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Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele elicits attenuated Chagas’ disease in mice
Edwin Garzon a, Margarida Coutinho Borges a, Anabela Cordeiro-da-Silva b,Valeria Nacife c, Maria de Nazareth Meirelles c, Eliane Guilvard a,
Marie France Bosseno a, Angel Gustavo Guevara d, Simone Frederique Breniere a,Ali Ouaissi a,*
a IRD UR 008 ‘Pathogenie des Trypanosomatidae’, Centre IRD de Montpellier, 911 Avenue Agropolis, BP 5045, 34032 Montpellier Cedex, Franceb Department of Biochemistry, Faculty of Pharmacy and Institute of Molecular and Cellular Biology, University of Porto, Portugal
c Department of Ultrastructure and Cellular Biology, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045-900, Rio de Janeiro, Brazild Laboratory of Clinical Investigations, Vozandes Community Services, Hospital Vozandes, Villalengua 267 y 10 de Agosto, Casilla 17-17-691 Quito,
Ecuador
Received 3 March 2003; received in revised form 10 May 2003; accepted 14 May 2003
Immunology Letters 89 (2003) 67�/80
www.elsevier.com/locate/
Abstract
The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas’ disease. We have previously
characterized a T. cruzi virulence factor named Tc52 sharing structural and functional properties with the thioredoxin and
glutaredoxin protein family. Single mutant parasite clones (Tc52�/�) exhibiting low virulence in vitro and in vivo were obtained by
targeted Tc52 gene replacement. In this report, we have extended our study to analyze the immune response and the disease
phenotype in Tc52�/�-infected BALB/c mice, during the acute and chronic phases of the disease. Significantly lower parasitemia
were found in Tc52�/�-infected mice, as compared to wild-type parasite (WT)-infected ones. However, the expansion of all classes
of lymphocytes and macrophages was similar for both clones. Furthermore, except for IgG2b levels which were higher in the case of
WT-infected mice, all classes of Ig presented no significant difference for WT and Tc52�/�-infected animals. Interestingly, a lack of
suppression of IL-2 production and of T-cell proliferation inhibition was observed in the case of spleen cells from Tc52�/�-infected
mice. Finally, the pattern of inflammation process was different and characterized as diffused in the case of Tc52�/�-infected mice,
or presenting numerous foci in the case of WT-infected mice. Localization of the Tc52 protein in tissue sections and infected heart
cell primary cultures by immunofluorescence and immunogold labeling, respectively, revealed the presence of Tc52 at the amastigote
surface and associated to aggregates within host cell vesicles. Taken together, these results reinforce the notion of Tc52 being a
virulence factor playing a role in the phenotype of the immune response associated to the infection and on the course of the disease.
# 2003 Elsevier B.V. All rights reserved.
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Keywords: Trypanosoma cruzi ; Tc52 mutant; IFN-g; IL-2; Pathology1. Introduction
Chagas’ disease (American trypanosomiasis), caused
by the hemoflagelle protozoan parasite Trypanosoma
cruzi , affects nearly 18 million people in endemic regions
of central and south America [1] and is transmitted by
haematophagous insects of the Reduviidae family. The
complex life cycle of T. cruzi includes different poly-
morphic forms in the insect vector and in the vertebrate
host. There are two parasite stages in the vector:
epimastigote and metacyclic trypomastigote, whereas
in the mammalian host, the parasite can adopt an
intracellular reproductive form (amastigote) or an
infective flagellate form (trypomastigote).
Previous studies have shown that the degree of
resistance to T. cruzi varied among mouse strains [2].
The parasite strain virulence is an additional factor,
which might potentially influence the outcome of T.* Corresponding author. Tel./fax: �/33-467-41-6331.
E-mail address: ali.ouaissi@montp.inserm.fr (A. Ouaissi).
0165-2478/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0165-2478(03)00112-3
cruzi infection [2]. Indeed, the infectivity of T. cruzi is a
complex function depending on the parasite ability to
penetrate cells and epithelia and to evade the host
immune response. Targeted deletion of T. cruzi genesallowed to identify parasite factors involved in the
phenotypic expression of T. cruzi virulence [3�/6]
although in vivo studies with respect to the ability of
mutant parasites to produce disease in experimental
hosts have not been performed.
We have previously characterized a T. cruzi gene
encoding a parasite-released 52 kDa-protein (Tc52) [7],
composed of two homologous domains and sharingsignificant homology with both glutathion S-trans-
ferases and small heat-shock proteins [8]. Biochemical
and enzymatic investigations revealed that Tc52 plays a
role in the regulation of the parasite intracellular thiol-
disulfide redox balance, by reducing glutathione dis-
ulfide bonds [9]. Moreover, immunological approaches
revealed that, like other proteins that belong to the
thioredoxin and glutaredoxin family [10], Tc52 couldexert multifunctional activities. Indeed, Tc52 showed
first, the ability to modulate the gene expression of
macrophage cytokines and iNOS , as well as NO
production [11], and second, to interfere with human
dendritic cell physiology through complex molecular
interactions. The deletion of both Tc52 allelic genes
resulted in a lethal event for T. cruzi [12]. However, the
parasite could tolerate the deletion of one Tc52 alleleresulting in the production of single mutant parasite
clones (Tc52�/�) displaying low in vitro and in vivo
virulence [13].
The aim of the present study was to examine the
immunological and histopathological changes occurring
in BALB/c mice infected with a single Tc52�/� mutant
as compared to the parental clone. Virulence data
including parasitemia, and presence of amastigote nestsin tissues were compared for both clones. A broad
immunological survey was performed, which includes
measurement of lymphocyte cellularity, T-cell prolifera-
tive response, cytokine secretion, Ig production, as well
as histological evaluation of the inflammatory re-
sponses. This comparative in vivo study provides new
clues for the understanding of the role of Tc52 protein in
the parasite�/host interactions.
