- Email: [email protected]
Azithromycin treatment is able to control the infection by two genotypes of Toxoplasma gondii in human trophoblast BeWo cells
Accepted Manuscript Azithromycin treatment is able to control the infection by two genotypes of Toxoplasma gondii in human trophoblast BeWo cells Mayara Ribeiro, Priscila Silva Franco, Janice Buiate Lopes-Maria, Mariana Bodini Angeloni, Bellisa de Freitas Barbosa, Angelica de Oliveira Gomes, Andressa Silva Castro, Rafaela José da Silva, Fernanda Chaves de Oliveira, Iliana Claudia Balga Milian, Olindo Assis Martins-Filho, Francesca Ietta, José Roberto Mineo, Eloisa Amália Vieira Ferro PII:
S0014-4894(17)30006-1
DOI:
10.1016/j.exppara.2017.08.004
Reference:
YEXPR 7436
To appear in:
Experimental Parasitology
Received Date: 4 January 2017 Revised Date:
28 July 2017
Accepted Date: 8 August 2017
Please cite this article as: Ribeiro, M., Franco, P.S., Lopes-Maria, J.B., Angeloni, M.B., de Freitas Barbosa, B., de Oliveira Gomes, A., Castro, A.S., da Silva, Rafaela.José., de Oliveira, F.C., Balga Milian, I.C., Martins-Filho, O.A., Ietta, F., Mineo, José.Roberto., Ferro, Eloisa.Amá.Vieira., Azithromycin treatment is able to control the infection by two genotypes of Toxoplasma gondii in human trophoblast BeWo cells, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.08.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Azithromycin treatment is able to control the infection by two genotypes of
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Toxoplasma gondii in human trophoblast BeWo cells
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Mayara Ribeiroa, Priscila Silva Francoa, Janice Buiate Lopes-Mariaa, Mariana Bodini
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Angelonia, Bellisa de Freitas Barbosaa, Angelica de Oliveira Gomesb, Andressa Silva
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Castroa, Rafaela José da Silvaa, Fernanda Chaves de Oliveiraa, Iliana Claudia Balga
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Miliana, Olindo Assis Martins-Filhoc, Francesca Iettad, José Roberto Mineoe, Eloisa
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Amália Vieira Ferroa,*
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a
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Embryology, Federal University of Uberlândia, Av. Pará, 1720, Uberlândia, CEP
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38400 902, Brazil
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b
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of Uberaba, Rua Frei Paulino, 30, Uberaba, CEP 38025 180, Brazil
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c
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Cruz, 30190-002 Belo Horizonte, MG, Brazil
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d
Department of Life Science, University of Siena, Aldo Moro Road 2, Siena, Italy
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e
Laboratory of Immunoparasitology, Department of Immunology, Microbiology and
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Parasitology, Federal University of Uberlândia, Av. Pará, 1720, Uberlândia, CEP
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38400 902, Brazil
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Departament of Structrual Biology, Institute of Biological Science, Federal University
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Laboratory of Chagas Disease, René Rachou Research Center, Fundação Oswaldo
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Laboratory of Immunophysiology of Reproduction, Department of Histology and
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* Corresponding author. Address: Laboratory of Immunophysiology of Reproduction,
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Department of Histology and Embryology, Federal University of Uberlândia, Av. Pará,
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1720, Uberlândia, CEP 38400 902, Brazil. Tel.: +55 34 3218 2240, Fax: + 55 34 3218
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2333. E-mail addresses: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
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Trophoblast infection by Toxoplasma gondii plays a pivotal role in the vertical
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transmission of toxoplasmosis. Here, we investigate whether the antibiotic therapy with
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azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control
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trophoblast infection by two Brazilian T. gondii genotypes, TgChBrUD1 or
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TgChBrUD2. Two antibiotic protocols were evaluated, as follow: i) pre-treatment of T.
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gondii-tachyzoites with selected antibiotics prior trophoblast infection and ii) post-
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treatment of infected trophoblasts. The infection index/replication and the impact of the
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antibiotic therapy on the cytokine milieu were characterized. It was observed that
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TgChBrUD2 infection induced lower infection index/replication as compared to
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TgChBrUD1. Regardless the therapeutic protocol, azithromycin was more effective to
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control the trophoblast infection with both genotypes when compared to conventional
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antibiotics. Azithromycin induced higher IL-12 production in TgChBrUD1-infected
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cells that may synergize the anti-parasitic effect. In contrast, the effectiveness of
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azithromycin to control the TgChBrUD2-infection was not associated with the IL-12
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production. BeWo-trophoblasts display distinct susceptibility to T. gondii genotypes and
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the azithromycin treatment showed to be more effective than conventional antibiotics to
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control the T. gondii infection/replication regardless the parasite genotype.
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Keywords: Trophoblast cell; Azithromycin; Toxoplasma gondii atypical strains;
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Cytokines
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ACCEPTED MANUSCRIPT 1. Introduction
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Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects
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a wide range of warm-blooded animals, including humans. Toxoplasmosis is typically
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asymptomatic in immunocompetent hosts, but the parasite is able to induce severe
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illness in immunocompromised patients and congenitally infected individuals (Dupont,
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et al., 2012, Ferguson, et al., 2012). There are three well-characterized lineages of T.
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gondii, known as types I, II and III. These lineages are predominantly found in Europe
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and North America, and each of them is constituted by a genotype that presents a high
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degree of clonality (Saeij, et al., 2005). In contrast, a different pattern is observed in
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South America and Africa, which are populated by lineages of T. gondii that have more
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diverse genotypes (Sibley, et al., 2009). In Brazil, there is high genetic diversity of T.
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gondii, including recombinant genotypes, and the three most common genotypes are #6,
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#8 and #11, previously designated as type BrI, BrIII and BrII, respectively (Carneiro, et
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al., 2013, Pena, et al., 2008). An analysis of mortality rates in infected mice indicated
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that Type BrI lineages are highly virulent, whereas Type BrIII are non-virulent and
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Type BrII are moderately virulent(Pena, et al., 2008). In Uberlândia city (Minas Gerais
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state, Brazil), two parasite isolates were found and named as TgChBrUD1 and
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TgChBrUD2. The TgChBrUD1 isolate had ToxoDB PCR-RFLP genotype #11 (also
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known as type BrII), and the TgChBrUD2 isolate had ToxoDB PCR-RFLP genotype #6
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(also known as type BrI Africa 1) (Lopes, et al., 2016). An in vivo study using Calomys
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callosus as a rodent model demonstrated that these Brazilian isolates were highly
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virulent for this host, and significant differences in the severity of infection related to
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the gender were observed (Franco, et al., 2014). The treatment of toxoplasmosis is
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indicated for pregnant and immunocompromised individuals (Montoya and Liesenfeld,
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2004). In women who are infected during pregnancy, spiramycin is used to prevent fetal
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ACCEPTED MANUSCRIPT infection because this antibiotic reaches high concentrations in placental tissues despite
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its low penetration of fetal tissues (Kaye, 2011). If fetal infection is confirmed, the
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mother should be treated with a combination of sulfadiazine, pyrimethamine and folinic
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acid (Montoya and Remington, 2008). Sulfadiazine and pyrimethamine are widely used,
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but these drugs are toxic and may cause adverse effects (Kaye, 2011, Montoya and
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Remington, 2008). In addition to causing side effects, these drugs might not be able to
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reduce the parasitism, as T. gondii has shown resistance to treatment with sulfadiazine
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in in vitro studies (Meneceur, et al., 2008).
