Acta Tropica 211 (2020) 105594

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Acta Tropica
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Genetic polymorphism in IL17RA induces susceptibility to Toxoplasma gondii T

infection in Brazilian pregnant women
Joelma Maria de Araujo Andradea,b,1, Claudio Bruno Silva de Oliveiraa,1,
Ywlliane da Silva Rodrigues Meurerc, Jéssica Emanuella Santanad,
Yngrid Gleyter Barbosa de Almeidad, Priscilla Vilela dos Santose, Débora Maria Soares de Souzae,
Guilherme de Paula Costae, André Talvanie, Gustavo Martelli Palominof,
Janaina Cristiana de Oliveira Crispim Freitasf, Valter Ferreira de Andrade-Netoa,
⁎

a
Laboratory of Malaria and Toxoplasmosis Biology/LABMAT, Department of Microbiology and Parasitology, Bioscience Center, Federal University of the Rio Grande do
Norte. Natal, Rio Grande do Norte, Brazil
b
Postgraduate Program of Biological Science, Bioscience Center, Federal University of the Rio Grande do Norte. Natal, Rio Grande do Norte, Brazil
c
Department of Psychology, Federal University of Paraíba, João Pessoa, Paraíba, Brazil
d
UNINASSAU Faculty, Natal, Rio Grande do Norte, Brazil
e
Laboratory of Immunobiology of Inflammation, DECBI/ICEB and Post-graduate Program of Health and Nutrition, Federal University of Ouro Preto, Minas Gerais, Brazil
f
Department of Clinical Analysis, School of Pharmacy, Federal University of the Rio Grande do Norte. Natal, Rio Grande do Norte, Brazil

ARTICLE INFO ABSTRACT

Keywords: Congenital toxoplasmosis is a parasitic disease caused by Toxoplasma gondii, an obligate intracellular parasite
Toxoplasma gondii which can cause fetal death/abortion and can induce damage in the brain and eyes of the infected babies. The
Pregnant women environmental and genetic factors associated with T. gondii and the maternal immune response, drive part of the
Polymorphism pathogenesis of congenital toxoplasmosis. Thus, in this study, we aimed to investigate the allelic and genotypic
IL17RA
frequencies of specific single nucleotide polymorphisms (SNPs) in the IL17A and IL17RA genes, as well as the
Cytokines
production of IL-17A, IL-33, and CCL2 in pregnant women, from the State of Rio Grande do Norte, Brazil, further
relating these along with the clinical parameters, to the toxoplasmosis infection. Through PCR-RFLP techniques,
two SNPs implicated in Th17 immune response, IL17A rs2275913 (G> A) and IL17RA rs4819554 (A> G)
modulation were evaluated in pregnant women, either infected or not infected by T. gondii. These women were
also evaluated in terms of plasma release of CCL2, IL-33, and IL-17A which relate to hypertension, number of
abortions, and ethnic pattern. The results showed that the G-allele of the SNP rs2275913 (IL17A) appeared to be
protective in this population, while the rs4819554 (IL17RA) SNP G allele was associated with greater suscept-
ibility to T. gondii infection [ρ value = 0.025; OR = 2.815 (1.118–7.089); CI = 95%]. None of the cytokines had
any influence on the analyzed parameters (abortion and hypertension). In conclusion, our data suggest an im-
munogenic evidence of susceptibility to T. gondii infection driven by the rs4819554 (IL17RA) SNP G allele in
Brazilian pregnant women. Further studies are needed to reinforce this trial marker in populations from distinct
geographical areas as well as to confirm the protective pattern related to the G-allele of the SNP rs2275913
(IL17A) in pregnant women.

