Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele elicits attenuated...

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.

�/�
Keywords: Trypanosoma cruzi ; Tc52 mutant; IFN-g; IL-2; Pathology
1. 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: [email protected] (A. Ouaissi).

0165-2478/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0165-2478(03)00112-3

mailto:[email protected]

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.


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).


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).


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.


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.


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).


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).


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.


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.


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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