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Both azithromycin and spiramycin belong to the macrolide group, a class of broad-
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spectrum antibiotics used to treat bacterial and intracellular parasite infections
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(Srivastava, et al., 2011). Macrolides lead to anti-inflammatory activity by inhibiting the
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synthesis of microbial toxins and other virulence factors (Steel, et al., 2012). Several in
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vivo and in vitro studies that used azithromycin to treat congenitally acquired
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toxoplasmosis were able to demonstrate reductions in T. gondii replication following
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treatment (Costa, et al., 2009, Franco, et al., 2011). Previous studies from our group
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have indicated that treatment with azithromycin is effective against T. gondii for both
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rodents and human cells that were infected with the virulent RH strain (type I) (Costa, et
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al., 2009, Franco, et al., 2011).
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BeWo cells are derived from a human choriocarcinoma and they have been
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characterized as an appropriate in vitro model of human trophoblast to investigate T.
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gondii infection in the maternal fetal interface by our group. In fact, we have
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investigated several aspects of the immunology of pregnancy using these cells as a
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trophoblast model (Barbosa, et al., 2015, Franco, et al., 2011).
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The aim of the present study was to verify whether the antibiotic therapy with
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azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control the T.
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ACCEPTED MANUSCRIPT gondii-trophoblast infection by two Brazilian T. gondii genotypes
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(TgChBrUD1/genotype#11/(type BrII) or TgChBrUD2/genotype#6/(type BrI/Africa1)
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and change the immunological microenvironment, using this well-established model of
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in vitro infection of BeWo human trophoblast cell lineage.
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ACCEPTED MANUSCRIPT 2. Material and Methods
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2.1. Cell culture and parasites
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BeWo trophoblast cells were obtained from the American Type Culture Collection
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(ATCC, Manassas, VA, USA) and cultured in RPMI-1640 media (Cultilab, Campinas,
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SP, Brazil) that was supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin
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(Sigma Chemical CO., St. Louis, USA) and 10% fetal bovine serum (Cultilab). The
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cells were maintained in a humidified incubator at 37°C and 5% CO2.
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Tachyzoites of the TgChBrUD1/genotype#11/(type BrII) and
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TgChBrUD2/genotype#6/(type BrI/Africa1) strains of T. gondii were maintained by
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serial passage in BeWo cells to obtain parasites in vitro for further experimental
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infections. The media that contained free tachyzoites was transferred to 15 mL tubes
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and centrifuged (400×g, 5min) at room temperature. Pellets were suspended in complete
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media, and the parasites were stained by Trypan blue and counted in a hemocytometer
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to establish infectious dose.
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2.2. Drugs
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Azithromycin (Biofarma, Uberlândia, MG, Brazil), spiramycin (Sigma), sulfadiazine
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(Sigma) and pyrimethamine (Sigma) were each prepared as a stock solution in DMSO
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at a concentration of 3.000 µg/mL. The stock solutions were diluted in complete media,
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and cells were treated with varying concentrations of the drugs, which were chosen
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based on previous studies (Franco, et al., 2011, Meneceur, et al., 2008).
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2.3. Drug treatments and cell viability
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Cell viability was evaluated using a tetrazolium salt colorimetric (MTT) assay. For this
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purpose, BeWo cells were cultured for 24 h in 96-well plates (5x104 cells/well/200 µL)
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ACCEPTED MANUSCRIPT at 37°C and 5% CO2.Then the cells were incubated with 200 µL/well of an antibiotic
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solution containing azithromycin or spiramycin in different concentrations (50, 100, 200
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or 400 µg/mL). For treatments using a combination of sulfadiazine and pyrimethamine,
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different ratios were selected, which included 50:1, 100:4, 200:8, 400:16, 600:24,
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800:32 or 1,000:40 µg/mL of sulfadiazine:pyrimethamine. As control, BeWo cells were
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not treated and only medium was added in the wells. After 24 h, the supernatants were
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removed, and the cells were washed and pulsed with 10 µL of MTT (Sigma). The plates
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were incubated under standard culture conditions; after 3 h, the formazan crystals that
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were produced by viable cells were solubilized by adding a solution of 10% sodium
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dodecyl sulfate (SDS) and 50% N, N-dimethylformamide. Optical density was
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determined at 570 nm using a plate reader (Titertek MultiskanPlus, Flow Laboratories,
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McLean, USA), and the results were expressed as percentage of viable cells relative to
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control (100%). Two independent experiments were performed in triplicate for each
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condition.
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2.4. Treatments and infection of BeWo cells
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BeWo cells were cultured on 13-mm glass coverslips in 24-well plates (5 x 104
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cells/well/200 µL) for 24 h at 37°C and 5% CO2. The cells were infected with
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tachyzoites of T. gondii (TgChBrUD1 or TgChBrUD2) at a ratio of three parasites per
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cell. After 24 h, the cells were treated with 100 µg/mL of azithromycin, 100 µg/mL of
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spiramycin or 100 µg/mL of sulfadiazine in combination with 4 µg/mL of
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pyrimethamine. As control, BeWo cells were infected and not treated, or uninfected and
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untreated. After 24 h of treatment, the supernatants were collected and stored at -80°C
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for cytokines measurement. The cells were fixed in 10% buffered formalin for 24 h,
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washed with phosphate-buffered saline (PBS) and stained with 1% toluidine blue for 3
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ACCEPTED MANUSCRIPT seconds. The coverslips were mounted on glass slides and examined under light
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microscopy to assess infection index (percentage of infected cells per 200 examined
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cells) and intracellular replication (mean number of parasites in 200 infected cells).26
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Three independent experiments were performed in triplicate for each condition.
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2.5. Treatments of T. gondii tachyzoites
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Free tachyzoites (1.5x105 parasites/well) were pre-treated with azithromycin (100
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µg/mL), spiramycin (100 µg/mL) or the combination of sulfadiazine and pyrimethamine
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(100 and 4 µg/mL, respectively) for 3 h at 37°C and 5% CO2. Next, the cells were
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infected with pre-treated parasites for 24 h and analyzed using light microscopy to
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assess intracellular replication and infection index, as described above. As control, T.
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gondii tachyzoites were not treated. Two independent experiments were performed in
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triplicate for each condition.
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2.6. Cytokine measurements
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Levels of IL-12p70, TNF, MIF, IL-6, IL-10 and TGF-β1 cytokines were measured in
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supernatants of untreated or treated cells using commercially available
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immunoenzymatic kits (BD Biosciences, New Jersey, USA and R&D Systems,
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Minneapolis, USA) according to the manufacturer’s recommendations. The results were
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expressed in pg/mL according to the standard curve.
