Acta Parasitologica

https://doi.org/10.2478/s11686-020-00188-0

ORIGINAL PAPER

Zinc Supplementation: Immune Balance of Pregnancy During

the Chronic Phase of the Chagas Disease
Cássia Mariana Bronzon da Costa1 · Marina del Vecchio Filipin1 · Fabrícia Helena Santello1 ·
Inara Fernanda Lage Gallo1 · Luiz Miguel Pereira1 · Fernando Barbosa Jr1 · José Clóvis do Prado Júnior1 ·
Ana Amélia Carraro Abrahão1

Received: 10 June 2019 / Accepted: 18 February 2020

© Witold Stefański Institute of Parasitology, Polish Academy of Sciences 2020

Abstract
Background  Chagas disease or American trypanosomiasis is caused by the protozoan Trypanosoma cruzi and is endemic of
the Americas. The control of the disease is restricted to toxic and potentially teratogenic drugs, which limit the use during
pregnancy. The use of food supplementation offers a safe and low-cost form to alleviate Chagas disease symptoms, mostly
in areas with alimentary risk. For example, zinc demonstrates positive effects in immune response, including in Chagas
disease during pregnancy.
Purpose  This study describes the innate response in pregnant rats chronically infected with T. cruzi and supplemented with
zinc.
Methods  Pregnant female Wistar rats, infected with T. cruzi, were treated with 20 mg/kg/day zinc sulfate and euthanized on
the 18th day. Samples (plasma, splenocytes, and peritoneal exudate) were collected and several immune parameters (nitric
oxide, RT1B, CD80/CD86, MCP-1, CD11b/c, NK/NKT, IL-2, IL-10, INF-cc, and apoptosis) evaluated.
Results  Under Zinc supplementation and/or T. cruzi infection, the gestation developed normally. Several innate immune
parameters such as RT1B, CD80/CD86, MCP-1 expressing lymphocytes, IL-2, and IL-17 were positively altered, whereas
nitric oxide, CD11b/c, NK/NKT, apoptosis, INF-γ, and corticosterone demonstrated a pro-pregnancy pattern.
Conclusion  Our results indicated that zinc has diverse effects on immune response during pregnancy. An anti-T. cruzi
immunity, as well as a pro-gestation response, were observed after zinc supplementation. The complete comprehension of
zinc supplementation in pregnancy will base an adequate strategy to alleviate Chagas disease symptoms and propagation,
especially for populations from endemic areas.

Keywords  Chagas disease · Pregnancy · Zinc · Chronic phase

Introduction

Trypanosoma cruzi is the causative agent of Chagas disease,

Electronic supplementary material  The online version of this
article (https​://doi.org/10.2478/s1168​6-020-00188​-0) contains
supplementary material, which is available to authorized users.
* Cássia Mariana Bronzon da Costa
marianabronzon@gmail.com
* Ana Amélia Carraro Abrahão
acarraro@fcfrp.usp.br
1
Department of Clinical Analysis, Toxicology and Food
Science, School of Pharmaceutical Sciences of Ribeirão
Preto, University of São Paulo, Ribeirão Preto 14040‑903,
Brazil
which afflicts millions of people of America. The disease is
transmitted after a bite and contact with contaminated feces
of Triatomine insects. Contaminated insect feces also infect
food and lead to oral transmission. Blood transfusion, organ
transplantation, and congenital transmission are related to
the expansion of the Chagas disease to non-endemic regions,
such as North America, Europe, Japan, and Australia [53].
Due to the importance of congenital transmission, the
dynamic of Chagas disease during pregnancy is a key point
to describe and control the parasite propagation. Moreover,
the classic anti-T. cruzi drugs (benznidazole and nifurtimox)
have been related to toxicity and/or teratogenicity [11, 39].