1. Introduction
Toxoplasma gondii is an intracellular parasite with tropism to the
human placenta, eyes, and the central nervous system
(Carruthers et al. 2007; Wohlfert et al., 2017). The disease, tox-
oplasmosis, when affecting immunocompetent individuals triggers
nonspecific symptoms such as muscle pain, swollen lymph nodes, fever,
and headache (Khan et al. 2006; Mendes et al. 2014; Silva et al. 2014),
whereas in immunosuppressed individuals, it can be fatal
(Liu et al. 2019; Van Bilsen et al. 2017).
The most severe clinical manifestation of T. gondii infection is
congenital toxoplasmosis, which usually does not cause considerable

⁎
Corresponding author. Laboratory of Malaria and Toxoplasmosis Biology/LABMAT, Department of Microbiology and Parasitology, Bioscience Center, Federal
University of the Rio Grande do Norte. Natal, Rio Grande do Norte, Brazil.
1
Equal contributors.

https://doi.org/10.1016/j.actatropica.2020.105594
Received 17 April 2020; Received in revised form 21 June 2020; Accepted 21 June 2020
Available online 26 June 2020
0001-706X/ © 2020 Elsevier B.V. All rights reserved.
J.M.d.A. Andrade, et al.
damage to the pregnant mother, but it does to the fetus, in the short or
long term, causing spontaneous abortion, stillbirth, hydrocephalus,
macro or microcephalus, cerebral calcifications, retinochoroiditis, and
ocular or central nervous system alterations (Gómez-Chávez et al. 2019;
Olariu et al. 2011).
The induction of a panel of Th1-profile inflammatory mediators,
such as IL-1, IFN-gamma, TNF, IL-6, IL-12, CCL2, CCL5, IL-17A and
others, is defined as a key event to elicit the anti-T. gondii response
(Neves et al. 2012; Dutra et al. 2013). Clinical manifestations can also
be triggered by the host inflammatory response, such as the active
ocular toxoplasmosis related to the decreased production of CCL-2 and
increased levels of IL-17A and IL-33, respectively (Rey et al. 2013;
Jones et al. 2010; Tong and Lu, 2015; Zhang et al. 2019). In addition,
the genetic modification from the single nucleotide polymorphisms
(SNPs) of inflammatory and regulatory cytokine genes have also been
associated to the T. gondii susceptibility and its related pathogenesis
(Wujcika et al. 2018).
Additionally, the diagnosis of a T. gondii infection in pregnant
women can be confusing due to false positives when only im-
munoglobulins are detected to analyze the mother's immune response
(Simon et al. 2020). We emphasize that in prenatal screenings, a false-
positive IgG test result in a pregnant woman can lead to stopping pre-
ventive measures, leading to the risk of an acute infection during
pregnancy. On the other hand, no immunologic or genetic marker exists
to apply in a clinical trial of pregnant women in the potential risk of T.
gondii infection. This marker would support clinicians in the manage-
ment of pregnant women living in areas of high risk of toxoplasmosis
and, consequently, minimize the consequences by increasing the sur-
viving rate of the fetus and health of the baby.
Since the city of Santa Cruz, in the Rio Grande do Norte state in
Brazil presents 66.2% of toxoplasmosis among all pregnant women
(Freitas et al. 2017), the aim of this cross-sectional study was to in-
vestigate the allele and genotypic frequencies of the IL17A and IL17RA
polymorphisms and the plasma markers (IL-17A, IL-33, and CCL2) in
pregnant women infected with T. gondii, with a clinical record of
abortion and hypertension, in the state of Rio Grande do Norte, Brazil.
2. Methodology
2.1. Population, sample collection, and processing
A total of 204 pregnant women visiting the “Maternidade Escola
Januário Cicco” in Natal, Rio Grande do Norte state, Brazil, between
2015 and 2017, were enrolled in the survey, with each providing
written Informed Consent Term. The population sample in this study
was classified according to their ethnic characteristics as white, black
and "pardo". The Brazilian census categories have always included the
term "pardo" or "parda" for the admixed population; miscegenation is a
pattern in Brazilian society.
Blood samples were obtained by peripheral blood puncture and
plasma samples were isolated, centrifuged at 700 G for 10 min and
stored at −80°C until further serological and molecular analysis.
2.2. Serological diagnosis of toxoplasmosis
The plasma anti-T. gondii IgG and IgM were measured by enzyme-
linked immunosorbent assay (ELISA) according to the method de-
