (Santa Cruz Biotechnology,
Santa Cruz, CA) revealed by FITC-labeled anti-mouse IgG
Ab.
Binding experiments
To study Tc52 binding to human and murine cells, Tc52 and the control proteins BSA and the streptococcal protein BB (27) were labeled with Alexa Fluor 488 dye (Alexa488; Molecular Probes, Eugene, OR). Cells (2 x 105 cells/well in 96-well V-bottom plates) were incubated for 20 min at 4°C in FACS buffer (RPMI medium/0.1% BSA) with Alexa488-labeled proteins. Results are expressed as MFI values. In neutralization experiments, DC were preincubated for 10 min in FACS buffer with different concentrations of Tc52 or of the control proteins before addition of nonsaturable amount of Alexa488-labeled Tc52 (2.5 µg/ml). Results are expressed as a percentage of inhibition defined as follows: A - B/A x 100 where A and B are the MFI obtained in the absence or presence of the unlabeled protein, respectively.
Human cytokine quantification
Day 6 human DC were stimulated as described and the
concentrations of IL-8, MCP-1, and MIP-1 were determined in the 16-h
cell-free culture supernatants by ELISA (sensitivity of 10, 5, and 10
pg/ml, respectively; R&D Systems). Results are expressed as picograms
per milliliter or as nanograms per milliliter (mean ± SD,
n = 4).
Primary allogeneic MLR
Day 6 DC were washed, recultured at 2.5 x 105 cells/5 ml/well in six-well culture plates in cytokine-containing CM, and either unstimulated or stimulated with 5 µg/ml Tc52 or 10 ng/ml LPS. After 4 days, DC were washed two times in CM, irradiated (3000 rad), and cultured in quintuplicate at 4 x 102 cells or 2 x 104 cells/200 µl/well in 96-well flat-bottom culture plates with 5 x 104 allogeneic T cells, purified as previously described. After 5 days, cells were pulsed during the last 16 h with [3H]thymidine (0.25 µCi/well; Amersham Pharmacia Biotech, Uppsala, Sweden). Results are expressed as cpm (mean ± SD of quintuplicate values) or in proliferation index defined as follow: A/B where A and B are the cpm values obtained in the presence or absence of Tc52, respectively.
Stimulation of TLR-transfected cells
Human TLR1–4 and CD14 cDNA were subcloned in the pCDNA3.1 vector (Invitrogen, Groningen, The Netherlands) and used to transfect 293 cells (American Type Culture Collection, Manassas, VA) by lipofection. Forty-eight hours post-transfection, cells were washed and recultured for 12 h in CM with 2% FCS for TLR1, TLR3, and TLR4 or without FCS for TLR2. After medium renewal, cells were either unstimulated or stimulated for 6 h with 5 µg/ml Tc52 or casein. IL-8 was quantified by ELISA in the 16-h supernatants. Results are expressed as nanograms per milliliter (mean ± SD, n = 3) or as the percentage of increase defined as follows: A - B/B x 100 where A and B are the concentrations of IL-8 obtained in the presence or absence of Tc52, respectively.
Immunization and experimental infection
Five-week-old male BALB/c mice (n = 8) were inoculated three times i.p. at 2-wk intervals with three doses of 10 µg of Tc52, in association with 30 µl of Bordetella pertussis and alum (BpAl) as adjuvant (4 IU B. pertussis and 1.25 mg aluminum hydroxide/500 µl; VAXICOQ; Institut Mérieux, Lyon, France). Control groups corresponded to untreated mice (n = 8) and mice immunized with BpAl alone (n = 8). Two weeks after the last immunization, mice were infected i.p. with 5000 bloodstream trypomastigotes of a T. cruzi TcY7 clone derived from the Y strain epimastigotes (16), parasitemia was measured in tail blood, and mortality was regularly recorded in two independent experiments as described (28).