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2.7. Statistical analysis
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All data were expressed as mean values ± SEM and differences between groups were
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assessed using One-Way ANOVA and either the Bonferroni or Dunnet post-test or
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Student’s t test when appropriate. All data were analyzed using GraphPad Prism 5.0
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(GraphPad Software Inc., San Diego, USA). Differences were considered statistically
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significant when P < 0.05.
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ACCEPTED MANUSCRIPT 3. Results
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3.1. The selected antibiotics induced minimal toxicity rate in BeWo cells when applied
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at low concentrations
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Azithromycin and spiramycin were not cytotoxic when added to BeWo cells up to a
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concentration of 200 µg/mL; however, they significantly decreased cell viability at 400
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µg/mL (P<0.05) (Fig. 1A-B). Additionally, the combination of sulfadiazine and
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pyrimethamine was not cytotoxic up to concentrations of 200 µg/mL and 8 µg/mL,
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respectively, and a significant reduction in cell viability was observed when cells were
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treated within a range that included 400 µg/mL of sulfadiazine plus 16 µg/mL of
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pyrimethamine to 1,000 µg/mL of sulfadiazine plus 40 µg/mL of pyrimethamine (Fig.
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1C). Based on these findings, further experiments were carried out using either 100
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µg/mL of azithromycin or spiramycin or 100 µg/mL of sulfadiazine plus 4 µg/mL of
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pyrimethamine.
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3.2. Azithromycin was more effective to control the trophoblast infection with distinct T.
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gondii genotypes as compared to conventional antibiotic therapy
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The infection index and intracellular replication of T. gondii were determined in BeWo
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cells that were treated with antibiotics (Fig. 2A-F). In comparing the TgChBrUD1 and
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TgChBrUD2 strains, it was found that exposure to TgChBrUD1 led to a higher
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percentage of infected BeWo cells (Fig. 2A, C, E) and a higher number of intracellular
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tachyzoites (Fig. 2B, C, E) (P<0.05). Treatment with either azithromycin or the
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combination of sulfadiazine and pyrimethamine reduced both infection index and
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intracellular replication in BeWo cells that were infected with TgChBrUD2 versus
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TgChBrUD1 (P<0.05) (Fig. 2A-B). Azithromycin was able to reduce the infection
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indexes and intracellular replication of both T. gondii strains in treated BeWo cells
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ACCEPTED MANUSCRIPT versus untreated cells (P<0.05) (Fig 2A-F), whereas treatment with spiramycin
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produced this effect only in TgChBrUD1-infected cells (P<0.05) (Fig. 2A-B). In
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contrast, treating cells with a combination of sulfadiazine and pyrimethamine was able
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to reduce the intracellular replication of only the TgChBrUD2 strain when compared to
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untreated cells (P < 0.05) (Fig. 2A-B).
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3.3 Pre-treatment of T. gondii tachyzoites with azithromycin is also able to control the
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trophoblast infection
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In order to verify the direct effect of drugs on T. gondii infection in BeWo cells, we pre-
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treated the parasites with the selected drugs before infect the cells. Our data showed that
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only azithromycin reduced the percentage of infected BeWo cells and the number of
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intracellular tachyzoites for both T. gondii strains, as compared to untreated parasites
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(P<0.05) (Fig. 2C-D). Pre-treating parasites with either spiramycin or the combination
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of sulfadiazine and pyrimethamine significantly reduced TgChBrUD2 replication
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compared to untreated parasites (Fig. 2D). When comparing TgChBrUD1 and
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TgChBrUD2, a higher infection index and number of intracellular tachyzoites was
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found in TgChBrUD1-infected cells, regardless of treatment (P<0.05) (Fig. 2C-D).
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3.4. Azithromycin induced higher IL-12 production in TgChBrUD1-infected cells that
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may synergize the anti-parasitic effect
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Cytokine production was evaluated in supernatants of BeWo cells that were infected
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with T. gondii and treated with azithromycin, spiramycin or the combination of
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sulfadiazine and pyrimethamine in comparison with controls (Fig. 3). IL-12 production
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was not significantly changed by any treatment in uninfected cells (Fig. 3A). Infection
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with TgChBrUD1 resulted in a significant increase in IL-12 production in untreated
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ACCEPTED MANUSCRIPT cells. The levels of this cytokine also increased in TgChBrUD1 infected cells and
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treated with azithromycin and spiramycin compared to untreated control. TgChBrUD1-
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infected BeWo cells that were either untreated or treated with azithromycin had higher
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levels of IL-12 production than cells treated with a combination of sulfadiazine and
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pyrimethamine (Fig. 3A). Combination of sulfadiazine and pyrimethamine was not able
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to change IL-12 production compared to untreated control. Additionally, infection with
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TgChBrUD2 resulted in a significantly increased level of IL-12 production in both
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untreated and spiramycin-treated cells compared to controls (P<0.05). However, after
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treatment with spiramycin, there was a significant increase in IL-12 regardless of the T.
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gondii strain used if compared to untreated controls (Fig. 3A).
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Uninfected BeWo cells had similar levels of TNF even after each of treatments (Fig.
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3B). When BeWo cells were infected with TgChBrUD1, TNF levels were reduced, even
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after azithromycin treatment when compared to uninfected and azithromycin-treated
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cells respectively, similar to what was observed in cells that were infected with this
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strain but remained untreated. In contrast, TgChBrUD2-infected BeWo cells did not
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show significant changes in TNF production after any treatment conditions, including
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untreated cells, compared to controls (Fig.3B). TgChBrUD2-infected cells when treated
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with combination of sulfadiazine and pyrimethamine showed higher TNF level if
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compared to untreated control. An increased level of TNF in TgChBrUD2-infected cells
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was also observed with the combination of sulfadiazine and pyrimethamine when
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compared to azithromycin (P < 0.05) (Fig. 3B).
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Azithromycin and spiramycin treatments in uninfected BeWo cells showed higher MIF
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production than untreated control (Fig 3C). TgChBrUD1-infection increased MIF, the
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same was observed after infection and any drug treatment when compared to control.
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An increased level of MIF in TgChBrUD2-infected cells and in TgChBrUD2-infected
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0.05) (Fig. 3C). Additionally, spiramycin treatment in TgChBrUD2 infected cells
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increased MIF level when compared to TgChBrUD2 infected and untreated cells. On
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the other hand, sulfadiazine and pyrimethamine treatment in TgChBrUD2 infected cells
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reduced MIF production (Fig. 3C).
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All treatments were able to reduced IL-6 production when compared to respective
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control, regardless T. gondii infection (P < 0.05) (Fig. 3D).
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IL-10 production was higher in uninfected and untreated BeWo cells compared to
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treated cells, regardless of the drug treatment (P<0.05) (Fig. 3E). However, when
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infected BeWo cells were treated with antibiotics, the levels of IL-10 significantly
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increased, regardless of the T. gondii strain and drug treatment used compared to each
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uninfected control (P<0.05) (Fig. 3E). On the other hand, TgChBrUD1-infected BeWo
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cells treated with spiramycin reduced significantly IL-10 release if compared to only
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infected cells (Fig. 3E).