scribed by Oliveira et al. (2016). Briefly, 96-well flat-bottomed plates
(Greiner Bio-One GmbH, Frickenhausen, Germany) were coated over-
night with T. gondii lysate antigen (TLA) in a sodium carbonate buffer.
Then, the plates were washed with phosphate-buffered saline (PBS)
supplemented with Tween 20 (PBS-T), and blocked with skim milk
(Molico-Nestlé®, Araçatuba, SP, Brazil), following which, plasma sam-
ples were added to their respective wells, and incubated for 1 h at 37°C.
After washing the plates, a diluted solution of anti-human IgG
(1:10,000; Anti-IgG) or IgM-HRP(1:1000; Anti-IgM) (Sigma-Aldrich, St.
2
Acta Tropica 211 (2020) 105594
Louis, MO, USA) was added to each well and incubated. For
IgG + samples, avidity was quantified as follows: urea (6 M) was added
to each well; thereafter, the plates were washed and incubated with 100
µL/well of OPD (3 mg) (1,2-Diaminobenzidine; Sigma-Aldrich Brasil
Ltda, SP, Brazil) diluted in citrate buffer (15 mL) containing H2O2 (3
µL); the reaction was stopped by adding 30 µL of 4 N H2SO4 to each well
and the absorbance was read at 492 nm. Sample results were compared
with positive and negative controls.
2.3. DNA extraction and polymorphisms analysis
In order to analyze the genetic polymorphisms in IL17A
(rs2275913) and IL17RA (rs4819554) genomic DNA was extracted from
the blood samples using the Salting Out method, described by
Salazar et al. (1998). The quantification and evaluation of DNA purity
were performed using a spectrophotometer (Nanodrop, Thermo Scien-
tific- Waltham, MA, USA). In this step, 98 samples were selected for
molecular analysis due to their higher concentration and purity.
The amplification of the target regions of the IL17A and IL17RA
genes occurred by polymerase chain reaction – restriction fragment
length polymorphism (PCR-RFLP), based on Gomes et al. (2016). The
reaction occurred at a final volume of 25μL/well, containing: 2.5µL of
10x PCR buffer minus Mg; 0.7µL of MgCl2 in 50 mM (IL17A) or MgSO4
in 25 mM (IL17RA); 0.5µL of dNTP mix 10 mM; 0.5µL of each primer:
SNP rs2275913 (F: 5′-AGGTACATGACACCAGAAGACC-3′; R: 5′- TGCC
CACGGTCCAGAAATAC-3′) (DNA Express, São Paulo, Brasil); SNP
rs4819554 (F: 5′-GGAAGAGAGGAGAGGCGAAT-3′; R: 5′- CACCCCTTT
GCCTGGTTCTG-3′); 0.2µL de Taq DNA Polymerase; between 3 and 5µL
of DNA test and Milli-Q water in sufficient quantity to achieve the final
volume. After, plates were positioned in the thermocycler (MyCycler
Thermal Cycler, Bio-Rad Laboratories, Hercules, CA, USA), and the
characterization among the SNPs was performed by the cleavage of the
amplicons, using RFLP (Table 1). Finally, the polymorphisms were
performed in a UV-transilluminator after electrophoresis in a 2%
Agarose gel colored with SYBR® Green (Life Technologies, USA)
(Fig. 1).
2.4. Immunological analysis
Circulating levels of the inflammatory mediators CCL2, IL-17A and
IL-33 in plasma, were quantified, in triplicate, using the enzyme-linked
immunosorbent assay (ELISA) commercial kits (Peprotech, Rocky Hill,
NJ, USA). Briefly, 96-well micro titer plates were coated with the ap-
propriate monoclonal capture antibodies diluted in PBS, incubated
overnight and washed with wash buffer (PBS, 0.05% Tween-20).
Nonspecific binding sites were blocked using blocking buffer (1% BSA
in PBS). Plates were rinsed with wash buffer and the samples were
added and incubated at room temperature. Plates were then washed
again and the appropriate biotinylated detection antibodies, which
were diluted in diluent buffer containing 0.05% tween-20 and 0.1%
BSA, were added for 2 h at room temperature. The plates were washed
and avidin horseradish peroxidase diluted in diluent buffer was added
to each well and incubated at room temperature. After the last washing
of the plates, chromogen substrate ABTS (2,2′-Alzino-bis(3-ethylben-
zothiazoline-6-sulfonic acid, Sigma-Aldrich Inc., St. Louis, MO, USA)
was added to the wells and the plates were incubated in the dark at
room temperature. The absorbance was read at 405 nm with wave-
length correction set at 650 nm in a spectrophotometer (Emax-
Molecular Devices).
2.5. Statistical analysis
Genotype and allele frequencies were obtained by direct counting.
The Hardy-Weinberg equilibrium was assessed by the chi-square
goodness-of-fit test. A comparison of the allelic and genotypic fre-
quency distributions in populations was performed using the Chi-square
J.M.d.A. Andrade, et al. Acta Tropica 211 (2020) 105594