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Thus, novel strategies are necessary to alleviate the effects
of the disease in pregnancy.
The gestation is characterized by genetic and hormonal
alterations, to continually provide nutrients to the embry-
onic development. During the gestational period, a maternal
immune tolerance is developed to accept the fetus [43]. The
metabolism is also altered during pregnancy, and an ade-
quate caption of nutrients is necessary. Imbalances in nutri-
tion during pregnancy represent a constant risk for mother
and fetus. According to the World Health Organization [53],
the adequate intake of nutrients is one of the determinants
of fetal growth, with repercussions on the weight and gesta-
tional age at birth. Therefore, among the diverse nutrients,
zinc has an important role in the immune response and has
been related to efficient control of viral, bacterial, and pro-
tozoal infections [9]. Our group has studied the effects of
zinc supplementation during the pregnancy in rats infected
with T. cruzi [12–14]. Zinc has a fundamental role in growth,
development and differentiation of tissues, and embryogen-
esis [22, 36, 49]. Moreover, clinical trials have demonstrated
that maternal zinc deprivation increases the risk of mortality,
growth retardation, and fetal malformations, including neu-
ral tube defects [25]. Zinc stimulates the immune response in
the acute phase of the Chagas disease, and several adaptive
parameters were improved in the chronic phase [12, 14]. In
this study, we described the innate immune response during
the chronic phase of Chagas disease and the role of the zinc
supplementation. Zinc improved several innate immune-
response parameters (RT1B, CD80/CD86, MCP-1, IL-2, and
IL-17). In parallel, other points indicated a pro-gestation pat-
tern (nitric oxide, CD11b/c, NK/NKT, apoptosis, INF-γ, and
corticosterone), balancing the pro-inflammatory response
induced against the infection. Therefore, zinc promotes the
immune response without compromising fetal development.
Although the results are promising, future assays will be
necessary to elucidate the mechanisms related to the zinc
supplementation during pregnancy associated with the Cha-
gas disease. Adequate nutrient intake is fundamental for the
development and health of the mother and fetus. Special
attention may be directed to Chagas disease-endemic areas,
where a food risk is also considered.
Material and Methods
Animals
Female Wistar rats (five animals/group) were used and
housed in the Facility House of the São Paulo University
Campus of Ribeirão Preto, with water and food at libitum.
The animal manipulation protocol was approved by the
local Ethics Committee (Protocol number 11.1.1210.53.2).
Four experimental groups were designed: non-infected/
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non-treated pregnant animals (PC), non-infected/zinc-treated
pregnant animals (PCZ), T. cruzi-infected/non-treated preg-
nant animals (PI), T. cruzi-infected/zinc-treated pregnant
animals (PIZ).
Pregnancy and T. cruzi Infection
The animals were infected intraperitoneally with 1 × 105 try-
pomastigotes forms of T. cruzi Y strain [42] and housed for
30 days. This period of infection (transition of the acute to
the chronic phase of Chagas disease) is characterized by the
decrease of trypomastigotes counting in peripheral blood
[28, 32]. On the 30th day after infection (when no trypomas-
tigotes were observed in peripheral blood), the female rats
were maintained with male counterparts, in a proportion of
2:1 per cage and the pregnancy was confirmed by the pres-
ence of the vaginal plug [12].
Zinc Supplementation and Euthanasia
On the first day of pregnancy, animals were treated with
20 mg/kg/day zinc sulfate (Sigma-Aldrich, Catalog number:
1724769) by oral gavage for 18 days [12]. At the final stage
of the pregnancy (18th day), the animals were anesthetized
with 2.5% tribromoethanol and euthanized by decapitation.
Fetuses and Placentas Analysis
After euthanasia, the fetuses and placentas were collected
and the weight and length/diameter measured using an ana-
lytic balance (Sartorius, Goettingen, Germany) and a digital
caliper (Mitutoyo, Kawasaki, Japan), respectively.
Quantification of Serum Zinc
Zinc quantification was adapted from a method previously
described [3] with little modification for rat serum sam-
ples analysis [13]. Briefly, serum was diluted (1:20) with a
solution containing 0.01% (v/v) T ­ riton® X-100, 0.5% (v/v)
−1
nitric acid, and 10 µg ­L of each one of Rh (as internal
standard). Analyses were carried out with an inductively
coupled plasma mass spectrometer equipped with a reaction
cell (ICP-MS ELAN DRCII, PerkinElmer, SCIEX, Norwalk,
CT) operating with high-purity argon (99.999; Praxaair, Bra-
zil). The sample introduction system was composed of a
quartz cyclonic spray chamber and a M ­ einhard® nebulizer
®
connected by ­Tygon tubes to the ICP-MS’s peristaltic pump
(set at 20 rpm). The ICP-MS was operated with Pt sampler
and skimmer cones purchased from Perkin Elmer.
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Peritoneal and Splenic Cells