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Finally, TGF-β1 production remained unchanged for all experimental conditions (Fig.
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3F).
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ACCEPTED MANUSCRIPT 4. Discussion
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Congenitally acquired toxoplasmosis has been associated with neonatal mortality and
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morbidity. Clinical symptoms observed at different degrees depend on the virulence of
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the parasite strain, the maternal immune response and the gestational age when infection
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occurs (Carlier, et al., 2012). Although the conventional therapy used to treat
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congenitally acquired toxoplasmosis has led to favorable outcomes, it can also cause
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several adverse effects (Montoya and Remington, 2008). In the present study, we
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evaluated the efficiency of using an alternative antibiotic (azithromycin) compared to
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conventional antibiotics (spiramycin and sulfadiazine/pyrimethamine) in human
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trophoblastic cells to control infection caused by Brazilian strains of T. gondii.
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Azithromycin and spiramycin affect BeWo viability at concentration of 600 and 800
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µg/mL, respectively, when the stock solution was prepared in sterile water (Franco, et
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al., 2011). Herein it was demonstrated that concentrations ranging from 50 to 200
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µg/mL of azithromycin, spiramycin or sulfadiazine/pyrimethamine, when diluted in
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DMSO, were not cytotoxic to BeWo cells. These results were due to the high solubility
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of these antibiotics in this vehicle. Thus, it is possible to use BeWo cells as an in vitro
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model for evaluating the effectiveness to use these drugs to treat T. gondii infection.
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In our study, the moderate TgChBrUD1 strain showed higher infection index and
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intracellular proliferation when compared to the virulent TgChBrUD2 strain. In a
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previous in vivo study, we observed that C. callosus females infected by TgChBrUD2
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presented higher parasitism in many types of tissues, when compared to TgChBrUD1-
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infected females (Franco, et al., 2014). However, it was detected higher mortality index
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in females infected by TgChBrUD1. Thus, even TgChBrUD1 has induced lower
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parasitism in these animals, this strain promoted higher mortality (Franco, et al., 2014).
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Probably this phenomenon is due to the exacerbated inflammatory response against
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ACCEPTED MANUSCRIPT TgChBrUD1, promoting high mortality. In addition, surprising results were also found
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by our group when comparing BeWo cells infected by T. gondii clonal high virulent
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strain (RH) or by clonal moderate virulent ones (ME49) (Angeloni, et al., 2009). It was
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observed a lower apoptosis index in BeWo cells infected by T. gondii high virulent RH
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strain when compared to type II moderate ME49 strain. Therefore, our findings about
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low or high infection index in BeWo cells during TgChBrUD2 or TgChBrUD1
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infection, respectively, could be associated with several immune mediators produced by
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these BeWo cells and future studies are necessary to address this hypothesis.
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Treatment with azithromycin reduced the infectivity and proliferation of both T. gondii
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strains included in this study, whereas treatment with spiramycin only decreased the
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infection index of BeWo cells infected with the TgChBrUD1 strain. Interestingly,
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treatment with the combination sulfadiazine/pyrimethamine affected only the
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intracellular proliferation of TgChBrUD2 and not its rate of infectivity. A previous
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study demonstrated that T. gondii strains might be resistant to sulfadiazine (Meneceur,
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et al., 2008). Likewise, the Brazilian strains that were assessed in the present study are
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likely resistant to conventional treatment. The reduction of parasitism that followed
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treatment with azithromycin was caused by the direct action of this drug on the parasites
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themselves, as this antibiotic inhibits protein synthesis by binding to the 50S subunit of
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the pathogen ribosome (Steel, et al., 2012). Both in vitro and in vivo assays have
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demonstrated that azithromycin has potent activity against clonal strains of T. gondii
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and can decrease the replication of the parasite in BeWo cells (Franco, et al., 2011).
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Furthermore, vertical transmission by clonal strains of T. gondii could also be controlled
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with azithromycin treatment in a murine model (Costa, et al., 2009).
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The balance between pro- and anti-inflammatory cytokines is important during the
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gestational period, and the inflammatory responses that are triggered by T. gondii
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ACCEPTED MANUSCRIPT infection could damage the developing fetus (Challis, et al., 2009). In the present study,
323
it was demonstrated that production of the pro-inflammatory cytokine IL-12 in BeWo
324
cells infected with TgChBrUD1 increased after treatment with either azithromycin or
325
spiramycin. This cytokine was also increased in TgChBrUD2-infected cells that were
326
treated with spiramycin. In contrast, TNF production was more closely related to the
327
parasite strain than to the drug treatment, since only TgChBrUD1-infected cells
328
exhibited decreased TNF production, and no changes were observed in cells that were
329
infected with the TgChBrUD2 strain. In addition, infection with both Brazilian strains
330
increased MIF expression.TNF, IL-12 and MIF have central roles in the immune
331
response against T. gondii since these cytokines can active signaling pathways that
332
induce the production of proinflammatory cytokines and are important in controlling the
333
parasite infection (Flores, et al., 2008, Pifer and Yarovinsky, 2011).Thus, reduction of
334
the parasitism after azithromycin treatment observed in the present study seems not to
335
be related with these cytokines and the increased of their levels may be associated with
336
mechanisms of the parasite evasion to the host immune system via the production of
337
virulence factors. Further studies are necessary to confirm this hypothesis. IL-10 and
338
TGF-β1 play important roles during the development of a semi-allogeneic fetus;
339
however, both cytokines have the potential to increase susceptibility to intracellular
340
pathogen infections (Challis, et al., 2009). In our previous study, we demonstrated that
341
T. gondii could invade and replicate within BeWo cells, especially when these cells
342
were stimulated with IL-10 and TGF-β1 (Barbosa, et al., 2008). Thus, the increased
343
production of IL-10 in BeWo cells infected by TgChBrUD1 or TgChBrUD2 strains
344
observed in this study could be related to mechanisms that parasite used to evade hosts
345
cells.
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ACCEPTED MANUSCRIPT IL-6 is expressed throughout pregnancy and plays a pivotal role in embryo implantation,
347
as this cytokine facilitates the growth and invasion of the trophoblast during pregnancy
348
(Sharma, et al., 2016). Furthermore, IL-6 plays an important role during T. gondii
349
infection, as it was demonstrated that BeWo secreted IL-6 even when infected by this
350
parasite (Barbosa, et al., 2015). Previous studies also demonstrated that IL-6 production
351
was significantly reduced after sulfadiazine and macrolides treatment(Bottari, et al.,
352
2015). Thus, our findings are in agreement with these previous studies, since the
353
treatments tested downmodulated IL-6 production, regardless infection.
354
Macrolides have anti-inflammatory and immunomodulatory actions. They impair the
355
activation of pro-inflammatory signaling pathways, such as NF-κB, and they are also
356
able to inhibit the phosphorylation of extracellular signal-regulated kinase (ERK), both
357
of which are important for the expression of pro-inflammatory cytokines (Srivastava, et
358
al., 2011). Our findings showed that azithromycin could inhibit the expression of pro-
359
inflammatory cytokines. Additionally, our results demonstrated that azithromycin could
360
directly affect the parasite itself, reducing the parasitism in BeWo cells.