test. The chance of each SNP genotype and allele with respect to the

GG: 297 and 217pb. GA: 514pb, 297pb and 217pb. AA: 514pb.
reactive (R)/nonreactive (NR) condition was estimated with odds ratio

GG: 430pb. GA: 430, 243 and 187pb. AA: 243 and 187pb.
(OR) with a 95% confidence interval (CI). Quantitative data are ex-
pressed as mean ± standard deviation (SDM). The Kruskal-Wallis test
was used for unpaired and nonparametric samples. Statistical analyses
were performed using the IBM SPSS Statistics program and the figures
were produced using GraphPad Prism v 6.0 (GraphPad Software, CA,
USA). Values of ρ ≤0.05 were considered statistically significant.
Fragments produced by each genotype

3. Results

In the studied population (204 women), 138 (67.64%) were positive

to T. gondii infection. Of these, 26 (12.74%) were reactive only for IgM,
of which 3 (1.47%) women had low avidity to IgG and 109 (53.43%)
women had high avidity to IgG. The mean age of the reactive group of
women was 30.5 ± 7.1 years, with a majority of them being “pardas”
(term given to racial miscegenation; a pattern in Brazilian population),
with no reports of symptoms related to T. gondii infection (Table 2).
We also characterized this population evaluating the T. gondii DNA
in the blood samples of 98 pregnant women with a positive prevalence
Restriction enzyme

in 71.42% of the cases. Among these, 13.26% were identified as being

in the acute phase of T. gondii infection.
EcoNI (XagI)

Regarding the rs2275913 (IL17A) SNP genotype pattern of pregnant

patients and categorized among reactive and non-reactive for T. gondii
PvuII

infection, the following distribution was obtained for reactive patients:

53.16% GG, 17.25%, and 1.02% AA; whereas between non-reactive
patients show 21.43% GG, 06.12% AG, and 01.02% AA (Table 3). When
we evaluate the stratified frequency among only reactive pregnant
Amplified product

patients, the following distribution was verified: 74.28% GG; 24.29%

AG; and 1.03% AA; whereas among non-reactive patients show fol-
lowing distribution: 75% GG; 21.43% AG; and 3.57% AA. Concerning
514pb
430pb

the IL-17A production, we do not observed significant effect despite

founded higher absolute production showed for AA genotype than the
other genotypes (Fig. 2).
About the allele frequency analysis from total sampled patients,
94°C for 5 min. 35 cycles: 94°C for 45 s, 60°C for 45 s, 72°C for 45 s; 72°C for 7 min
94°C for 5 min. 35 cycles: 94°C for 45 s, 60°C for 45 s, 72°C for 45 s; 72°C for 7 min