2. Materials and methods
2.1. Experimental infections, parasitemia and tissue
collection
The T. cruzi TcY7 clone derived from the Y strain
epimastigote [14], here referred to as wild-type (WT), aswell as a mutant clone derived from TcY7 and carrying
a targeted deletion of one Tc52 protein-encoding allele
(Tc52�/�) [13], were used throughout the study.
Epimastigotes were grown at 28 8C in liver tryptose
medium (LIT) supplemented with 10% fetal calf serum
(FCS, Gibco BRL, Lyon, Fance), 100 U/ml penicillin
and 100 mg/ml streptomycin (Sigma Chemical Co., St.Louis, MO). First, blood stream trypomastigotes (BT)
were obtained from BALB/c mice (IFFA-CREDO,
Lyon, France) infected intraperitoneally (i.p.) with 1-
month-old epimastigote cultures, (106 parasites per
animal). Massive production of trypomastigotes was
then carried out in vitro through infection of L929
fibroblast cultures and parasites collected by centrifuga-
tion were kept in liquid nitrogen until further use [15].Before an experimental infection, frozen parasites (WT
or Tc52�/�) were passaged in mice (105 per animal) and
BT were collected by intracardiac puncture 15 days
post-infection.
BALB/c mice (8-week-old males of 20�/25 g corporal
weight) were used in this study: Parasitemia surveys
during the acute phase, was conducted in two indepen-
dent experiments using 6 (first experiment) or 8 (secondexperiment) mice per group infected intraperitoneally
with 103 BT of either WT or Tc52�/� parasites in 0.5
ml RPMI 1640 medium. A group of 60 animals were
used for immunological and histopathological studies
during the acute and chronic phases. Uninfected mice
were used as controls. Animal procedures were carried
out in the IRD-approved facilities (license no. 34-18 to
A. Quaissi).Mice were analyzed at 7, 16, 23, 27 days (acute phase),
as well as 360 days (chronic phase) after infection.
Parasitemia was determined on individual 5 ml-blood
samples collected from the mouse tail vein and observed
under a light microscope for BT counting as described in
previous reports [15]. For immunological and histo-
pathological studies, groups of 6 WT- or Tc52�/�-
infected mice were sacrificed at the different time-pointsafter determination of individual parasitemia. For each
group, spleen and serum were collected from six animals
(four in the chronic phase due to mouse death), as well
as heart and quadriceps from three mice. Sera collected
7, 16, 23, 27 and 360 days post-infection were kept at �/
80 8C until use.
2.2. Spleen cell culture and proliferation assays
Spleens from WT and Tc52�/�-infected mice were
collected at 7, 16, 23, 27, and 360 days post-infection,
and gently dissected to obtain suspensions of dissociated
cells. After three washes in phosphate buffer saline
(PBS, pH 7.4) supplemented with 2% FCS, cells were
counted, and adjusted to 107 viable nucleated cells per
ml (trypan-blue-excluding). Spleen cells were then
cultured (2�/105 cells/well) for 48 h in 200 ml RPMImedium containing 10% FCS and supplemented with 2
mM glutamine (Sigma Chemical Co., St. Louis, MO),
penicillin and streptomycin (100 U/ml and 100 mg/ml,
E. Garzon et al. / Immunology Letters 89 (2003) 67�/8068
respectively), 0.05 mM 2-mercaptoethanol (2-ME), 20
mM HEPES (Gibco BRL) and 10% FCS and supple-
mented or not (control) with anti-CD3 monoclonal
antibody (Pharmigen) at 1.25 mg/ml. Proliferative activ-
ity was evaluated after a 16 h-pulse with 1 mCi of [3H]
thymidine (Amersham, Arlington Heights, IL). Pulsed
spleen cells were harvested on a glass filter using an
automated multiple sample harvester (Perkin�/Elmer,
Courtaboeuf, France), and then air-dried. Incorporation
of [3H] thymidine was then determined by liquid
scintillation counting. Assays were carried out in
triplicates and the mean of counts per minute (cpm)
incorporated by stimulated and unstimulated (controls)
was recorded.
2.3. Immunofluorescence and flow cytometry analysis
Spleen cells prepared as above were washed three
times with fluorescence-activated cell sorter (FACS)
buffer (0.1% FCS and 0.11% sodium azide in PBS)
and then distributed into a 96-well microtiter culture
plate (200 ml/well) at a concentration of 5�/106 cell/ml.
The cells were incubated with saturating concentration
(1:100 (v/v) dilution) of fluorescein isothiocyanate
(FITC)-conjugated rat anti-mouse CD8 (Ly-2) mAb
(Pharmingen, San Diego, CA) or FITC-conjugated rat
anti-CD4 (L3T4) mAb (Pharmingen), FITC-conjugated
goat anti-mouse IgM (Southern Biotechnology, Birmin-
ghan, AL) or FITC-conjugated rat anti-mouse macro-
phage F4/80 antigen (Caltag laboratories, Burlingame,
CA). After a 30 min-incubation on ice, the cells were
washed three times (244�/g , 5 min, 4 8C) in FACS
buffer. Annexin assays were performed with the Phar-
mingen kit according to the manufacturer’s instructions.
Briefly, cells were washed once with binding buffer (10
mM HEPES buffer containing 0.14 mM NaCl and 2.5
mM CaCl2, pH 7.4). A total of 2�/105 cells were
incubated in 100 ml binding buffer containing 2 ml of
annexin-V-FITC for 15 min at room temperature.