The peritoneal cells were collected by injection of cold
RPMI 1640 medium (10 mL, Sigma-Aldrich, USA) in the
peritoneal cavity of animals. The suspension was centrifuged
at 410g for 15 min and the pellet suspended in 2 mL of
RPMI 1640 medium. The splenic cells were harvested by the
mechanical disaggregation of spleen followed by a passage
in a 70-μm nylon cell strainer (Falcon, USA). The disaggre-
gated cells were washed with a hypotonic buffer (160 mM
NH4Cl, 10 mM Tris–HCl, pH 7.4) and suspended in RPMI
1640. The peritoneal and splenic cells were counted in a
hemocytometer, and the cellular viability verified after dilu-
tion (1:2) in a 0.4% trypan blue solution (Sigma-Aldrich
catalog number: T8154). For general use, the cells were
diluted to 2 × 106 cells/mL.
Nitric Oxide Quantification
Peritoneal cells were distributed (2 × 105/well) in 96-well
plates (TPP, Switzerland) and incubated with 1  μg/mL
lipopolysaccharide (LPS, Sigma-Aldrich, catalog num-
ber: L2654) for 48 h, 37 °C, 5% ­CO2. After incubation, the
plates were centrifuged at 1500g, for 4 min, 20 °C and the
supernatant (50 μL/well) transferred to a new 96-well plates.
The nitrite measurement was performed according to the
Griess reaction protocol [5]. The samples were incubated
with 100 μL of Griess solution (1% sulfanilamide, 0.1%
N-(1-Naphthyl) ethylenediamine in 5% phosphoric acid) for
15 min at room temperature and the plates read in an ELISA
reader at 540 nm (Synergy H1, Biotek, USA). The nitrite
concentrations were calculated by linear regression using a
sodium nitrite curve (500–7.8 μM, serially diluted).
Phenotypic Analysis of Peritoneal and Splenic Cells
A similar protocol was adopted for phenotypic analysis of
peritoneal and splenic cells, according to [12, 14]. Briefly,
2 × 106 cells in 12 × 75 mm round-bottomed polystyrene
tubes (Falcon, USA) were fixed with 3.5% paraformalde-
hyde for 15 min and washed with phosphate-buffered saline
(PBS). The cells were suspended in Fc receptor block-
ing (anti-CD32, BD Pharmingen) for 20 min, at 4 °C and
washed with PBS. The samples were suspended in Cell
Staining Buffer (BD Pharmingen) and labeled with anti-rat
RT1B/PercP, anti-rat CD11bc/PECy7, anti-rat CD86/FITC,
anti-rat CD80/PE (peritoneal cells) or anti-rat CD3/APC,
anti-rat CD161-/FITC (splenic cells) for 30 min, at 4 °C. The
cells were washed with PBS and suspended in Cell Stain-
ing Buffer for flow cytometry analysis. All antibodies were
purchased from BD Biosciences Pharmingen, CA, USA.
The data acquisition was performed in a FACS Canto flow
cytometer (BD Biosciences, California, USA) equipped with
the FACSDiva software (BD Biosciences, California, USA).
T. cruzi Protein Extract
For evaluation of the apoptosis under specific stimulation,
T. cruzi protein extracts were used. Trypomastigote forms
(Y strain) were cultivated in cell cultures (LLCMK2 line-
age). The culture supernatants were collected and filtered in
a 5 μm filter syringe to separate cells and trypomastigotes.
The parasites (1 × 108/mL) were washed with PBS and sus-
pended in RPMI. The suspension was sonicated (two soni-
cation cycles at 20 kHz, 30 W in a QSonic sonicator, Cole
Parmer, USA), centrifuged for 10 min, at 10,000g, 4 °C and
the supernatant filtered in 0.22 μm filter syringe. The T. cruzi
protein extract was quantified by the Bradford protein assay
and diluted to 3 mg/mL.
Intracellular MCP‑1 Detection
Splenocytes were distributed in 96-well plates (Corning Inc.,
USA) and incubated with 500 ng/mL ionomycin (Sigma-
Aldrich, USA) and 50 ng/mL phorbol 12-myristate 13-ace-
tate (PMA, Sigma-Aldrich, USA) for 4 h at 37 °C in 5%
­CO2. After incubation, cells were cultured with brefeldin
A (10 μg/mL, BD Pharmingen) for 2 h, 37 °C in 5% C ­ O 2,
washed in Stain Buffer BSA (BD Pharmingen) and blocked
with Fc block (anti-rat CD32, BD Pharmingen). Surface
staining was performed with anti-rat CD3/FITC, anti-rat
CD4/PECy7, and anti-rat CD8/PerCP (BD Pharmingen).
After surface staining, cells were fixed and permeabilized
with Cytofix/Cytoperm solution (BD Pharmingen), washed
with Perm/Wash solution (BD Pharmingen) and intracel-
lularly labeled with anti-rat MCP-1/PE. The labeled cells
were washed in Perm/Wash solution, fixed in 1% paraform-
aldehyde in PBS and analyzed by flow cytometry in a FACS
Canto flow cytometer (Becton Dickinson Immunocytometry
Systems), using FACSDiva software.
Annexin V and Propidium Iodide
For early and late apoptosis, splenic cells (2 × 105/well)
were distributed in 96-well plates in RPMI 1640. The
cells were incubated for 24  h, at 37  °C, 5% ­C O 2 with
0.3 mg/mL T. cruzi protein extract or RPMI 1640 (con-
trol). In parallel, cells were incubated under the same
conditions for 15 min, however, without T. cruzi protein
extract. The cells were transferred to 12 × 75 mm round-
bottomed polystyrene tubes, centrifuged for 5 min, 2500g,
4 °C and washed with PBS. The samples were suspended
in 1:100 Annexin V-FITC diluted in binding buffer (BD
Biosciences Pharmingen) and incubated for 15  min,
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at room temperature in the dark. Before the cytometry Results