361
Strain diversity is an evolutionary strategy that can impact biological characteristics that
362
are related to biomedical parameters, such as virulence and drug resistance. The results
363
of several previous studies have indicated that diverse T. gondii strains exist in
364
particular countries, as Brazil (Lopes, et al., 2016, Pena, et al., 2008). However, these
365
studies have primarily focused on genotyping these strains. Experimental models to
366
study specific interactions between the hosts and the parasites of recombinant T. gondii
367
strains have not yet been established, and many details of these interactions need further
368
clarification. In this context, the present study showed that BeWo cells are more
369
susceptible to infection with the TgChBrUD1 T. gondii strain and that this could be
370
related to the decreased production of TNF that is caused by this strain. Additionally,
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ACCEPTED MANUSCRIPT azithromycin treatment was more effective to reduce the infection indexes and
372
intracellular replication of both Brazilian T. gondii strains in trophoblastic cells versus
373
conventional therapy. Therefore, azithromycin might be considered as an appropriate
374
alternative treatment for cases in which congenitally acquired toxoplasmosis is caused
375
by recombinant strains of T. gondii.
376 377
Conflicts of Interest
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All authors have no conflict of interest to declare.
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Acknowledgments
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This work was supported by Brazilian Research Agencies (FAPEMIG, CNPq and
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CAPES).
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ACCEPTED MANUSCRIPT Figure legends
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Fig. 1. Viability of BeWo cells after treatment with increasing concentrations of
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azithromycin (AZ) (A), spiramycin (SPI) (B) or the combination of sulfadiazine and
387
pyrimethamine (SDZ/PYR) (C). Cells were cultured in 96-well-plates (5x104 cells/200
388
µL/well) and after 24 h in culture they were treated with varying concentrations of the
389
drugs. Untreated cells were assessed as controls (100% viability). The numbers of
390
viable cells were analyzed using the MTT assay. Data are expressed as mean values ±
391
SEM of the percentages of viable cells compared to controls and are representative of
392
two independent experiments that were conducted in triplicate. *Comparison between
393
treated cells and control (P<0.05).
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Fig. 2. T. gondii infection rate (A) and intracellular replication (B) in BeWo cells,
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following post-treatment with azithromycin (AZ), spiramycin (SPI) or a
397
sulfadiazine/pyrimethamine combination (SDZ/PYR) and T. gondii infection rate (C)
398
and intracellular replication (D) in BeWo cells infected with pre-treated parasites, Cells
399
were cultured in 24-well plates (5x104 cells/200 µL/well) and infected after 24h in
400
culture with either the TgChBrUD1 or TgChBrUD2 strain of T. gondii (ratio of 3
401
parasites per 1 cell). The infected cells were treated with either 100 µg/mL AZ or 100
402
µg/mL SPI or 100/4 µg/mL SDZ/PYR. After 24h, the cells were fixed and stained by
403
toluidine blue, and the percentage of infected cells and number of intracellular parasites
404
per 200 infected cells were analyzed. Data are expressed as mean values ± SEM and are
405
representative of three independent experiments that were conducted in triplicate.
406
*
407
(P<0.05). #Comparison between TgChBrUD1- and TgChBrUD2-infected cells
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(P<0.05).
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Comparison between infected/treated cells and control (infected/untreated cells)
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ACCEPTED MANUSCRIPT Photomicrographs of BeWo cells after TgChBrUD1 infection (E), TgChBrUD1
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infection and azithromycin treatment (F), TgChBrUD2 infection (G) and TgChBrUD2
411
infection and azithromycin treatment (H). Arrows indicate parasitophorous vacuoles.
412
Toluidine blue staining.
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Fig. 3. IL-12 (A), TNF (B), MIF (C), IL-6 (D), IL-10 (E) and TGF-β (F) measurements
415
by ELISA in cell-free supernatants of BeWo cells that were either not infected or
416
infected with the TgChBrUD1 or TgChBrUD2 strains of T. gondii and subsequently
417
treated with either azithromycin (AZ), spiramycin (SPI) or a combination of
418
sulfadiazine and pyrimethamine (SDZ/PYR). Data are expressed as mean values ± SEM
419
and are representative of two independent experiments that were conducted in triplicate.
420
*
421
(uninfected/untreated or uninfected/treated cells) (P<0.05). #Comparison between
422
different treatments in uninfected or infected cells (P<0.05).
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References
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Franco, P. S., Gomes, A. O., Barbosa, B. F., Angeloni, M. B., Silva, N. M., Teixeira-Carvalho, A., Martins-Filho, O. A., Silva, D. A. O., Mineo, J. R., and Ferro, E. A. V., 2011. Azithromycin and spiramycin induce anti-inflammatory response in human trophoblastic (BeWo) cells infected by Toxoplasma gondii but are able to control infection. Placenta 32, 838-844. Franco, P. S., Ribeiro, M., Lopes-Maria, J. B., Costa, L. F., Silva, D. A. O., de Freitas Barbosa, B., de Oliveira Gomes, A., Mineo, J. R., and Ferro, E. A. V., 2014. Experimental infection of Calomys callosus with atypical strains of Toxoplasma gondii shows gender differences in severity of infection. Parasitology Research 113, 2655-2664. Kaye, A., 2011. Toxoplasmosis: Diagnosis, Treatment, and Prevention in Congenitally Exposed Infants. Journal of Pediatric Health Care 25, 355-364. Lopes, C. S., Franco, P. S., Silva, N. M., Silva, D. A. O., Ferro, E. A. V., Pena, H. F. J., Soares, R. M., Gennari, S. M., and Mineo, J. R., 2016. Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil. Epidemiology and Infection 144, 1865-1875. Meneceur, P., Bouldouyre, M. A., Aubert, D., Villena, I., Menotti, J., Sauvage, V., Garin, J. F., and Derouin, F., 2008. In Vitro Susceptibility of Various Genotypic Strains of Toxoplasma gondii to Pyrimethamine, Sulfadiazine, and Atovaquone. Antimicrobial Agents and Chemotherapy 52, 1269-1277. Montoya, J. G. and Liesenfeld, O., 2004. Toxoplasmosis. The Lancet 363, 19651976. Montoya, Jose G. and Remington, Jack S., 2008. Clinical Practice: Management ofToxoplasma gondiiInfection during Pregnancy. Clinical Infectious Diseases 47, 554-566. Pena, H. F. J., Gennari, S. M., Dubey, J. P., and Su, C., 2008. Population structure and mouse-virulence of Toxoplasma gondii in Brazil. International Journal for Parasitology 38, 561-569. Pifer, R. and Yarovinsky, F., 2011. Innate responses to Toxoplasma gondii in mice and humans. Trends in Parasitology 27, 388-393. Saeij, J. P. J., Boyle, J. P., and Boothroyd, J. C., 2005. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends in Parasitology 21, 476-481. Sharma, S., Godbole, G., and Modi, D., 2016. Decidual Control of Trophoblast Invasion. American Journal of Reproductive Immunology 75, 341-350. Sibley, L. D., Khan, A., Ajioka, J. W., and Rosenthal, B. M., 2009. Genetic diversity of Toxoplasma gondii in animals and humans. Philosophical Transactions of the Royal Society B: Biological Sciences 364, 2749-2761. Srivastava, P., Vardhan, H., Bhengraj, A. R., Jha, R., Singh, L. C., Salhan, S., and Mittal, A., 2011. Azithromycin Treatment Modulates the Extracellular Signal-Regulated Kinase Mediated Pathway and Inhibits Inflammatory Cytokines and Chemokines in Epithelial Cells from Infertile Women with RecurrentChlamydia trachomatisInfection. DNA and Cell Biology 30, 545-554. Steel, H. C., Theron, A. J., Cockeran, R., Anderson, R., and Feldman, C., 2012. Pathogen- and Host-Directed Anti-Inflammatory Activities of Macrolide Antibiotics. Mediators of Inflammation 2012, 1-17.