Propidium iodide (0.5 mg/ml) was added before analysis
to exclude dead cells. Flow cytofluorometric analysis
was done in a fluorescence-activated cell sorter (Becton
Dickinson, Mountain View, CA). Detectors for forward
(FSC) and side (SSC) light scatter were set on a linear
scale, whereas logarithmic detectors were used for all
three fluorescence channels (FL-1, FL-2 and FL-3).
Compensation for spectral overlap between FL channels
was performed for each experiment using single-color-
stained cell populations. Instrument settings were saved
to disk and used again with slight modifications if
necessary in related experiments. All data were collected
ungated to disk and were analyzed using CellQuest
software.
2.4. Immunoglobulin enzyme-linked immunosorbent
assay (ELISA)
Sera collected 7, 16, 23, 27 and 360 days post-infection
were diluted in PBS containing 1% gelatin. Flat-bottom
96-well microtiter Immune Plate Maxisorp (Nunc,
Rochester, NY) were coated overnight at 4 8C with
unlabelled goat anti-mouse Ig (5 mg/ml) or parasite
extracts (14) in 50 ml carbonate buffer, pH 8.5. After
washing in PBS pH 7.4 containing 0.1% Tween-20 (PBS-
T), plates were blocked for 1 h at 37 8C with 1% gelatin
in PBS (200 ml/well) and incubated (2 h at 37 8C) with
serial dilutions of each serum. After washing with PBS-
T, plates were incubated for 1 h at room temperature
with peroxidase-labelled goat anti-mouse immunoglo-
bulin isotypes (anti-IgM, anti-IgG1, anti-IgG3, anti-
IgG2a and anti-IgG2b; Southern Biothechnology) and
developed with o -phenildiamin (OPD, Sigma) in citrate
buffer. The optical densities were recorded at 492 nm.
The concentration of each isotype was determined by
comparison to a standard curve generated with unla-
belled purified isotypes.
2.5. Cytokine sandwich-ELISA
The cytokine concentration in supernatants of spleen
cells stimulated with 1.25 mg/ml of anti-CD3 mAb after
a 48 h-incubation, at 37 8C under 5% CO2 was
determined by two-site sandwich ELISA. Flat-bottom
96-well microtiter plates (Nunc) were coated with
unlabelled rat antibodies to the following mouse cyto-
kines: IFN-g (R4-6A2 cell line), IL-2 (JES6-1A12 cell
line), IL-4 (BVD4-1D11 cell line) and IL-10 (JES5-2A5
cell line) in 50 ml carbonate buffer, pH 8.5, overnight at
4 8C. The plates were washed in PBS-T, blocked with
1% bovine serum albumin (BSA) in PBS (200 ml/well;
Sigma) for 2 h at room temperature, washed and then
incubated with each supernatant for 2 h at room
temperature. After washing with PBS-T, the plates
were incubated for 1 h at room temperature with
biotinylated-labeled rat antibodies to the following
mouse cytokines: INF-g (XMG1.2 cell line), IL-2
(JES6-5H4 cell line), IL-4 (BVD6-24G2 cell line) and
IL-10 (SXC-1 cell line). Specific antibody for interleu-
kins were provided by Pharmingen. After washing in
PBS-T, the plates were incubated for 1 h at room
temperature with streptavidin-peroxidase (Sigma Che-
mical Co., St. Louis, MO) and developed with OPD in
citrate buffer. Optical densities were measured at 450
nm. The concentration of specific interleukins was
determined by comparison to a standard curve gener-
ated with different recombinant interleukins: rIFN-g,
rIL-2, rIL-4, rIL10 (R&D System’s, Abingdon, UK).
E. Garzon et al. / Immunology Letters 89 (2003) 67�/80 69
2.6. Histopathological analysis
The hearts and quadriceps collected from three
different mice at various times post-infection weretreated as follow. Hearts were divided longitudinally in
two parts. For every mouse, heart right auricle and
ventricle were analyzed. All tissues were fixed in 4%
paraformaldehyde (PFA) and routinely processed for
paraffin embedding for conventional histology. Sections
(5 mm thick) were H&E-stained. For each tissue, and for
every animal, a series of nine sections was analyzed by
light microscopy. Sections were chosen as three groupsof three successive sections, distant from one another by
50 mm in the paraffin block. Uninfected BALB/c mice
were used as controls.
Tc52 tissue distribution in heart was determined by
immunohistochemistry using serial sections containing
amastigote nests. Sections were successively incubated
in: (a) anti-Tc52 immune-serum diluted 1:20 (v/v) in
PBS containing 0.5% BSA for 16 h at room temperature;and (b) fluorescein-conjugated rabbit anti-mouse IgG
(Eugene, Oregonc., USA) diluted 1:200 (v/v) in PBS for
30 min at 37 8C. All incubations were performed in a
moist chamber and were followed by three washes in
PBS. Preimmune serum was used for controls. Slides
were counterstained with Evan’s blue diluted 1:10 000
(v/v) in PBS and mounted in a 1:1 (v/v) glycerol/PBS
solution. Sections were examined by epifluorescencemicroscopy.
2.7. Mouse heart muscle cells infection and
immunoelectron microscopy study
Primary cultures of mouse heart muscle cells (HMC)
were purified as previously described [16]. Animal
procedures were carried out in accordance with the
guidelines established by the FIOCRUZ Committee ofEthics for the use of animals, resolution 246/99. BT
forms of T. cruzi Y strain obtained from Swiss Webster
mice at the peak of the parasitemia as described in Ref.