An additional series of BALB/c mice (12 animals per group) were immunized following the scheme described above to perform a kinetic of Ab and cellular response. Two weeks after the last immunization, mice were infected i.p. with 103 trypomastigotes to keep alive all the mice during the acute phase. Three mice were bled and sacrificed at 5, 15, and 40 days postinfection and their spleen cells were stimulated in vitro with Tc52 protein.
T cell proliferation assay
Spleens were aseptically removed from anesthetized BALB/c mice and spleen cell suspensions were prepared as described (20). Cultures of spleen cells were set up in triplicate in microculture plates (Nuclon, Roskilde, Denmark) as follows: cell suspension (1 x 105 in RPMI 1640 medium, containing 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml gentamicin) were cultured with Tc52 native protein (an optimal dose of 5 µg/ml was used throughout the experiments) in a total volume of 200 µl culture medium. After culture for 3 days at 37°C under humidified atmosphere of 5% CO2 in air, 0.5 µCi [3H]methylthymidine per well was added. Sixteen hours later, cells were harvested using a multiple automated cell harvester onto filters, dried, placed in scintillation fluid, and counted in a scintillation counter.
IgG subclass determination
This was performed as reported in a previous study (28). Briefly, flexible polyvinylchloride microtitration plates (Dynatech, Alexandria, VA) were coated with Tc52 at concentration of 5 µg/ml in carbonate buffer (pH 9.6) and incubated overnight at 4°C. The unbound Ag was discarded and plates were blocked with 200 µl of PBS containing 1% BSA and 0.05% Tween 20 for 2 h at 37°C. Further washing steps were conducted and the following peroxidase-labeled rabbit Abs to mouse Ig subclasses were added: anti-IgG1 (1/2000), anti-IgG2a (1/500), anti-IgG2b (1/1000), anti-IgG3 (1/500), and anti-IgM (1/500). All the Abs were diluted in PBS/0.05% Tween 20 and incubated for 1 h at 37°C. After five washes, 100 µl o-phenyldiamine dihydrochloride was added as substrate and the reaction was allowed to proceed for 20 min at 37°C before being stopped with 1 N HCl. The absorbance was read at 492 nm by an ELISA reader.
Statistical analysis
Statistical significance was analyzed by a paired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Day 6 human immature DC were exposed to Tc52 and the cell surface
expression of activation markers was analyzed by FACS. The expression
of the costimulatory molecule CD86 (Table I
) and the maturation marker CD83 (Table I and Fig. 1A) is undetectable
on immature DC (<6% positive cells). Tc52 induces CD83 and CD86
expression on a percentage of cells that increases dose-dependently.
Its effect is significant (p < 0.05) at 0.08
µg/ml (14 ± 3% of CD86-positive cells, mean ± SD,
n = 3) (Fig. 1A) and maximal at 5 µg/ml,
the highest concentration tested (all the cells being CD83- and
CD86-positive) (Table I and Fig. 1A). Tc52-treated DC also
express high levels of CD54 and HLA-DR (Table I), and present large
veils by phase contrast microscopy (data not shown). As controls,
casein does not induce CD83 and CD86 expression, whereas LPS
up-regulates CD54, CD83, CD86, and HLA-DR on DC (Table I and Fig. 1A).
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A). In contrast, heat treatment abolishes Tc52- but not
LPS-induced CD83 expression on DC (Fig. 1A). These data
eliminate the possibility of involvement of contaminating
endotoxin in Tc52-induced DC maturation.
Because Tc52 enhances accessory and costimulatory molecule expression
on DC, we tested whether it up-regulates DC costimulatory properties.
In primary MLR assays, Tc52-treated DC stimulate allogeneic T cells
more efficiently than untreated ones (proliferation index of 3.5
± 0.6, with 104 DC/ml; mean ± SD,
n = 4) (Fig. 1B).