analysis, propidium iodide (PI, BD Biosciences Pharmin-
gen) was added to the suspensions. The cells under early Zinc Serum Levels and Effects on Fetuses
(Annexin ­V + ­P I −) and late apoptosis (Annexin V
­ + ­P I +) and Placentas
were detected after analysis in a FACS Canto flow cytom-
eter (BD Biosciences, California, USA) equipped with the The T. cruzi infection and/or the zinc supplementation did
FACSDiva software. not alter the weight and length/diameter of fetuses and pla-
centas, compared to the non-infected and non-treated control
(Suppl. Fig. 1). The zinc serum levels were constant among
Cytokine Quantification groups, and no statistical difference was observed. The zinc
concentrations in sera were 2.8 ± 0.5 μg/mL, 3.5 ± 0.7 μg/
Concentrations of IFN-γ, IL-2, and IL-17 were measured mL, 6.6 ± 3.7 μg/mL, and 5.4 ± 0.9 μg/mL for PC, PCZ, PI,
by specific two-site enzyme-linked immunosorbent assay and PIZ, respectively. (Suppl. Fig. 2).
(ELISA) according to the manufacturer’s specifications.
For quantification, reference standard curves were uti-
Nitric Oxide Production and Antigen Presentation
lized. All kits were purchased from R&D Systems (Min-
neapolis, MN, USA). The samples were processed indi-
During the analyzed period of T. cruzi infection (30th–48th
vidually (five animals/group) and plates read at 450 nm
days post-infection), the nitric oxide production was unal-
in an ELISA reader (Synergy H1, Biotek).
tered among the groups (Suppl. Fig. 3). However, the antigen
presentation was responsive to infection and/or zinc supple-
mentation (Fig. 1). The percentage of CD11b/c+ cells from
Corticosterone Detection
peritoneum decreased after T. cruzi infection (PI), compared
to the non-infected/non-treated group (PC). Although the
The corticosterone levels from plasma samples were
zinc supplementation (PIZ) reverted this condition in rela-
determined using the Corticosterone ELISA kit (Abcam,
tion to the infected group, the proportion of the CD11b/c+
catalog number: 108821). The measurement was per-
cells was lower compared to the PC group (Fig. 1a). The
formed following the manufacturer’s instructions, and
­MHCII+ cells were similar among infected (PI) and non-
the plates read in an ELISA reader (Synergy H1, Biotek)
infected animals (PC or PCZ), whereas the infected and
at 450 nm. The corticosterone levels were quantified in
zinc-treated group (PIZ) demonstrated a significant eleva-
relation to a standard curve provided by the manufacturer.
tion (Fig. 1b).