[17] were used to infect HMCs at a multiplicity of
infection of 10 per host cell. After a 24 h-incubation,
HMC cultures were washed in Ringer’s solution and
fresh medium was added to the cultures. HMCs were
collected at various times post-infection, and treated forelectron microscopy (EM). Post-embedding EM meth-
odology procedure was performed as previously de-
scribed [18]. Briefly, normal and T. cruzi -infected cells
were fixed for 1 h at 4 8C in 0.1 M cacodylate buffer at
pH 7.2 containing 4% PFA, 0.01% glutaraldehyde (GA),
3.5 M sucrose, and 0.2% picric acid. After fixation and
successive washes, free aldehyde groups were quenched
by incubating cell cultures (30 min, 4 8C) in PBScontaining 0.5 M NH4Cl. Washed cells were dehydrated
in methanol before infiltration with Lowicryl K4M
(Polysciences. Inc., Warrinfton, PA). Lowicryl embed-
ding was performed in an appropriate UV light con-
tainer kept for 5 days at �/20 8C and 2 days at 22 8Cfor polymerization. Antibody labeling was carried out
using the method previously described [19]. Gridssupporting cell thin sections were successively incubated
in: (a) Tris buffer saline (TBS); (b) anti-Tc52 mouse
immune serum (1 h); and (c) anti-mouse IgG goat
antibodies conjugated to 10 nm gold particles (30 min).
All antibody dilutions were performed in TBS supple-
mented with 2% BSA and 1% Tween. Specimens were
observed under a EM-10 Electron Microscope (Carl
Zeiss).
2.8. Statistical analysis
The comparison between two mean values for two
unpaired series of data was statistically analyzed by a
two-tailed t-test assuming equal variance using the Excel
2000 program. Probability values of B/0.05 were
considered significant.
3. Results
3.1. In vivo infectivity of WT and Tc52�/� parasites
Previous studies showed that several single mutant
parasite clones carrying a targeted deletion of Tc52
protein encoding allele (Tc52�/�) had similar pheno-types when comparing their in vitro and in vivo
virulence [13]. Therefore, one representative Tc52�/�
clone was selected together with the WT control in order
to examine the outcome of T. cruzi infection in BALB/c
mice. The expression of the Tc52 polypeptide controlled
by western-blot quantification using serial dilutions of
WT and Tc52�/� clone, is $/twofold lower in the
Tc52�/� mutant than in WT parasites (data notshown). Thus, mice were inoculated i.p. with 103 blood
stream trypomastigotes of each clone, the resulting
parasitemia being measured over the course of infection.
Results typical from two independent experiments are
shown in Fig. 1A. The patterns of infection with either
the WT or Tc52�/� clone greatly differed. Indeed,
although the prepatency periods (16 days post-infection)
were rather similar for both clones, a major differencewas observed at day 16 in the level of parasitemia, which
was significantly higher in the case of WT-infected mice
(P B/0.05 for both experiments). In both groups, after
30 days of infection the parasitemia levels were micro-
scopically undetectable.
3.2. Spleen cell subsets in T. cruzi -infected mice
The numbers and the characteristics of cell popula-
tions in the spleen of mice infected with the WT or
Tc52�/� parasites were analyzed over the infection.
E. Garzon et al. / Immunology Letters 89 (2003) 67�/8070
Groups of six animals were sacrificed at each time point
(7, 16, 23, 27 and 360 days post-infection) after
parasitemia was ascertained (Fig. 1B). A highly sig-
nificant increase ranging from three- to 12-fold in the
total number of spleen cells was observed from day 16 to27 in mice infected with either WT or Tc52�/�, as
compared to normal mice (data not shown). These large
increases in cell numbers were seen for all three spleen
lymphocyte classes (CD4�, CD8� T-cells, and B-cells)
and macrophages (Figs. 2 and 3). Further examination
of the T-lymphocyte subsets in the spleens of mice
revealed that over the experimental period, the
CD4�:CD8� ratio varies from 1.98 to 4.45 for non-infected controls, whereas ratios for WT and Tc52�/�-
infected mice vary from 0.93 to 3.38 and 1.78 to 4.26,
respectively (Fig. 2). It is worth noting that at 23 and 27
days post-infection, the CD4�:CD8� ratio largely
decreases in the case of WT-infected mice (0.93 and
0.98, respectively) as the result of a sharp increase in the
number of CD8� cells, while the number of CD4� cells
remains constant. Numbers of B-cells from both WTand Tc52�/�-infected mice similarly increased from the
16th day post-infection (P B/0.01; Fig. 3). However,
increases in spleen macrophage counts were observed at
23 and 27 days post-infection with numbers significantly
higher in the case of Tc52�/�-infected mice, as com-
pared to WT-infected mice (P B/0.01). In the chronic
phase, spleen cells from WT as well as Tc52�/�-infected
mice still presented higher numbers of CD4�, CD8� T-cells, and B-cells than spleen cells from normal mice.
3.3. Lack of immunosuppression during the acute phase
of Tc52�/�-infection
Severe immunosuppression is known to develop
during the acute phase of the Chagas’ disease [20]. In
the murine model of T. cruzi infection, these effects have
been attributed to a wide range of mechanisms, among
which, reduced lymphoproliferation in response to
mitogens and decreased IL-2 production [20]. Therefore,
we investigated the ability of spleen T-cells from WTand Tc52�/�-infected mice to respond to anti-CD3
stimulation. As shown in Fig. 4, when total spleen cells
were stimulated with anti-CD3 mAb, no T-cell prolif-
eration occurred for WT-infected mice, whereas the level
of proliferation in the case of Tc52�/�-infected mice
was of the same order of magnitude than that observed
for normal spleen cells. These observations indicated
that the mutant Tc52�/� failed to induce the down-regulation of the T-cell proliferation characteristic of the
murine acute phase of T. cruzi infection.