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*
SDZ/PYR ( µg/mL)
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600/24
80
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0
200/8
400
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Figure 1
Cell viability (%)
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SPI (µg/mL)
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*
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Figure 2 Post-treatment of T. gondii-infect Bewo trophoblast cells B
60 #
#
*
40
Intracellular replication
Infection index (%)
#
* *
20
0 AZ
SPI
TgChBrUD2 #
#
1,000 #
*
*
500
0
SDZ/PYR
Control
AZ
*
*
SPI
SDZ/PYR
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Control
TgChBrUD1
1,500
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A
D
60 #
#
40
#
*
0 Control
SPI
1,000
G
# #
750
#
*
500
* *
*
250
0 Control
SDZ/PYR
F
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E
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*
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Infection index (%)
#
Intracellular replication
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Pre-treatment of T. gondii tachyzoites
H
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Figure 3
A
B
#
50
Medium AZ SPI SDZ/PYR
20
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#
25
#
*
*
#
* §
*
10
*
0
0 TgChBrUD2
#
#
*
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*
*
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E
*
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#
600
#
#
400
# #
200
TgChBrUD2
TgChBrUD1
TgChBrUD2
20
0 Control
F 15000
#
#
TgChBrUD1
40
TgChBrUD2
#
#
IL-6 (pg/ml)
TgChBrUD1
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*
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*
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IL-10 (pg/mL)
# #
TGF-β (pg/mL)
D
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800
Control
Produção de MIF (pg/ml)
C
TgChBrUD1
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#
*
SC
#
#
§
TNF (pg/mL)
IL-12 (pg/mL)
*
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# *
*
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*
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ACCEPTED MANUSCRIPT Highlights
• Azithromycin reduced infection by Brazilian T. gondii genotypes in BeWo cells; • Azithromycin was more effective than classical antibiotic therapy;
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• Azithromycin is an appropriate alternative therapy for congenital toxoplasmosis.
S0014-4894(17)30006-1
DOI:
10.1016/j.exppara.2017.08.004
Reference:
YEXPR 7436
To appear in:
Experimental Parasitology
Received Date: 4 January 2017 Revised Date:
28 July 2017
Accepted Date: 8 August 2017
Please cite this article as: Ribeiro, M., Franco, P.S., Lopes-Maria, J.B., Angeloni, M.B., de Freitas Barbosa, B., de Oliveira Gomes, A., Castro, A.S., da Silva, Rafaela.José., de Oliveira, F.C., Balga Milian, I.C., Martins-Filho, O.A., Ietta, F., Mineo, José.Roberto., Ferro, Eloisa.Amá.Vieira., Azithromycin treatment is able to control the infection by two genotypes of Toxoplasma gondii in human trophoblast BeWo cells, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.08.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT 1
Azithromycin treatment is able to control the infection by two genotypes of
2
Toxoplasma gondii in human trophoblast BeWo cells
3
Mayara Ribeiroa, Priscila Silva Francoa, Janice Buiate Lopes-Mariaa, Mariana Bodini
5
Angelonia, Bellisa de Freitas Barbosaa, Angelica de Oliveira Gomesb, Andressa Silva
6
Castroa, Rafaela José da Silvaa, Fernanda Chaves de Oliveiraa, Iliana Claudia Balga
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Miliana, Olindo Assis Martins-Filhoc, Francesca Iettad, José Roberto Mineoe, Eloisa
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Amália Vieira Ferroa,*
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a
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Embryology, Federal University of Uberlândia, Av. Pará, 1720, Uberlândia, CEP
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38400 902, Brazil
13
b
14
of Uberaba, Rua Frei Paulino, 30, Uberaba, CEP 38025 180, Brazil
15
c
16
Cruz, 30190-002 Belo Horizonte, MG, Brazil
17
d
Department of Life Science, University of Siena, Aldo Moro Road 2, Siena, Italy
18
e
Laboratory of Immunoparasitology, Department of Immunology, Microbiology and
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Parasitology, Federal University of Uberlândia, Av. Pará, 1720, Uberlândia, CEP
20
38400 902, Brazil
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Departament of Structrual Biology, Institute of Biological Science, Federal University
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Laboratory of Chagas Disease, René Rachou Research Center, Fundação Oswaldo
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Laboratory of Immunophysiology of Reproduction, Department of Histology and
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* Corresponding author. Address: Laboratory of Immunophysiology of Reproduction,
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Department of Histology and Embryology, Federal University of Uberlândia, Av. Pará,
24
1720, Uberlândia, CEP 38400 902, Brazil. Tel.: +55 34 3218 2240, Fax: + 55 34 3218
25
2333. E-mail addresses: [email protected]
2
ACCEPTED MANUSCRIPT ABSTRACT
27
Trophoblast infection by Toxoplasma gondii plays a pivotal role in the vertical
28
transmission of toxoplasmosis. Here, we investigate whether the antibiotic therapy with
29
azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control
30
trophoblast infection by two Brazilian T. gondii genotypes, TgChBrUD1 or
31
TgChBrUD2. Two antibiotic protocols were evaluated, as follow: i) pre-treatment of T.
32
gondii-tachyzoites with selected antibiotics prior trophoblast infection and ii) post-
33
treatment of infected trophoblasts. The infection index/replication and the impact of the
34
antibiotic therapy on the cytokine milieu were characterized. It was observed that
35
TgChBrUD2 infection induced lower infection index/replication as compared to
36
TgChBrUD1. Regardless the therapeutic protocol, azithromycin was more effective to
37
control the trophoblast infection with both genotypes when compared to conventional
38
antibiotics. Azithromycin induced higher IL-12 production in TgChBrUD1-infected
39
cells that may synergize the anti-parasitic effect. In contrast, the effectiveness of
40
azithromycin to control the TgChBrUD2-infection was not associated with the IL-12
41
production. BeWo-trophoblasts display distinct susceptibility to T. gondii genotypes and
42
the azithromycin treatment showed to be more effective than conventional antibiotics to
43
control the T. gondii infection/replication regardless the parasite genotype.