which include both reactive (positive patients) and non-reactive (ne-

gative patients) groups, the G allele was present in 79.35% of the cases,
while the A allele was observed in 20.65% of the cases (p<0.05).
For SNP rs4819554 (IL-17RA) we observed the following genotypic
frequencies for total number of pregnant patients: 48.42% AA, 34.74%
AG, and 16.84% GG. The stratified frequency of total sampled followed
the distribution in reactive patients: 30.61% AA, 27.55% AG, and
13.27% GG; and non-reactive patients: 19.39% AA, 06.12% AG, and
03.06% GG (Table 3). Among only reactive patients the following dis-
tribution was verified: 67.86% AA; 21.43% AG; and 10.71% GG.;
whereas among non-reactive patients shown following distribution:
42.86% AA; 38.57% AG; and 18.57% AA. Regarding the allelic fre-
quency from total sampled patients, it was around 62.6% for allele A
and around 37.4% for allele G (p<0.05).
Additionally, for both positive and negative individuals, the odds
ratio of the presence of these alleles and/or genotypes was also verified.
Thus, we found for SNP rs4819554, that the allele G (AG + GG) exhibit
more effect in the reactive group and was associated with a greater
susceptibility to T. gondii infection [ρ value = 0.025; OR = 2.815
PCR conditions

(1.118–7.089); CI = 95%], an observation not found in the non-re-

active group (Table 3).
Finally, the release of CCL2, IL-33, and IL-17A cytokines did not
reveal significant differences between the reactive and non-reactive
groups, when the medical record of abortion and hypertension was
PCR-RFLP characteristics.

considered (Fig. 3 and 4).

IL17RA (rs4819554)
IL17A (rs2275913)

4. Discussion

This study allowed us to determine that the prevalence of human

Table 1

toxoplasmosis in pregnant women in the Northeast region of Brazil

SNP

remained homogeneous over the last decade, in accordance with

3
J.M.d.A. Andrade, et al. Acta Tropica 211 (2020) 105594

Fig. 1. Electrophoresis gel representing the digestion

products after PCR-RFLP of fragments of theIL17RA
(A) and IL17A (B) genes. For IL17RA the genotypes
have the following weight pattern: GG (430 bp); AA
(243 and 187 bp); while the AG (430, 243 and 187 bp).
For IL17A, the following molecular weights are ob-
served: GG genotype (297 and 217 bp); AG (514, 297
and 217 bp); AA (514 bp) did not appear in this re-
action. M, 100 bp molecular-weight marker.

Table 2
Demographic characteristics of the pregnant women investigated.
Characteristic Reactive Nonreactive X2 or U Value ρ value

N° Sample 71.43% (70) 28.57% (28)

Age (years) 30.5 ± 7.1 28.5 ± 7.8 838a 0.226
Ethnic characteristics
White 12.24% (12) 1.02% (1) 3844b 0.147
Black 1.02% (1) 1.02% (1)
“Parda” 58.16% (57) 26.53% (26)
Abortion
Yes 44.90% (44) 20.41% (20) 0.649c 0.487
No 26.53% (26) 8.16% (8)
Hypertension
Yes 33.67% (33) 14.29% (14) 0.065c 0.798
No 37.76% (37) 14.29% (14)

Categorical variables are expressed as a percentage of the sample. Ages are

expressed as mean ± standard deviation of the sample. Fig. 2. IL-17A production distributed from toxoplasmosis patients carrying the
a
ρ value was calculated by the Mann-Whitney U test for unpaired samples. genotype variants of the SNP rs2275913 (IL17A). IL-17 production was de-
b
Fisher's exact test. termined by ELISA. The bars represent the mean ± SD. P values were calculated
c
Chi-square test for categorical variables. by the Kruskal-Wallis (ANOVA) test for unpaired and non-parametric samples.