3.4. Lack of suppression of IL-2 production in Tc52�/�
mutant
The restoration of T-cell responsiveness observed in
the acute phase of the Tc52�/�-infection, as compared
to the WT-infection, brought us to determine the ex-
vivo cytokine production by spleen cells upon stimula-
tion with anti-CD3 mAb. Spleen cells from non-
infected, WT and Tc52�/�-infected mice (collected 7and 16 days post-infection), were cultured in the
presence of anti-CD3 mAb for 48 h. The concentrations
of IFN-g, IL-2, IL-10 and IL-4 were measured in culture
supernatants by comparison to a recombinant cytokine
standard curve using ELISA. As shown in Table 1, a
striking difference between Tc52�/� and WT-infected
mice was observed for IL-2 production. Indeed, spleen
cells from animals infected with WT parasites showed amajor decrease (starting at day-7) in their capacity to
secrete IL-2 upon stimulation with anti-CD3 mAb, as
compared to normal spleen cells (P B/0.01), but retained
Fig. 1. In vivo infectivity of WT and Tc 52�/� parasites. (A) Mice (6�/8 animals per group) were challenged with 103 WT or Tc52�/� blood stream
trypomastigotes (BT) per animal. BT counts were determined in triplicates at 7, 16, 23, and 27 days post-infection by microscopic inspection of
individual blood samples. One representative experiment from two is depicted. (B), groups of six animals were sacrificed for immunological and
histogical studies at each time-point after parasitemia determination. BT counts for WT and Tc52�/� mutant clones are represented by black
triangles and open squares, respectively. Results are expressed as the arithmetic mean (9/SD).
E. Garzon et al. / Immunology Letters 89 (2003) 67�/80 71
their ability to produce IFN-g. In contrast, the amounts
of IL-2 secreted by spleen cells from Tc52�/�-infected
mice were similar to those observed in the case of
normal spleen cells (P �/0.05). Furthermore, a highly
significant increase of IL-10 release was observed 7 dayspost-infection in mice infected with WT parasites, as
compared to normal control mice (P B/0.001). Finally, a
significant decrease in IL-4 secretion was observed in
WT and Tc52�/�-infected mice spleen cells when
compared to control spleen cells from normal mice
(P B/0.05).
3.5. Comparable concentrations of serum
immunoglobulins in mice infected with T. cruzi WT or
Tc52�/� parasites
Sera collected from mice at various times after
infection were examined by ELISA to determine im-
munoglobulin concentrations. Except for the IgG2b
isotype, all classes of Ig presented no significant
difference in serum concentrations, for WT and
Tc52�/�-infected animals (Fig. 5). Therefore, it wasof interest to evaluate the parasite specific immunity,
particularly whether the lower parasite burden could
contribute to an enhanced host immune specific re-
sponse. The results obtained showed that the levels of
specific antibodies to T. cruzi antigens were very low in
both WT and Tc52�/�-infected mice (data not shown),
in agreement with previous reports demonstrating that
during T. cruzi infection a minority of antibodies were
parasite-specific [21].
3.6. Numbers of spleen cells undergoing apoptosis during
T. cruzi infections in mice
Several studies have suggested that apoptosis may be
related to the alterations observed in the immune system
during experimental T. cruzi infections [22]. In an
attempt to determine the extent of apoptosis during T.
cruzi infection, a kinetic study of Annexin V binding
was conducted using spleen cells from both WT and
Tc52�/�-infected mice collected 7, 16, 23, 27 and 360
days post-infection. Increased numbers of apoptotic
cells were observed after 16 days of infection in the
spleen cell populations from both WT and Tc52�/�-
infected mice, as compared to the baseline level of
control mice. Nonetheless, levels of apoptosis were
significantly lower in the case of Tc52�/�-infected
mice at 16 and 23 days post-infection (Fig. 6).
Fig. 2. Analysis of spleen CD4� and CD8� lymphocyte populations during experimental infection. Data are arithmetic means (9/SD) of six mice
per group analysed individually and are representative of two independent experiments. Spleen cells from normal mice (NSC); Tc 52�/�-infected
mice (Tc52�/� ISC); and WT-infected mice (WT ISC). Asterisks indicate time points at which differences are statistically significant between each
group of infected mice and control mice (*P B/0.05; ** P B/0.01; *** P B/0.001).
E. Garzon et al. / Immunology Letters 89 (2003) 67�/8072
3.7. Analysis of inflammatory lesions in heart tissues
Heart and skeletal muscle histology was carried out to
examine whether the deletion of one Tc52 gene allele
could affect the extent of the parasite load in tissues and
their associated damage. Tissue sections obtained from
groups of three WT or Tc52�/�-infected mice, sacri-
ficed during the acute (7, 16, 23, 27 days) and chronic
phases (360 days) of T. cruzi infection, were examined
for the detection of parasite nests, and the presence of
inflammatory cells.
A small number of amastigote nests (pseudocytes),
were found in the heart tissue of both groups of mice.
Indeed, a total of 3 nests versus 2, for a total number of
15 mice per group (135 sections), were observed during
the experimental period for the WT and Tc52�/�
clones, respectively. Furthermore, despite numerous
examinations of the skeletal muscle tissue (135 sections
per group), parasite nests could not be detected regard-
less of the parasite clone considered.