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Keywords: Trophoblast cell; Azithromycin; Toxoplasma gondii atypical strains;
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Cytokines
47
3
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49
Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects
50
a wide range of warm-blooded animals, including humans. Toxoplasmosis is typically
51
asymptomatic in immunocompetent hosts, but the parasite is able to induce severe
52
illness in immunocompromised patients and congenitally infected individuals (Dupont,
53
et al., 2012, Ferguson, et al., 2012). There are three well-characterized lineages of T.
54
gondii, known as types I, II and III. These lineages are predominantly found in Europe
55
and North America, and each of them is constituted by a genotype that presents a high
56
degree of clonality (Saeij, et al., 2005). In contrast, a different pattern is observed in
57
South America and Africa, which are populated by lineages of T. gondii that have more
58
diverse genotypes (Sibley, et al., 2009). In Brazil, there is high genetic diversity of T.
59
gondii, including recombinant genotypes, and the three most common genotypes are #6,
60
#8 and #11, previously designated as type BrI, BrIII and BrII, respectively (Carneiro, et
61
al., 2013, Pena, et al., 2008). An analysis of mortality rates in infected mice indicated
62
that Type BrI lineages are highly virulent, whereas Type BrIII are non-virulent and
63
Type BrII are moderately virulent(Pena, et al., 2008). In Uberlândia city (Minas Gerais
64
state, Brazil), two parasite isolates were found and named as TgChBrUD1 and
65
TgChBrUD2. The TgChBrUD1 isolate had ToxoDB PCR-RFLP genotype #11 (also
66
known as type BrII), and the TgChBrUD2 isolate had ToxoDB PCR-RFLP genotype #6
67
(also known as type BrI Africa 1) (Lopes, et al., 2016). An in vivo study using Calomys
68
callosus as a rodent model demonstrated that these Brazilian isolates were highly
69
virulent for this host, and significant differences in the severity of infection related to
70
the gender were observed (Franco, et al., 2014). The treatment of toxoplasmosis is
71
indicated for pregnant and immunocompromised individuals (Montoya and Liesenfeld,
72
2004). In women who are infected during pregnancy, spiramycin is used to prevent fetal
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74
its low penetration of fetal tissues (Kaye, 2011). If fetal infection is confirmed, the
75
mother should be treated with a combination of sulfadiazine, pyrimethamine and folinic
76
acid (Montoya and Remington, 2008). Sulfadiazine and pyrimethamine are widely used,
77
but these drugs are toxic and may cause adverse effects (Kaye, 2011, Montoya and
78
Remington, 2008). In addition to causing side effects, these drugs might not be able to
79
reduce the parasitism, as T. gondii has shown resistance to treatment with sulfadiazine
80
in in vitro studies (Meneceur, et al., 2008).
81
Both azithromycin and spiramycin belong to the macrolide group, a class of broad-
82
spectrum antibiotics used to treat bacterial and intracellular parasite infections
83
(Srivastava, et al., 2011). Macrolides lead to anti-inflammatory activity by inhibiting the
84
synthesis of microbial toxins and other virulence factors (Steel, et al., 2012). Several in
85
vivo and in vitro studies that used azithromycin to treat congenitally acquired
86
toxoplasmosis were able to demonstrate reductions in T. gondii replication following
87
treatment (Costa, et al., 2009, Franco, et al., 2011). Previous studies from our group
88
have indicated that treatment with azithromycin is effective against T. gondii for both
89
rodents and human cells that were infected with the virulent RH strain (type I) (Costa, et
90
al., 2009, Franco, et al., 2011).
91
BeWo cells are derived from a human choriocarcinoma and they have been
92
characterized as an appropriate in vitro model of human trophoblast to investigate T.
93
gondii infection in the maternal fetal interface by our group. In fact, we have
94
investigated several aspects of the immunology of pregnancy using these cells as a
95
trophoblast model (Barbosa, et al., 2015, Franco, et al., 2011).
96
The aim of the present study was to verify whether the antibiotic therapy with
97
azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control the T.
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(TgChBrUD1/genotype#11/(type BrII) or TgChBrUD2/genotype#6/(type BrI/Africa1)
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and change the immunological microenvironment, using this well-established model of
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in vitro infection of BeWo human trophoblast cell lineage.
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2.1. Cell culture and parasites
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BeWo trophoblast cells were obtained from the American Type Culture Collection
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(ATCC, Manassas, VA, USA) and cultured in RPMI-1640 media (Cultilab, Campinas,
106
SP, Brazil) that was supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin
107
(Sigma Chemical CO., St. Louis, USA) and 10% fetal bovine serum (Cultilab). The
108
cells were maintained in a humidified incubator at 37°C and 5% CO2.
109
Tachyzoites of the TgChBrUD1/genotype#11/(type BrII) and
110
TgChBrUD2/genotype#6/(type BrI/Africa1) strains of T. gondii were maintained by
111
serial passage in BeWo cells to obtain parasites in vitro for further experimental
112
infections. The media that contained free tachyzoites was transferred to 15 mL tubes
113
and centrifuged (400×g, 5min) at room temperature. Pellets were suspended in complete
114
media, and the parasites were stained by Trypan blue and counted in a hemocytometer
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to establish infectious dose.
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2.2. Drugs
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Azithromycin (Biofarma, Uberlândia, MG, Brazil), spiramycin (Sigma), sulfadiazine
119
(Sigma) and pyrimethamine (Sigma) were each prepared as a stock solution in DMSO
120
at a concentration of 3.000 µg/mL. The stock solutions were diluted in complete media,
121
and cells were treated with varying concentrations of the drugs, which were chosen
122
based on previous studies (Franco, et al., 2011, Meneceur, et al., 2008).
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2.3. Drug treatments and cell viability
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Cell viability was evaluated using a tetrazolium salt colorimetric (MTT) assay. For this
126
purpose, BeWo cells were cultured for 24 h in 96-well plates (5x104 cells/well/200 µL)
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ACCEPTED MANUSCRIPT at 37°C and 5% CO2.Then the cells were incubated with 200 µL/well of an antibiotic
128
solution containing azithromycin or spiramycin in different concentrations (50, 100, 200
129
or 400 µg/mL). For treatments using a combination of sulfadiazine and pyrimethamine,
130
different ratios were selected, which included 50:1, 100:4, 200:8, 400:16, 600:24,
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800:32 or 1,000:40 µg/mL of sulfadiazine:pyrimethamine. As control, BeWo cells were
132
not treated and only medium was added in the wells. After 24 h, the supernatants were
133
removed, and the cells were washed and pulsed with 10 µL of MTT (Sigma). The plates
134
were incubated under standard culture conditions; after 3 h, the formazan crystals that
135
were produced by viable cells were solubilized by adding a solution of 10% sodium
136
dodecyl sulfate (SDS) and 50% N, N-dimethylformamide. Optical density was
137
determined at 570 nm using a plate reader (Titertek MultiskanPlus, Flow Laboratories,
138
McLean, USA), and the results were expressed as percentage of viable cells relative to
139
control (100%). Two independent experiments were performed in triplicate for each
140
condition.