previous studies (Barbosa et al., 2009; Freitas et al. 2017). Besides,
using a single nucleotide polymorphism (SNP), we pointed out a no-
velty with this study, showing that, for the SNP rs4819554 (IL17RA),
the allele G (AG + GG) was more prevalent on the T. gondii reactive
women, suggesting higher susceptibility to this parasite.
One hypothesis for this prevalence of toxoplasmosis in Brazilian
pregnant women in the Northeast region of Brazil is the long-time ex-
position to environmental risk factors of T. gondii infection
(Mendes et al. 2014). We observed a prevalence of IgM (with high
avidity to the anti-T. gondii IgG) among reactive pregnant women. This
data indicates that the infection occurred before the pregnancy period,
minimizing the risk to the fetus (Pleyer et al., 2014). However, 21% of
the volunteers presented circulating IgM antibodies (with low avidity to
the anti-T. gondii IgG) which necessitates special monitoring by diag-
nosis and drug therapy to prevent potential congenital damages
(Barbosa et al., 2009; Dupont et al., 2012).
With the exception of serological tests (IgM and IgG) to diagnose T.
gondii infection in the current times, still no biomarkers exist to diag-
nose pregnant women or to identify those with more predisposition to
the infection. In this present study, we proposed a relationship between
the Th17 gene polymorphisms and the clinical status of the congenital
toxoplasmosis. We did not find a correlation between the rs2275913
(IL-17A) SNP polymorphisms and the abortion and hypertension re-
cords in T. gondii positive and negative women; however, the GG

Table 3
Genotypes and alleles data for rs2275913 (IL17A) and rs4819554 (IL17RA) SNPs in the reactive (positive patients) and nonreactive (negative patients) groups.
IL17A X2 P OR IL17RA X2 P OR
Reactive Non reactive (IC95%) Reactive Non reactive (IC95%)

Genotype
a b
AA 01.02% 01.02% 0.902 0.797 2.476 (0.148 - 4.194) 30.61% 19.39% 5.002 0.082 2.850
AG 17.25% 06.12% 27.55% 06.12% (0.992 – 8.184)
GG 53.16% 21.43% 13.27% 03.06%
b b
AA+AG 18.27% 07.14% 0.005 0.942 1.038 (0.378 – 2.850) 58.16% 25.51% 0.904 0.342 0.526
GG 53.16% 21.43% 13.27% 03.06% (0.138 – 2.010)
AA 01.02% 01.02% 0.415 0.498a 2.556 (0.154 – 42.333) 30.61% 19.39% 5.000 0.025 b
2.815
AG+GG 70.41% 27.55% 40.82% 09.18% (1.118 – 7.089)
Allele
A 14.87% 5.78% 2.000 0.999b 1.006 (0.378 – 2.681) 43.52% 19.08% 2.345 0.126 b
0.513
G 57.03% 22.32% 30.53% 6.87% (0.217 – 1.215)

Categorical variables are expressed as a percentage of the sample. ρ value was calculated by aFisher's exact test and bChi-square test for categorical variables. Odds
ratio were used to evaluate the size effect of the association between variables.

4
J.M.d.A. Andrade, et al. Acta Tropica 211 (2020) 105594

Fig. 3. Production of CCL2 (A), IL-33 (B), and IL-17A (C) cytokines in non-reactive and reactive pregnant women for T. gondii infection with (black bar) or without
history of abortion (white bar) Bars represent the median with interquartile range. P values were calculated by the Mann-Whitney-U test for unpaired and non-
parametric samples.