Histopathological study of WT and Tc52�/�-in-
fected mice was carried out on H&E-stained sections
from heart and skeletal muscle (Fig. 7). Despite low
numbers of amastigote nests in the heart tissue, numer-
ous foci of inflammatory reactions were constantly
observed in WT-infected mice (Fig. 7B). Only a diffused
Fig. 3. Analysis of spleen B lymphocyte and macrophage populations during experimental infection. Data are arithmetic means (9/SD) of six mice
per group analysed individually and are representative of two independent experiments. Spleen cells from normal mice (NSC); Tc 52�/�-infected
mice (Tc52�/� ISC); and WT-infected mice (WT ISC). Asterisks indicate time points at which differences are statistically significant between each
group of infected mice and control mice (*P B/0.05; ** P B/0.01; *** P B/0.001).
Fig. 4. Ex-vivo spleen cell proliferation as measured by [3H]-thymidine
incorporation (cpm). Spleen cells from normal mice (NSC) and both
WT (WT ISC) and Tc 52�/� (Tc 52�/�ISC)-infected mice at 23 days
post-infection were stimulated ex-vivo with anti-CD3 mAb. Data are
arithmetic means (9/SD) of triplicate cultures of six mice per group
analysed individually. Differences between WT ISC and NSC or
Tc52�/� ISC, respectively, are statistically significant (P B/0.01). No
statistically significant difference was observed between NSC and
Tc52�/� ISC (P �/0.1). The results are from a representative
experiment of two carried out independently.
E. Garzon et al. / Immunology Letters 89 (2003) 67�/80 73
inflammation process was observed in all the heart
sections of Tc52�/�-infected mice (Fig. 7C), which was
absent in non-infected controls (Fig. 7A). In contrast,
although parasites could not be detected in the skeletal
muscle, comparable inflammation was seen in both WT
(Fig. 7E) and Tc52�/�-infected mice (Fig. 7F) as
indicated by increasing numbers of infiltrating cells, as
compared to controls (Fig. 7D). Sections containing WT(Fig. 8C and D) or Tc52�/� (Fig. 8E and F) amastigote
nests, were used to localize reactive Tc52-epitopes in
heart sections using the indirect immunofluorescence
technique and the same dilution of anti-Tc52 immune
serum. Positive immunostaining was detected within
infected cells, at the surface and in the cytoplasm of
amastigotes (Fig. 8D and F). In addition, some granular
staining, which does not colocalize with an intracellularparasite (as indicated by Evans blue counterstaining,
Fig. 8C and E), was observed in the cytoplasm of
infected cells (Fig. 8D and F). Interestingly, a significant
decrease in the level of immunologically detectable Tc52
molecule was observed in the case of Tc52�/�-infected
mice (Fig. 8F) when compared to WT-infected ones
(Fig. 8D). Thus, these observations are in agreement
with the results of Western blot measurements [13] andsupport the notion that Tc52�/� mutants are produ-
cing, in vivo, Tc52 molecule at levels lower than those of
WT parasites.
3.8. Immunoelectron microscopy study of Tc52
localization on sections of T. cruzi -infected mouse heart
muscle cells
To assess the exact location of Tc52 peptide epitopes,
we conducted immunoelectron microscopy studies onsections of mouse heart muscle cells infected with T.
cruzi trypomastigotes and treated with anti-Tc52 mouse
immune serum. As shown in Fig. 9A, the gold particles
are mainly associated with the amastigote membrane
and cytoplasm. The gold-labelled positive sites were also
abundant on lumenal tightly apposed face of host cell
cytoplasm. Interestingly, the Tc52 reactive epitopes
could also be seen on the membrane of muscle cells
and form aggregates accumulating inside cytoplasmic
vesicles (Fig. 9B and insert). Non-specific binding was
very low as determined by incubation with pre-immuneserum followed by gold-conjugated goat antibodies to
mouse Ig (data not shown).
4. Discussion
In previous works, we have characterized a T. cruzi
protein named Tc52 that exerts a regulatory effect on
the host inflammatory and immune responses [24�/26].
We have also shown that monoallelic disruption of theTc52 gene resulted in less virulent T. cruzi clones [13].
Although initial molecular and immunological ap-
proaches suggested that Tc52, among other factors,
could participate in the phenotypic expression of T.
cruzi virulence, the question remained open on how the
lower virulence of T. cruzi mutant could influence the
nature of the immune response during an infection. To
address this question, we have selected a T. cruzi WTclone together with a representative Tc52�/� single
mutant parasite which produced $/twofold less Tc52
protein than the WT parasite parental clone, and
examined in vivo the host immune responses and
histopathological symptoms during the course of T.
cruzi infection in BALB/c mice.
Distinct patterns of infection were observed following
the inoculation of 103 parasites of either WT orTc52�/� clones. Parasitemia were always higher in
the case of mice infected with WT parasites. Interest-
ingly, this criteria was stable over time, in agreement
Table 1
Cytokine measurements in supernatants cultures of anti-CD3 stimulated spleen cells from normal, WT and Tc52�/� infected mice
IFN-g (pg/ml) IL-2 (pg/ml) IL-10 (pg/ml) IL-4 (pg/ml)
Non-infected (n�/6) 6334.479/1998.84 441.769/59.37a 650.449/150.89b 1578.439/262.19
Infected days post-infection
7th Day
WT (n�/6) 3239.799/1074.96 273.049/56.52a 3302.079/221.48b 974.649/59.34
Tc52�/� (n�/6) 2082.029/312.25 428.679/49.66 248.209/182.27 626.399/139.71
16th day
WT (n�/6) 6761.959/2770.59 51.799/21.06a 1099.929/276.16 484.169/55.80
Tc52�/� (n�/6) 6587.599/1900.39 347.799/142.19 1116.299/283.36 298.549/7.07
The levels of IFN-g, IL-2, IL-10, IL-4 were determined in the supernatants of spleen cells (2�/105 cells/well) from normal and infected BALB/c
mice cultured during 48 h. We have determined by ELISA the levels of these interleukins in comparison with a standard curve using the recombinant
ILs. Values are means9/standard deviations for triplicate cultures of spleen cells from mice analyzed individually.a A difference in IL-2 levels (P B/0.01) was observed between cultures from normal and WT infected mice after anti-CD3 stimulation, 7 and 16
days post-infection respectively.b A significant difference in IL-10 levels (P B/0.001) was observed between cultures from normal and WT infected mice after anti-CD3
stimulation.