During the maturation process, DC produce numerous cytokines involved
in leukocyte recruitment and activation (8). We thus
tested whether Tc52 may affect chemokine production by DC. Tc52 induces
a dose-dependent production of IL-8, MCP-1, and MIP-1 by immature
DC. This effect is significant at 0.4 µg/ml
(p < 0.05), maximal at 5 µg/ml (Fig. 1C), and unaffected by polymyxin B (data not shown). In
contrast to LPS, Tc52 does not induce TNF- production by immature DC
(data not shown). Finally, Tc52 dose-dependently induces NF-B
translocation (Fig. 1D). As a positive control, LPS-induced
DC maturation is also associated with translocation of the nuclear
factor NF-B (29). Together these data show that Tc52 is
a DC maturation factor.
Tc52 binds to human and murine DCs
Because Tc52 induces human DC maturation, we then analyzed by FACS
whether a binding of Tc52 to DC was detectable. Results show that
Alexa488-labeled Tc52 binds to immature human DC
(Fig. 2A) in a manner that is
dose dependent and partly saturable (Fig. 2B). Tc52 binding
to DC is significant at 2.5 µg/ml (p < 0.05)
and maximal at 20 µg/ml, the highest concentration tested (MFI of
19 ± 3 and 34 ± 5, respectively, mean ± SD,
n = 6) (Fig. 2B). No binding of the
Alexa488-labeled control proteins, BSA, and
streptococcal protein BB (27) (Fig. 2, A and
B), or tetanus toxoid (data not shown) is observed.
Alexa488-labeled Tc52 binding to immature DC is
partly competed by unlabeled Tc52 (inhibition of 72 ± 7% with 20
µg/ml Tc52, mean ± SD, n = 3) but not by the
control proteins BB (Fig. 2C) or BSA (data not shown). Tc52
binding is also detected on human monocyte-derived macrophages but not
on human peripheral blood T cells (Fig. 2D). We then
extended these results to murine APC. Day 5 bone marrow-derived murine
DC bind Tc52 in a dose-dependent manner (data not shown) with a maximum
at 20 µg/ml (Fig. 2D). As expected (21), a
binding of Tc52 to thioglycolate-elicited macrophages is also observed
(Fig. 2D). Together, these data suggest the existence of
Tc52-binding element(s) on human and murine APCs.
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Innate cell activation by pathogens usually involves members of the TLR family (4). Therefore, we tested the involvement of these signaling molecules in Tc52-mediated cell activation.
To evaluate the involvement of TLR4, bone marrow-derived immature DC
from wild-type C3H/HeN mice and from LPS-resistant C3H/HeJ mice (which
carry a mutant of TLR4) (30) were stimulated with Tc52.
Levels of IL-6 produced by DC from C3H/HeN and C3H/HeJ in response to
increasing concentrations of Tc52 were similar (0.71 ± 0.1 and
0.77 ± 0.09 ng/ml, respectively, at 5 µg/ml Tc52, mean ±
SD, n = 3) (Fig. 3A). As expected, LPS-induced
IL-6 production is greatly reduced in C3H/HeJ compared with C3H/HeN
mice (Fig. 3A). In parallel, Tc52 enhances the percentage of
CD11c-positive DC expressing high levels of MHC class II
I-Ak molecule from both C3H/HeN and C3H/HeJ mice
(after 48 h of treatment with or without 5 µg/ml Tc52, the
percentage of I-Ak-high DC were as follows:
52 ± 11 and 18 ± 9%, respectively, in the case of DC from
CH/HeN mice; and 43 ± 13 and 22 ± 9%, respectively, in the
case of C3H/HeJ mice (mean ± SD, n = 3). Together
these observations show that Tc52 favors murine DC activation through a
TLR4-independant pathway and again allow to exclude an effect of
residual contaminating endotoxin in Tc52-induced DC maturation.
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B). Nontransfected 293 cells do not produce
IL-8 either constitutively or in response to Tc52 (Fig. 3B).