Statistical Analysis Antigen‑Presenting Cell Costimulation (CD80/CD86)

The values were plotted as mean ± standard deviation (SD). The antigen-presenting cells (CD11b/c +) that express
The groups were analyzed by one-way ANOVA followed by costimulatory proteins (­ CD80 +/CD86 +) were increased
the Tukey post test. All statistical analyses were performed in infected animals treated with zinc (PIZ). The cells
in the GraphPad Prism 5.0 software (La Jolla, CA, USA). (CD11b/c + CD80 + ) were unaltered in infected (PI) or

Fig. 1  Antigen cell presentation. Female Wistar rats were infected anized. The peritoneal cells were collected, processed, labeled with
with T. cruzi and after 30 days submitted to mating. After pregnancy anti-rat CD11b/c or anti-rat RT1B, and analyzed by flow cytometry. a
confirmation, the animals were treated with zinc for 18 days and euth- CD11b/c+ cells. b ­RT1B+ cells. *p < 0.05

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non-infected/zinc-treated (PCZ) animals, whereas were
significantly elevated in the infected and zinc-treated
group (PIZ) compared to the control (PC) (Fig. 2a). The
(CD11b/c+CD86+) cells from infected animals (PI) were
decreased in relation to the control (PC). When ani-
mals were zinc treated (PIZ), the proportion of (CD11b/
c+CD86+) cells was elevated compared to infected or con-
trol groups (Fig. 2b).
Detection of NK ­(CD161+) and NKT ­(CD3+CD161+)
Cells
The splenic cells labeled with anti-rat CD161 were unal-
tered among all groups (Fig. 3a). However, NKT cells
­(CD3+CD161+) were responsive to T. cruzi infection and
zinc supplementation. The T. cruzi infection increased the
­CD3+CD161+ cells when compared to the control (PC).
The zinc supplementation (PIZ) reversed this pattern
by decreasing the C ­ D3 +CD161 + cells to control levels
(Fig. 3b).
Fig. 2  CD80+/CD86+-expressing cells. Female Wistar rats were
infected with T. cruzi and after 30  days submitted to mating. After
pregnancy confirmation, the animals were treated with zinc for
18  days and euthanized. The peritoneal cells were collected, pro-
Fig. 3  NK and NKT cells. Female Wistar rats were infected with T.
cruzi and after 30  days submitted to mating. After pregnancy con-
firmation, the animals were treated with zinc for 18 days and eutha-
Detection of MCP‑1‑Expressing Lymphocytes
The MCP-1-expressing lymphocytes (­CD4 + or C ­ D8 +)
were elevated under T. cruzi infection in relation to the
non-infected/non-treated group (PC). For ­CD4+MCP-1+
cells, the zinc supplementation (PIZ) increased the MCP-
1-expressing cells compared to the infected animals (PI)
(Fig.  4a), whereas the ­CD8+MCP-1+ lymphocytes were
unaltered (Fig. 4b).
Apoptosis
The early and late apoptosis of cells incubated for 15 min
demonstrated the opposite patterns. Whilst the early apop-
tosis was unaltered after T. cruzi infection and/or zinc sup-
plementation; the late apoptosis was reduced in relation to
the control (C) (Fig. 5a, b; Suppl. Fig. 4). The infection with
T. cruzi decreased the early and late apoptosis in cells incu-
bated for 24 h with T. cruzi extract or RPMI. When supple-
mented with zinc, the apoptosis (early or late) from infected
animals was increased, however, only in cells incubated
cessed, and labeled with anti-rat CD11b/c. The CD11b/c+ cells were
also labeled with anti-rat CD80 or anti-rat CD86 and analyzed by
flow cytometry. a CD11b/c+CD80+ cells. b CD11b/c+CD86+ cells.
*p < 0.05
nized. The splenic cells were collected, processed and labeled with
anti-rat ­CD161+ and/or anti-rat ­CD3+ and analyzed by flow cytom-
etry. a ­CD161+ cells. b ­CD3+CD161+ cells. *p < 0.05
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Fig. 4  MCP-1 expressing lymphocytes evaluation. Female Wistar
rats were infected with T. cruzi and after 30  days submitted to mat-
ing. After pregnancy confirmation, the animals were treated with
zinc for 18 days and euthanized. The splenic cells were collected and
incubated with ionomycin and PMA for cell secretion. The secretion
process was blocked with brefeldin A and the cells incubated with
for 24 h (under T. cruzi stimulation); the early apoptosis