E. Garzon et al. / Immunology Letters 89 (2003) 67�/8074
with other reports showing long term maintenance of
pathogenicity of individual T. cruzi clones [27]. Despite
highly different parasitemia levels, the WT and
Tc52�/� clones induced similar increase in lymphocyte
subpopulations in the spleen, as known to occur in T.
cruzi mice infections [21]. In relation to the humoral
response elicited during murine T. cruzi infection, IgG2
and IgG1 isotypes are considered to be important
factors in resistance [21]. Our results agree with those
observations since chronically infected mice which have
Fig. 5. Kinetic study of isotype profiles of Abs from normal mice (NSC), WT (WT ISC) and Tc 52�/� (Tc52�/� ISC) infected mice. The levels of
total antibodies in sera from Tc 52�/�-infected mice were determined by ELISA and compared with those observed in the case of normal and WT-
infected mice. At each time point, six animals per group were sacrificed and their sera analysed individually. Ig concentrations (mg/ml) for WT and
Tc52�/�-infected animals are represented by black and open squares, respectively, whereas black triangles represent normal mice. Results are
expressed as arithmetic mean of the Ig concentrations (9/SD).
E. Garzon et al. / Immunology Letters 89 (2003) 67�/80 75
cleared their parasitemia showed an isotypic profile of
IgG1�/IgG2a�/IgG2b. A striking finding was the low
levels of IgG2b observed in the case of Tc52�/�-
infected mice when compared to WT-infected ones.
However, recent investigations in our laboratory using
two naturally occurring, genetically distant T. cruzi
clones exhibiting different virulence in vivo, showed low
levels of IgG2b in the case of the low virulent clone
(Garzon et al. unpublished observations). Whether this
is a general characteristic of clones with low virulence
await further investigations.
It has been known for many years that spleen cells
from acutely infected mice are unable to produce IL-2 in
vitro in response to mitogens [28,29]; and this could
account for the transient state of immunosuppression
that occurs during T. cruzi acute infection. Moreover,
several investigators have reported the involvement of T.
cruzi secreted molecules in the development of immu-
nosuppression phenomenon [30]. In the present study,
an interesting and unexpected finding was the associa-
tion between the down-regulation of Tc52 production
by the parasite and the lack of suppression of IL-2
secretion and anti-CD3-induced T-cell proliferation.
However, others have suggested that the state of
immunosuppression could be a consequence of the
intense B- and T-cell polyclonal activations observed
during the early stages of infection [21]. Although this
latter mechanism could participate in the immunosup-
pression observed during the acute phase of Chagas’
disease, it is unlikely that this could apply to the
observations reported in this study. Indeed, the expan-
sion of all classes of lymphocytes as well as macrophages
was observed in both WT and Tc52�/�-infected mice,
and all the indications we have collected so far appear to
point that the lack of the transient immunosuppression
state occurred only in the case of Tc52�/�-infected
mice.
In previous studies, we have demonstrated that Tc52
when delivered extracellularly or intracellularly to the
macrophages, induced increased IL-10 gene expression
[25,26]. In the present report, significant increased
production of IL-10 was observed in the case of spleen
cells from WT-infected mice, whereas the levels observed
in the case of Tc52�/�-infected mice were comparable
to those of normal mice spleen cells. Therefore, these
results demonstrate, for the first time, that Tc52 play a
role in IL-10 cytokine regulation during in vivo T. cruzi
infection. It is noteworthy that murine IL-10 can
Fig. 6. Spleen cell apoptosis in WT or Tc 52�/�-infected mice. Extensive apoptosis is observed in spleen cells from both WT (WT ISC) and
Tc52�/�-infected mice (Tc52�/� ISC) when compared to spleen cells from normal mice (NSC). Asterisks indicate time points at which differences
are statistically significant between each group of infected mice and control mice (**P B/0.01).
E. Garzon et al. / Immunology Letters 89 (2003) 67�/8076
downregulate the host immune response by decreasing
the production of IL-2 [31] and inhibiting mitogen
driven T-cell proliferation [32]. Therefore, it is reason-
able to suggest that the reduction of Tc52 production by
gene targeting which in turn downregulate the IL-10
synthesis could be among the mechanisms participating
in the immunoregulatory mechanisms leading to the
control of IL-2 production.
Cell death by apoptosis has been reported to be a
prominent feature during experimental infection with T.
cruzi , a phenomenon that might also play a role in
immunosuppression and parasite persistence in infected
hosts [33]. In our study, we found increased numbers of
spleen cells undergoing apoptosis in both WT and
Tc52�/�-infected mice after 16 post-infection, although
significantly higher in the case of WT-infected mice at 16
and 23 days post-infection (P B/0.05) as well as during
the chronic phase (360 days post-infection, PB/ 0.05).
Taking into account the recent data showing that
interaction of apoptotic cells with macrophages infected
Fig. 7. Histopathological analysis of heart (A, B and C) and skeletal muscle (D, E and F) of normal (A, D), WT (B, E) and Tc 52�/�-infected mice
(C, F). Heart and skeletal muscle collected 16 days after infection were processed for light microscopy. H&E-stained sections were examined with
20�/ objective. Clusters of inflammatory cells were evidenced in the heart of WT-infected mice.