As previously reported (2, 32), 293 cells transfected with
TLR2 or TLR4 are constitutively activated and produce IL-8 (Fig. 3B). Interestingly, transfection of TLR2, but not of TLR1,
TLR3, or TLR4, confers responsiveness to Tc52 (increase of 238 ±
56%, mean ± SD, n = 3). As expected
(33), LPS, used as control, enhances IL-8 production by
TLR4-transfected cells (increase of 256 ± 35%, using 5 µg/ml
LPS) (data not shown). In additional experiments using the human U373
astrocytoma cell line, which expresses TLR1, TLR3, and TLR4 but not
TLR2 (32), we verified that TLR2 transfection confers
responsiveness to Tc52 (data not shown). Finally, DC generated from
bone marrow cells from TLR2-/- mice do not
produce IL-6 in response to Tc52 (Fig. 3C), thereby
confirming TLR2 involvement in Tc52-induced cell activation. Tc52-GSH binding site is involved in Tc52-induced DC maturation but not in Tc52 binding to DC
Tc52 interacts with GSH and exerts a trypanothione-glutathione thioltransferase activity (18, 20). Therefore, we examined whether a physiological concentration of exogenous GSH could affect Tc52-induced DC maturation and/or Tc52 binding to DC.
To evaluate the involvement of GSH in Tc52-induced DC maturation, Tc52
was added to human immature DC in the presence or absence of 10 mM
reduced GSH, and CD83 expression (Fig. 4A) as well as IL-8 production
(Fig. 4B) were analyzed. GSH significantly prevents
Tc52-induced CD83 expression (the percentage of inhibition of 91
± 11 and 47 ± 9 of the effect of 0.4 and 5 µg/ml Tc52,
respectively; mean ± SD, n = 3) (Fig. 4A) as well as IL-8 production by DC (the percentage of
inhibition of 97 ± 8 and 54 ± 7 of the effect of 0.4 and 5
µg/ml Tc52, respectively) (Fig. 4B). In contrast,
methionine used as a negative control does not affect Tc52-induced CD83
expression or IL-8 production by DC (Fig. 4, A and
B).
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A) or CD86 (data not shown) or produce IL-8 (Fig. 4B). When treated with Tc52, they still differentiate into
mature DC; they acquire CD83 (Fig. 4A) and CD86 (data not
shown) expression and produce IL-8 (Fig. 4B). These data
suggest that Tc52 induces DC maturation by acting directly on DC via
its active GSH binding site.
Because Tc52 signals via TLR2, we examined the possible involvement of
Tc52-GSH binding site in Tc52 signaling via TLR2. TLR2-transfected 293
cells were stimulated with Tc52 in the absence or presence of GSH and
IL-8 production was measured. Results show that GSH prevents
Tc52-induced IL-8 production (decrease of 80 ± 9%, mean ±
SD, n = 3), whereas no inhibitory effect was observed
with methionine (Fig. 4C). Together, these data suggest that
Tc52-GSH binding site is involved in Tc52-induced DC maturation and
signaling via TLR2.
Finally, we evaluated whether Tc52-GSH binding site is involved in Tc52
binding to DC. Alexa488-labeled Tc52 was
incubated with immature human DC in the presence of GSH. At any
concentration tested, we failed to prevent Tc52 binding to DC (data not
shown). Together, these data suggest that two different regions of Tc52
interact with DC. While Tc52-GSH binding site seems to be involved in
DC maturation via TLR2, the substrate binding site(s) might interact
with Tc52-binding structure(s) on DC surface (Fig. 5).
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DC are the only APCs able to initiate a specific immune response. The above data, showing that Tc52 binds to and activates DC, are in agreement with our previous observations showing that mice immunized with Tc52 develop a potent Ab- and cellular-specific immune response (21). Because Tc52 is crucial for parasite survival, we tested whether the anti-Tc52 immune response may affect parasite development.