was superior concerning the control (C) (Fig. 5c–f; Suppl.
Fig. 4). For cells incubated for 24 h, the zinc supplementa-
tion in healthy animals (PCZ) maintained apoptosis com-
pared to the control (C), except the early apoptosis in non-
stimulated cells (RPMI) (Fig. 5c; Suppl. Fig. 4).
Cytokine Quantification
The infection with T. cruzi (PI) demonstrated distinct pat-
terns in the INF-γ, IL-2, and IL-17 levels. The infection
elevated INF-γ and IL-17 detection in serum, whereas the
IL-2 levels were unaltered compared to the control (C)
(Fig. 6). The treatment of infected animals with zinc (PIZ)
decreased the INF-γ levels, reaching the control parameters
(C) (Fig. 6a). For IL-2 and IL-17, the treatment of infected
animals (PIZ) increased the levels of these cytokines in rela-
tion to all groups (PC, PCZ, or PI) (Fig. 6b, c).
Corticosterone Quantification
The T. cruzi infection decreased the corticosterone levels
compared to the controls (PC and PCZ), and the pattern was
maintained after zinc supplementation (PIZ) (Fig. 7).
Discussion
The pregnancy is a special condition in which the mater-
nal immune system is regulated to tolerate and allows fetal
development [54]. This immune balance reacts differently
to pathogens and exogenous molecules compared to males
and non-pregnant females [31]. The control of Chagas dis-
ease in pregnant models is limited due to the toxicity of
the benznidazole and nifurtimox [11, 39], which urges the
necessity for novel forms to alleviate the deleterious effects
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anti-rat CD32. The cells were washed and labeled with anti-rat CD3,
anti-rat CD4, and anti-rat CD8. For the detection of secreted MCP-
1, the cells were permeabilized, labeled with anti-rat MCP-1 and
analyzed by flow cytometry. a ­CD4+MCP-1+ cells. B ­CD8+MCP-1+
cells. *p < 0.05
of the pathogen and/or the treatment. Thus, immunomodu-
latory molecules have an interesting potential, which may
increase the response against pathogens as well as the side
effects caused by drugs. Due to the low zinc toxicity at nor-
mal intakes (as applied in this study), the element has the
potential for use during pregnancy. As observed, the sup-
plementation does not alter the zinc serum levels, the weight
or length of fetuses and placentas. Moreover, all pregnan-
cies occurred normally, and no fetal malformations were
detected. Our data indicate that zinc supplementation is safe
for use during pregnancy, even under T. cruzi infection.
The chronic phase of the Chagas disease and/or zinc sup-
plementation altered several parameters in pregnant rats.
In this study, one of our aims was directed to antigen-pre-
senting cells from peritoneal exudate. The innate response
induced by T. cruzi infection decreased the dendritic cells
as well as the cells expressing MHC II molecules. These
cells are pivotal for the lymphocyte and phagocyte stimula-
tion and T. cruzi down-regulates APCs to evade of immune
response [21, 50]. Moreover, macrophages and dendritic
cells are important for the maternal–fetal balance, propiti-
ating the immunological tolerance as well as the activation
of a particular response against pathogens [2, 31]. The zinc
supplementation in infected animals increased the propor-
tion of these cells compared to the infected ones. In parallel,
the nitric oxide (NO) is unaltered among groups. Despite the
effectiveness of NO for parasite control [34, 52], an excess
of this molecule leads to tissue damage, including cardio-
myopathy [7]. Indeed, during the conversion of acute to the
chronic stage of Chagas disease (20–60 days post-infection),
the parasite proliferation is usually controlled by the immune
system and a regulated nitric oxide production is welcomed
to avoid maternal–fetal disturbance. For an adequate T-cell
activation and proliferation, the APC’s surface proteins
CD80 and CD86 interact with CD28 expressed in most T
lymphocytes [10]. The CD80/86-CD28 costimulation has a
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Fig. 5  Early and late apoptosis of splenic cells. Female Wistar rats
were infected with T. cruzi and after 30  days submitted to mating.