genotype was the most frequent, followed by the AG and AA genotypes
in positive pregnant women, suggesting a genotypic characteristic of
this population. A similar pattern was previously found in pregnant
women from other states of Brazil (Saraiva et al. 2013) as well as in
Argentina (Rolandelli et al. 2017), another South American region.
However, when the geographical focus was changed to European
countries (Stappers et al. 2014), to China (Geng et al., 2015), to Mon-
golia (Shuang et al. 2015) and to Japan (Hayashi et al. 2013), the most
common genotype was AG, followed by GG and AA.
In addition, in our study, the G allele was the most prevalent in
pregnant women from the Northeast region of Brazil. This is note-
worthy because this allele is associated with a lower production of IL-
17A, thus acting as a possible protector against inflammatory damage;
while allele A is associated with severe inflammatory diseases
(Espinoza et al. 2011). This fact could explain why reactive pregnant
women are usually asymptomatic. Thus, for toxoplasmosis, the pre-
sence of the G allele could be a protective condition, which could be
reinforced by the fact that pregnant women with the AA genotype
present higher IL-17A production than GG and AG genotype carriers
(Espinoza et al. 2011; Corrêa et al. 2012; Rolandelli et al. 2017).
However, when we quantified the presence of allele A between reactive
and non-reactive pregnant women, no differences were found. There-
fore, in these women the allele A for rs2275913 (IL17A) does not in-
terfere in the predisposition to T. gondii infection; in contrast with what
was observed with Trypanosoma cruzi infection (Rodriguez et al. 2015)
and could also be implicated in the development of chronic cardio-
myopathy in the studied Latin American population
(Strauss et al. 2020). Additionally, patients infected with Leishmania
braziliensis, carrying the A allele maintain higher parasite loads
(Albuquerque et al. 2019), reinforcing the genetic susceptibility find-
ings for trypanosomatids.
Alternatively, in the analysis of rs4819554 (IL17RA) SNP, both G
and A alleles were previously related to severe kidney diseases due
increased IL-17RA protein expression (Kim et al. 2012). In contrast,
there are controversies regarding the involvement of polymorphisms in
members of the IL-17 family in the pathogenesis of cerebral malaria
(CM). Thus, in spite of the observation that IL-17A aggravates in-
flammation associated with various neurophysiological disorders, stu-
dies demonstrate that in populations of African children, the presence
of IL-17A and IL-17F increases antiparasite immunity during infection
by P. falciparum and then protects against severe disease. However,
IL17RA polymorphism modulate susceptibility to CM while there is
evidence that IL-17F protects against CM (Marquet et al. 2016).
Here, we noted that the G allele influenced the positive pregnant
women group although the highest frequency was found for genotype
AA and allele A and this pattern was associated to the absence of hy-
pertension or abortion. It is recognized that the lack of adaptability of
the T. gondii to the host can lead to severe disease (Oliveira, 2020) and
that genetic and immune responses of infected women are essential
evidences to define the course of the infection.
The mammalian genome is often influenced by infections, where the
extraordinary genomic complexity of lymphocyte receptors and the
complex set of mammalian immune functions are indicators of millions
of years of pathogen pressure (Müller and Howard, 2016). The G allele
implies the lower expression of IL17RA due to its lower binding affinity
to polymerase during transcription (Kim et al. 2012) in reactive women
and this lower expression decreases the activity of its ligand, IL-17A,
with consequent lower inflammatory activity and reduction of the

Fig. 4. Production of cytokines CCL2 (A), IL-33 (B), and IL-17A (C) in non-hypertensive (white bar) and hypertensive (black bar) pregnant women negative and
positive for T. gondii infection. Bars represent the median with interquartile range. P values were calculated by the Mann-Whithney test for unpaired and non-
parametric samples.