As shown in Fig. 6, mice immunized with
Tc52-BpAl and challenged with bloodstream trypomastigotes presented
reduced parasitemia levels when compared with control mice. At day 35,
no parasites could be detected in the blood of Tc52-immunized and
surviving mice, suggesting that the parasitemia was controlled in the
case of Tc52-immunized mice. Furthermore, results indicate that mice
immunized with Tc52-BpAl present a significant reduction in mortality
when compared with control mice (Table II). In this group, one of the animals
died up to 28 days postinfection. Five of eight mice survived and
were maintained until 120 days postinfection (data not shown). In
contrast, all mice in control groups died between days 22 and 35 after
infection. The second experiment showed a similar pattern of survival.
Six of eight mice survived in Tc52-BpAl-immunized mice, whereas
significant mortality occurred in the case of control nonimmunized or
adjuvant-immunized mice.
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, Tc52-immune mice showed higher levels of total anti-Tc52 Abs than
adjuvant-immunized or nonimmunized mice. Isotype and IgG subclass
analysis showed the production of IgG1, IgG2a, IgG2b, and IgG3. IgM was
produced at low levels in all the groups (data not shown). Furthermore,
the capacity of spleen cells to respond to Tc52 Ag was also examined.
As shown in Fig. 8, the proliferative
responses of Tc52-immunized and -infected mice remained higher than
those of adjuvant-immunized or nonimmunized and infected mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We report that the T. cruzi Tc52, a released protein
belonging to thiol-disulfide oxidoreductase family, binds to human DC
and macrophages but not to T cells. The binding is dose
dependent, partly saturable, and competed by unlabeled Tc52, thereby
suggesting that Tc52-binding structure(s) exist on DC. Tc52 also
induces DC maturation as assessed by an up-regulation of costimulatory
molecule and inflammatory chemokine expression. Although T.
cruzi releases LPS-like substances (11), their
involvement in Tc52-induced DC maturation is unlikely, as the effect of
Tc52 is unaffected by polymyxin B but abolished by heat treatment.
Moreover, in contrast to LPS, Tc52 does not induce TNF- production
by DC (data not shown) and does not signal via TLR4. Previously, Tc52
has been reported to induce NO and proinflammatory monokine production
by macrophages (21). Together these data show that Tc52
binds to and activates DC and macrophages.
Molecules of the GST and thioredoxin families are ubiquitous housekeeping enzymes present in many parasites and crucial for parasite survival. Using a gene knockout approach, we also demonstrated that Tc52 is crucial for parasite survival and virulence (16). Although the enzymatic activity is responsible for general cell functions, GSTs also have a variety of important roles in immunity, including modulation of T lymphocyte function, cell growth and differentiation, and catalysis of the covalent changes involved in the protein folding (35). It is likely that GSTs can elicit in vivo complex cellular interactions that may modulate host immune response. As an example, the dimeric form of GST present in the excretory-secretory products of Fasciola hepatica modulates T cell proliferation in vitro and NO production by normal peritoneal macrophages (36).
Innate cell activation usually involves signaling molecules of the TLR family (4). Using TLR-transfected cells, we demonstrate that Tc52 signals via TLR2. Interestingly, it has been reported that activation via TLR2 contributes to the killing of intracellular pathogens via NO-dependent or -independent pathways (37). As Tc52 induces NO production by macrophages (21), it is tempting to speculate that innate cells, and especially macrophages, by producing NO upon contact with Tc52, may contribute to limit the division of intracellular amastigotes. Together these data show that the Tc52 GST-related molecule, which is crucial for parasite survival, binds to and activates DC and macrophages and signals via TLR2. As a consequence, this class of proteins might constitute a new type of PAMPs. Whether Tc52 may also interact with NK cells and granulocytes that also contribute to the immune defense against T. cruzi awaits further investigations.
In the case of T. cruzi, recent investigations have shown
that GPI anchors and glycoinositolphospholipids (GIPLs) from T.
cruzi are potent activators of human and mouse macrophage TLR2
(38). Interestingly, the TLR2 activation by GPI led to the
synthesis of IL-12 and TNF-, a profile similar to that observed when
using LPS as a triggering agent (2). In contrast, TLR2
activation by Tc52 resulted in IL-8, MCP-1, and MIP-1 production,
whereas no secretion of TNF- occurred. This different pattern of
cytokine production allowed exclusion of a potential involvement of
contaminating GPI anchors in Tc52-dependent activation of TLR2.