After pregnancy confirmation, the animals were treated with zinc for
18  days and euthanized. The splenic cells were collected, processed
and incubated with T. cruzi extract or RPMI for 15  min or 24  h,
37 °C, 5% ­CO2. The cells were washed and incubated with Annexin
V and propidium iodide (PI) for 15  min and 5  min, respectively,
crucial role in T. cruzi control and animals lacking functional
CD28 and/or CD80/86 are highly susceptible to the acute
phase of the Chagas disease [29]. However, T-cell prolifera-
tion is decreased after zinc supplementation in pregnant rats
infected with T. cruzi [14]. Thus, novel assays are necessary
to clarify the relation between CD80/CD86 expression and
T-cell proliferation during pregnancy in animals infected
with T. cruzi. Although zinc decreases the T-cell prolifera-
tion, MCP-1-expressing T lymphocytes are elevated after
T. cruzi infection and zinc supplementation. MCP-1/CCL2
chemokine is secreted by a large number of cells in inflam-
matory processes for monocyte recruitment to infected
before the analysis by flow cytometry. a Early apoptosis of cells
incubated for 15  min in RPMI. b Late apoptosis of cells incubated
for 15 min in RPMI. c Early apoptosis of cells incubated for 24 h in
RPMI. d Late apoptosis of cells incubated for 24 h in RPMI. e Early
apoptosis of cells incubated for 24 h in T. cruzi extract. f Late apopto-
sis of cells incubated for 24 h in T. cruzi extract. *p < 0.05
tissues [26, 41]. Studies using Toxoplasma gondii and Myco-
bacterium tuberculosis indicate that the MCP-1 absence
reduces the ability to control infections [4, 37]. Likewise,
MCP-1 is produced in large quantities in the heart of T.
cruzi-infected mice to increase parasite destruction. MCP-1
knockout mice develop higher parasitemia and mortality
when compared to wild-type (WT) counterparts [33]. Simi-
larly, NK and NKT cells contribute to the control of chronic
infections, including Chagas disease [17]. The depletion
of NK and NKT cells in mice also increases the T. cruzi
parasitemia and decreases the survival rate [6, 38]. In preg-
nancy, high levels of NK cells are related to complications
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Fig. 6  INF-γ, IL-2, and IL-17 quantification. Female Wistar rats
were infected with T. cruzi and after 30  days submitted to mating.
After pregnancy confirmation, the animals were treated with zinc for
18  days and euthanized. The sera were collected, and the levels of
INF-γ, IL-2, and IL-17 quantified by the Rat Quantikine ELISA Kit
(R&D Systems), using specific standard curves. *p < 0.05
during pregnancy such as preeclampsia and hypertension
[8]. However, the mechanisms concerning NK and NKT
role in the immune response against T. cruzi or pregnancy
remain unclear. T. cruzi infection induces T-cell apoptosis
as a mechanism to the propagation and immune system eva-
sion. Intense apoptosis induced in Chagas disease interferes
with T-cell activation and production of pro-inflammatory
cytokines [16]. However, in our model of infection, the Cha-
gas disease decreased the percentage of apoptosis in cells
incubated with T. cruzi antigens, indicating an anti-parasite
pattern. During a normal pregnancy, the lymphocyte apop-
tosis is elevated compared to non-pregnancy models. The
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Fig. 7  Corticosterone plasma levels. Female Wistar rats were infected
with T. cruzi and after 30 days submitted to mating. After pregnancy
confirmation, the animals were treated with zinc for 18  days and
euthanized. The plasmas were collected, and the corticosterone levels
quantified using a Corticosterone ELISA kit (Abcam). *p < 0.05
elevation of apoptosis contributes to the elimination of anti-
paternal T cells, preventing deleterious immune responses
towards the fetus [1, 24]. On the other side, patients with
recurrent spontaneous abortion demonstrate significantly
lower T-cell apoptosis compared to fertile women [20]. Our
results indicate that the T. cruzi infection decreased the cell
apoptosis, mostly when cells were incubated with T. cruzi
antigens. However, the zinc supplementation of infected
animals improved the early and late apoptosis in cells incu-