E. Garzon et al. / Immunology Letters 89 (2003) 67�/80 77
with T. cruzi fuels parasite growth in a manner
dependent of prostaglandins, transforming growth fac-
tor (TGF-b) and polyamine biosynthesis [34], it is
tempting to speculate that the reduced cell death by
apoptosis in the case of Tc52�/�-infected mice, could in
someway play a role in the reduction of parasite load in
vivo.
Therefore, it is unlikely that this process could be
relevant to the lack of immunosuppression in Tc52�/�-
infected mice. Furthermore, a dramatic decline in the
spleen CD4�:CD8� ratio in WT-infected mice at 23
and 27 days post-infection resulted from of a sharp
increase in the number of CD8� cells. This was not seen
at any time point in Tc52�/�-infected mice. Although,
investigators have shown that the immunosuppressed
state of the animals was unaffected by CD8� cell
depletion [35], our data suggest indirectly that a
subpopulation of CD8�/ cells could exert detrimental
immunosuppressive effects. Indeed, it is interesting to
remind that infection with T. cruzi elicits over time the
generation of T cells suppressive to T and B cell
mitogenic responses as shown in a previous report
[36]. Therefore, it is likely that normal Tc52 production
in vivo (WT parasites) would favor the expansion of
CD8�/ and CD4�/ cells at similar level whereas reduc-
tion of Tc52 synthesis (Tc52�/�) resulted in a decrease
of CD8�/ expansion but did not affect the increased
cellularity of CD4�/ cells. Further studies on the
functionality of purified T cell subpopulations from
WT and Tc52�/�-infected mice will be needed to clarify
this point.
Despite a limited number of amastigote nests in
tissues of both WT and Tc52�/�-infected mice, histo-
pathological examination of tissues sections revealed
two patterns of inflammation characterized as diffused
in the case of Tc52�/�-infected mice or presenting
numerous foci in the case of WT-infected mice. Loca-
lization of the Tc52 protein in tissue sections and
Fig. 8. Expression of Tc52 epitopes in the hearts of T. cruzi WT parasite-infected mice. Tissue sections from PFA-fixed, paraffin-embedded hearts
from uninfected (A, B), WT-infected (C, D), or Tc 52�/�-infected (E, F) mice showing amastigote nests (arrowheads C, E) were analyzed. Sections
were treated with a mouse immune serum to Tc52 native protein diluted 1:20 [7], followed by a fluorescein-labeled rabbit anti-mouse IgG (B, D, F),
and counterstained with Evan’s blue (A, C, E). A strong positive immunoreactivity and a granular antigen staining could be seen around amastigotes
(arrowheads D, F) and in the periphery of infected cells (asterisks D, F). Sections were photographed by light and epifluorescence microscopy with a
40�/ objective.
E. Garzon et al. / Immunology Letters 89 (2003) 67�/8078
infected heart cell primary cultures by immunofluores-
cence and immunogold labeling, respectively, showed
for the first time, that Tc52 protein is expressed by
amastigotes present inside the cardiac myocytes and
forms aggregates in the cytoplasm of infected cells. The
Tc52 expression level is much higher in WT than in
Tc52�/�-infected mice. Therefore, altogether our re-
sults suggest that the Tc52 protein could be involved in
the heart inflammatory processes characteristic of the
Chagas’ disease. Taking into account our previous data
showing that Tc52 induces an increased macrophage
iNOS gene expression and NO production, one could
hypothesize that during the chronic phase, in situ
production of low amounts of Tc52 (not neutralized
by antibodies) by amastigotes inside T. cruzi -infected
cardiac myocytes, which have a iNOS , induces local
production of NO and nitrotyrosine formation which
might participate in myocardial dysfunction and heart
failure [25]. In fact, 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 [12], suggesting that Tc52 could beamong candidate molecules for the development of
vaccinal strategies against Chagas’ disease [23].
In summary, this study reinforces the notion that
biological characteristics of individual parasite clones
may significantly influence the nature of the immune
response associated to the infection. Furthermore, the
data may suggest that the chronic pathology may not be
due to the polyclonal lymphocyte activation character-istic of acute infection but likely to parasitism and
disruption of immunoregulatory mechanisms of the
host. The lack of suppression of IL-2 secretion and
anti-CD3-induced T-cell proliferation and the lack of
increased IL-10 secretion associated with reduced in-
flammation during the acute as well as the chronic phase
sustain the difference in the outcome of infection.
Therefore, it seems reasonable to assume that attenuat-ing the immunosuppression process would, in some way,
improve the outcome of chronic Chagas’ disease.
Acknowledgements
These investigations received financial support from
Institut de Recherche pour le Developpement (IRD),
Institut National de la Sante et de la Recherche
Mediacle (INSERM) and ‘Cooperation Scientifique et
Technologique Luso-Francaise: Project No. 706 C3’. E.
Garzon is a recipient of a fellowship from Departement
Soutien Formation pour les Communautes du Sud
(IRD). M. Borges is funded by ‘Fundacao para aCiencia e Tecnologia’, Portugal Grant PRAXIS XXI/
BD/20093/99. Both E. Garzon and M. Borges have
contributed equally to this study. We acknowledge Dr
Joelle Simony-La Fontaine, Michele Radal and Sylvie
Roques from the Pathology department of the Val
d’Aurelle Hospital in Montpellier for their precious
help in histology studies, as well as Dr Evelyne Bachere
from the laboratory of ‘Defense et Resistance chez lesInvertebres Marins’ (UMR5098 CNRS-IFREMER-
University of Montpellier II) for access to epifluores-
cence microscopy. We are also grateful to Daniel
Moukouanga and Nathalie Barougier, the IRD ani-
mal-care personnel.
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