5
J.M.d.A. Andrade, et al.
likelihood of worse clinical conditions. In addition, it is important to
highlight that the majority of the volunteers participating in our study
presented themselves as being “pardos”, following the high rate of ra-
cial miscegenation in this region (BRASIL; IBGE, 2011), a factor that
could contribute to the presence of atypical forms of toxoplasmosis in
this population.
NaCl intake is recognized as one of the main causes of hypertension
(Ha, 2014). This consumption, even at intermediate levels, has already
been interfering with the immune course of toxoplasmosis in a murine
experimental model (Oliveira et al. 2018). Additionally,
Kleinewietfeld et al. (2013) reported that high levels of NaCl lead to an
increase in IL-17A production. Thus, the stable levels of IL-17A ob-
served in this study, even in toxoplasmosis-reactive populations with a
medical history of hypertension, allow us to propose that in this study,
high blood pressure is a hemodynamic consequence of many factors and
is not associated with toxoplasmosis in pregnant women. Another result
observed in this study is that there was no relationship between IL-17A
production and abortion, as the levels of blood IL-17A were stable in
both toxoplasmosis-reactive and non-reactive women with a history of
previous abortion.
It is important to note that toxoplasmosis is a typically chronic
disease, where levels of some cytokines tend to decrease throughout
infection (Miller et al. 2009). By recognizing the importance of the host
immune response to control and define the course of the infection, in
this study we verified that the majority of the subjects was in a chronic
stage of infection, defined by the pattern of the IgM/IgG relation. This
might be one of the reasons why we did not find differences between
soluble inflammatory mediators (IL-17A, CCL2 and IL33) in reactive
and non-reactive pregnant women, not even when clinical conditions
(hypertension and abortion) were considered in the analysis.
Dupont et al. (2012) reported that some pathogens induce IL-10 pro-
duction as an immune evasion mechanism. This regulatory cytokine is
produced during and after the acute phase of infection and possesses a
controlling role on the inflammatory response.
Therefore, our data suggest an immunogenic evidence of suscept-
ibility to T. gondii infection driven by the rs4819554 (IL17RA) SNP G
allele in Brazilian pregnant women. In this context, this work provides
useful perspectives for future studies of evolutionary parasitology,
which are essential for analyzing parasite-host interactions, the me-
chanisms of evasion of the immune system of the host, as well as genetic
polymorphisms in both the host and the parasite, since these promote
phenotypic changes that may influence the broad symptomatologic and
clinical spectrum observed in human toxoplasmosis in the Rio Grande
do Norte state, in the Northeastern region of Brazil.
Ethical approval
The samples used in this study, as well as the relevant information
about the research subjects, were authorized for publication after ap-
proval by the Research Ethics Committee of the Federal University of
Rio Grande do Norte, which occurred with the number 1314,667.
Authors’ contributions
JMAA and CBSO performed to the data analysis, immunogenic
analyses and manuscript writing; YSRM conducted the data analysis,
manuscript production and statistical analysis; JES and YGBA per-
formed the phlebotomy and initial sample processing; PVS, DMSS, GPC
and AT contributed to the immunological analysis, manuscript pro-
duction and text review; GMP and JCOCF contributed to genetic ana-
lysis; VFAN responsible for conceptualization, funding acquisition,
writing-review & editing, contributed to data analysis and manuscript
production.
6
Acta Tropica 211 (2020) 105594
Funding
This work was supported by Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (FAPEMIG), Universidade Federal do Rio
Grande do Norte (UFRN) and Universidade Federal de Ouro Preto
(UFOP), Brazil. VFAN (Process # 306,036/2019–3) and AT (Process #
305,634/2017–8) are CNPq Research Productivity Scholarship and GPC
is a CNPq PDJ scholarship.
Credit author statement
Joelma Maria de Araujo Andrade and Claudio Bruno Silva de
Oliveira: performed to the data analysis, immunogenic analyses and
manuscript writing; Ywlliane da Silva Rodrigues Meurer: conducted
the data analysis, manuscript production and statistical analysis;
Jéssica Emanuella Santana and Yngrid Gleyter Barbosa de
Almeida: performed the phlebotomy and initial sample processing;
Priscilla Vilela dos Santos Débora Maria Soares de Souza
Guilherme de Paula Costa and André Talvani: contributed to the
immunological analysis, manuscript production and text review;
Gustavo Martelli Palomino and Janaina Cristiana de Oliveira
Crispim Freitas: contributed to genetic analysis; Valter Ferreira de
Andrade-Neto: responsible for conceptualization, funding acquisition,
writing-review & editing, contributed to data analysis and manuscript
production.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
We are grateful to the Department of Pharmacology, Paulista School
of Medicine, USP, the Laboratory of Immunobiology of Inflammation,
DECBI/ICEB/UFOP and the Department of Clinical Analysis, School of
Pharmacy/UFRN for all support during experimental procedures. We
would like to thank Editage (www.editage.com) for English language
editing. The authors would like to thank the Brazilian Research Support
Agencies (CNPq, CAPES and FAPEMIG)
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