Furthermore, this provides, to our knowledge, the first evidence that
the interaction of a protozoan parasite-derived molecule that belongs
to the thiol-disulfide oxidoreductase family with TLR2 leads to
selective release of inflammatory chemokines, a pattern distinct
from the classical profile observed in the case of LPS and related
molecules such as LPS, GPI, and GIPLs.
Although TLR2 is a transmembrane protein (4), we failed to show a direct binding of Tc52 to TLR2. No significant up-regulation of Alexa488-labeled Tc52 binding to 293 or U373 cell lines was detectable after transfection with TLR2. These data suggest that, while TLR2 is involved in Tc52-mediated signaling, other cell surface structure(s) may play a role in Tc52 binding to DC. In agreement with this observation, numerous authors also failed to detect a direct binding of bacteria that signals via TLR to TLR-transfected cells (39). Our observation suggests that a distinct type of innate receptors, some involved in binding/internalization and others involved in signaling, may cooperate to activate innate cells upon contact with Tc52. This is in agreement with data recently obtained with PAMPs, from yeast (40) or bacteria (2). The identification of Tc52-binding structure(s) on DC awaits further investigations.
Although the molecular mechanisms associated with Tc52 interaction with DC surface remains unclear, our data show that the GSH binding site of Tc52 is involved in Tc52-induced DC activation and signaling via TLR2 but is not involved in binding to DC. A common feature of GSTs is that they are composed of an N-terminal region also named glutathione binding site (G site) and a nonspecific hydrophobic C-terminal region (H site), which accommodates the electrophilic substrate (i.e., bilirubin, steroids, carcinogens, and some organic compounds). Therefore, the two Tc52 domains could have evolved toward different specificities, one retaining the capacity to bind glutathione and TLR2, the other being involved in the interaction with a DC surface structure. Further studies using truncated Tc52 or synthetic peptides derived from its primary sequence will allow dissection of the structural domains of Tc52 involved in binding to and activation of DC.
Although recent investigations have shown that T.
cruzi-conditioned medium could impair DC maturation and secretion
of IL-12 and TNF- (22), others have demonstrated that
T. cruzi transsialidase, a parasite-secreted protein,
activates mouse DC at least partly via CD43 ligation (23).
In fact, it is well known that T. cruzi secretes a number of
molecules comprising LPS-like substances (11), GPI, and
GIPL (38), which have the potential to activate rather
than inhibit DC. DC have a central role in initiating specific immune
response and numerous vaccinal strategies tend to target vaccinal Ags
to DC (1, 41). Thus, the identification of
parasite-derived molecules that may activate DC is of interest for
vaccine development. We show that the parasite-released protein, Tc52,
induces inflammatory monokine and chemokine production by macrophages
and DC and induces DC maturation. These in vitro data suggest that in
vivo Tc52 may favor local recruitment and activation of leukocyte and
then DC migration to the lymph node, where they initiate specific B and
T cell immune responses.
In agreement with the present evidence of activation of DC by Tc52, active immunization with Tc52 stimulated both arms of the immune system and induced a significant level of protection in terms of parasitemia and mortality in mice. In relation to the protective humoral response elicited during T. cruzi infection, IgG2 and IgG1 isotypes are considered to be important factors in resistance (9). Our results agree with those observations because Tc52-immunized mice showed an isotypic profile of IgG1>IgG2b>IgG2a for Abs targeted against the Tc52 Ag. In fact, it has been shown that not all classes of Abs are effective in transfer of immunity; IgG2a and IgG2b were efficient, whereas IgM and IgG1 were not (9). In recent investigations, we examined Tc52 distribution in sections of heart tissues from T. cruzi-infected mice and found that Tc52 was heavily expressed at the membrane of amastigotes and inside myocardial fibers (E. Garzon and A. Ouaissi, unpublished observations). Therefore, it is likely that the immune response to Tc52 Ag could participate in the induction of an immunoprotection by different mechanisms, including the Ab complement-mediated lysis and Ab-dependent cell-mediated cytotoxicity.