bated with T. cruzi antigens, compared to the infected and
non-supplemented group. Thus, zinc contributes to balanced
apoptosis, maintaining the immune response as well as the
protection of mother and fetus.
INF-γ, a pro-inflammatory cytokine, is produced by
macrophages, NK, and T cells to stimulate the antigen-pre-
senting cells, NO production, NK activity, and B-cell pro-
liferation [40, 48]. This molecule acts for the control of the
parasite propagation and is also related to cardiac damage
[7, 46]. Moreover, INF-γ induces a potent TH1 response,
which leads to fetal loss and preterm labor in the mouse
model [45]. Therefore, the control of INF-γ production after
zinc supplementation contributes to a pro-pregnancy course.
Otherwise, IL-2 is improved after zinc supplementation indi-
cating a pro-inflammatory response in pregnancy. Although
IL-2 administration inhibits pregnancy in a mouse model
[47], this cytokine has an important effect on controlling T.
cruzi infection. IL-2 released by macrophage stimulates a
protective immune response against T. cruzi, inducing the
production of INF-γ by T and NK cells [18]. Once INF-γ
is downregulated in pregnant rats infected with T. cruzi and
treated with zinc, there is a balance between anti-pathogen
and pro-pregnancy responses mediated by IL-2 and INF-γ
respectively. The production of IL-17 is elevated in T. cruzi
infection and accentuated after zinc supplementation. IL-17
is a pro-inflammatory cytokine which induces the production
Acta Parasitologica
of IL-6, TNF-α, GM-CSF, and induces neutrophil migration
[23, 55]. The cytokine controls T. cruzi infection by trapping
parasites in the endolysosomal compartment [19] and IL-17
KO mice are highly susceptible to acute Chagas disease [30].
However, an elevated proportion of IL-17 secreting cells
have been correlated with complicated pregnancies char-
acterized by miscarriage, preterm birth, and preeclampsia
[15]. Thus, novel assays are required to elucidate the role
of IL-17 in a model combining the period of conversion of
acute to the chronic phase of Chagas disease and pregnancy.
Corticosterone, an important stress hormone [27], is sensi-
tive to T. cruzi infection and/or zinc supplementation. High
levels of corticosterone induce immune suppression [44] and
improve the rat susceptibility to T. cruzi [35]. In pregnancy,
the elevation of corticosterone levels limits the fetal growth,
reducing the amino acid supply and density of blood vessels
in the placenta [51]. Thus, zinc demonstrates an anti-T. cruzi
and pro-pregnancy patterns, maintaining low levels of corti-
costerone in pregnant rats infected with T. cruzi.
This study reveals several immune aspects of zinc supple-
mentation in pregnant Wistar rats in an experimental model
of the Chagas disease. Our results indicate that the immune
response induced by T. cruzi during pregnancy has a distinct
profile compared to non-pregnant models. Zinc supplemen-
tation promoted an immune response with potential for the
T. cruzi control in parallel to a pro-gestation response. Thus,
the element contributes to the immune regulation, which is
fundamental to the special condition of pregnancy. However,
new assays are necessary to describe the mechanisms of zinc
immune regulation during pregnancy, guiding a consistent
strategy for food supplementation in patients affected by the
Chagas disease.
Acknowledgements  We would like to thank FAPESP for the fellow-
ship to C.M.B. da Costa (Grant number: 2013/04205-6). We also thank
Fabiana Rosetto Morais and Vanessa Cristina de Oliveira Souza for the
flow cytometry and HPLC assistance, respectively.
Author Contributions  CMBDC, JCDPJ, and AACA designed the
experiments, wrote, and revised the manuscript. CMBDC, MVF, FS,
and IFLG carried out the experimental work. LMP and FBJ analyzed
the data, wrote, and revised the manuscript. All authors read and
approved the final manuscript.
Compliance with Ethical Standards  14. da Costa CMB, Del Vecchio FM, Santello FH, Pereira LM, Toldo
Conflict of interest  The authors declare that they have no conflict of
interest.
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