Furthermore, taking into account the efficiency of naked DNA vaccination in stimulating specific immune responses against the vector-encoded target Ag, we performed preliminary immunization experiments in BALB/c mice using pcDNA plasmid carrying Tc52-encoding gene, and found a high reduction in parasitemia and efficient protection in mice challenged with trypomastigotes (A. Guevara and A. Ouaissi, unpublished observations). In some conditions, DC present exogenous Ags in the MHC class I pathway and initiate specific CD8+ responses (42). As different observations pointed to the immunoprotective role played by CD8+ T lymphocytes in T. cruzi infection (43), further studies are currently under investigation to evaluate whether Tc52 is cross-presented by DC and elicits CTLs.
Parasites can elicit a complex series of cellular interactions that result in specific immune response or suppression depending on the immunoregulatory balance in the host. Molecules of the GST and thioredoxin family are crucial for parasite survival and are currently considered targets for the development of vaccine strategies and/or drug design (44). Inhibition of GSTs and enzymes could be a strategy to fight against a large number of diseases, such as cancers and parasite infections. For instance, the Schistosoma mansoni GST (28 kDa) is a promising vaccine candidate in human schistosomiasis (45). Immunization of cattle with Schistosoma bovis GST induced a decreased number of S. bovis, eggs which are believed to be the cause of pathology (46). In addition, a significant degree of protection against fascioliasis could be achieved by immunization of sheep with F. hepatica GST (47).
Our study provides, to our knowledge, the first evidence that a T. cruzi Tc52 protein belonging to the GST superfamily binds to and activates human DC, inducing their maturation and secretion of chemokines. These observations add the Tc52 to the list of T. cruzi Ag that trigger the host innate immune system, and suggest that Tc52 could be a promising candidate for the development of vaccinal strategies against Chagas disease.
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2 Address correspondence and reprint requests to Dr. Ali Ouaissi, Institut de Recherche pour le Développement UR 008, 911 Avenue Agropolis, BP 5045, 34032 Montpellier, France. E-mail address: ali.ouaissi{at}montp.inserm.fr
3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; MCP-1, monocyte chemoattractant protein-1; MIP, macrophage-inflammatory protein; GIPL, glycoinositolphospholipid; GSH, reduced glutathione; PAMP, pathogen-associated molecular pattern; CM, complete medium; BpAl, Bordetella pertussis and alum; MFI, mean fluorescence intensity.
Received for publication December 6, 2001. Accepted for publication April 9, 2002.
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) or presence
(
) of 5
µg/ml polymyxin B. In some experiments, Tc52 and LPS were heated at
100°C for 20 min before incubation with DC (
). CD83 expression was
analyzed by FACS after 3 days. Results are expressed as the percentage
of positive cells (mean ± SD, n = 3).
B, Tc52 enhances the T cell stimulatory properties of
DC. Immature DC were either untreated or exposed to 5 µg/ml Tc52 or
10 ng/ml LPS. After 3 days, DC were irradiated and cultured with
freshly isolated allogeneic T cells from two donors (
),
washed, and analyzed by FACS. Results are expressed as MFI (mean
± SD, n = 6). C, DC were incubated
with the indicated concentrations of BB or Tc52 for 10 min before
addition of 2.5 µg/ml Alexa488-labeled Tc52. Results are
expressed as the percentage of inhibition (mean ± SD,
n = 3). D, Human monocyte-derived
macrophages and DC, human purified peripheral blood T cells, murine
bone marrow-derived DC, and thioglycolate-elicited macrophages were
incubated for 20 min with 20 µg/ml Alexa488-labeled Tc52,
washed, and analyzed by FACS. Results are expressed as MFI (mean
± SD) of three separate experiments.
) or with
adjuvant alone (