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2.4. Treatments and infection of BeWo cells
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BeWo cells were cultured on 13-mm glass coverslips in 24-well plates (5 x 104
144
cells/well/200 µL) for 24 h at 37°C and 5% CO2. The cells were infected with
145
tachyzoites of T. gondii (TgChBrUD1 or TgChBrUD2) at a ratio of three parasites per
146
cell. After 24 h, the cells were treated with 100 µg/mL of azithromycin, 100 µg/mL of
147
spiramycin or 100 µg/mL of sulfadiazine in combination with 4 µg/mL of
148
pyrimethamine. As control, BeWo cells were infected and not treated, or uninfected and
149
untreated. After 24 h of treatment, the supernatants were collected and stored at -80°C
150
for cytokines measurement. The cells were fixed in 10% buffered formalin for 24 h,
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washed with phosphate-buffered saline (PBS) and stained with 1% toluidine blue for 3
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microscopy to assess infection index (percentage of infected cells per 200 examined
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cells) and intracellular replication (mean number of parasites in 200 infected cells).26
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Three independent experiments were performed in triplicate for each condition.
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2.5. Treatments of T. gondii tachyzoites
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Free tachyzoites (1.5x105 parasites/well) were pre-treated with azithromycin (100
159
µg/mL), spiramycin (100 µg/mL) or the combination of sulfadiazine and pyrimethamine
160
(100 and 4 µg/mL, respectively) for 3 h at 37°C and 5% CO2. Next, the cells were
161
infected with pre-treated parasites for 24 h and analyzed using light microscopy to
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assess intracellular replication and infection index, as described above. As control, T.
163
gondii tachyzoites were not treated. Two independent experiments were performed in
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triplicate for each condition.
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2.6. Cytokine measurements
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Levels of IL-12p70, TNF, MIF, IL-6, IL-10 and TGF-β1 cytokines were measured in
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supernatants of untreated or treated cells using commercially available
169
immunoenzymatic kits (BD Biosciences, New Jersey, USA and R&D Systems,
170
Minneapolis, USA) according to the manufacturer’s recommendations. The results were
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expressed in pg/mL according to the standard curve.
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2.7. Statistical analysis
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All data were expressed as mean values ± SEM and differences between groups were
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assessed using One-Way ANOVA and either the Bonferroni or Dunnet post-test or
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Student’s t test when appropriate. All data were analyzed using GraphPad Prism 5.0
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ACCEPTED MANUSCRIPT 177
(GraphPad Software Inc., San Diego, USA). Differences were considered statistically
178
significant when P < 0.05.
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3.1. The selected antibiotics induced minimal toxicity rate in BeWo cells when applied
182
at low concentrations
183
Azithromycin and spiramycin were not cytotoxic when added to BeWo cells up to a
184
concentration of 200 µg/mL; however, they significantly decreased cell viability at 400
185
µg/mL (P<0.05) (Fig. 1A-B). Additionally, the combination of sulfadiazine and
186
pyrimethamine was not cytotoxic up to concentrations of 200 µg/mL and 8 µg/mL,
187
respectively, and a significant reduction in cell viability was observed when cells were
188
treated within a range that included 400 µg/mL of sulfadiazine plus 16 µg/mL of
189
pyrimethamine to 1,000 µg/mL of sulfadiazine plus 40 µg/mL of pyrimethamine (Fig.
190
1C). Based on these findings, further experiments were carried out using either 100
191
µg/mL of azithromycin or spiramycin or 100 µg/mL of sulfadiazine plus 4 µg/mL of
192
pyrimethamine.
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3.2. Azithromycin was more effective to control the trophoblast infection with distinct T.
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gondii genotypes as compared to conventional antibiotic therapy
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The infection index and intracellular replication of T. gondii were determined in BeWo
197
cells that were treated with antibiotics (Fig. 2A-F). In comparing the TgChBrUD1 and
198
TgChBrUD2 strains, it was found that exposure to TgChBrUD1 led to a higher
199
percentage of infected BeWo cells (Fig. 2A, C, E) and a higher number of intracellular
200
tachyzoites (Fig. 2B, C, E) (P<0.05). Treatment with either azithromycin or the
201
combination of sulfadiazine and pyrimethamine reduced both infection index and
202
intracellular replication in BeWo cells that were infected with TgChBrUD2 versus
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TgChBrUD1 (P<0.05) (Fig. 2A-B). Azithromycin was able to reduce the infection
204
indexes and intracellular replication of both T. gondii strains in treated BeWo cells
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ACCEPTED MANUSCRIPT versus untreated cells (P<0.05) (Fig 2A-F), whereas treatment with spiramycin
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produced this effect only in TgChBrUD1-infected cells (P<0.05) (Fig. 2A-B). In
207
contrast, treating cells with a combination of sulfadiazine and pyrimethamine was able
208
to reduce the intracellular replication of only the TgChBrUD2 strain when compared to
209
untreated cells (P < 0.05) (Fig. 2A-B).
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3.3 Pre-treatment of T. gondii tachyzoites with azithromycin is also able to control the
212
trophoblast infection
213
In order to verify the direct effect of drugs on T. gondii infection in BeWo cells, we pre-
214
treated the parasites with the selected drugs before infect the cells. Our data showed that
215
only azithromycin reduced the percentage of infected BeWo cells and the number of
216
intracellular tachyzoites for both T. gondii strains, as compared to untreated parasites
217
(P<0.05) (Fig. 2C-D). Pre-treating parasites with either spiramycin or the combination
218
of sulfadiazine and pyrimethamine significantly reduced TgChBrUD2 replication
219
compared to untreated parasites (Fig. 2D). When comparing TgChBrUD1 and
220
TgChBrUD2, a higher infection index and number of intracellular tachyzoites was
221
found in TgChBrUD1-infected cells, regardless of treatment (P<0.05) (Fig. 2C-D).
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3.4. Azithromycin induced higher IL-12 production in TgChBrUD1-infected cells that
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may synergize the anti-parasitic effect
225
Cytokine production was evaluated in supernatants of BeWo cells that were infected
226
with T. gondii and treated with azithromycin, spiramycin or the combination of
227
sulfadiazine and pyrimethamine in comparison with controls (Fig. 3). IL-12 production
228
was not significantly changed by any treatment in uninfected cells (Fig. 3A). Infection
229
with TgChBrUD1 resulted in a significant increase in IL-12 production in untreated
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231
treated with azithromycin and spiramycin compared to untreated control. TgChBrUD1-
232
infected BeWo cells that were either untreated or treated with azithromycin had higher
233
levels of IL-12 production than cells treated with a combination of sulfadiazine and
234
pyrimethamine (Fig. 3A). Combination of sulfadiazine and pyrimethamine was not able
235
to change IL-12 production compared to untreated control. Additionally, infection with
236
TgChBrUD2 resulted in a significantly increased level of IL-12 production in both
237
untreated and spiramycin-treated cells compared to controls (P<0.05). However, after
238
treatment with spiramycin, there was a significant increase in IL-12 regardless of the T.
239
gondii strain used if compared to untreated controls (Fig. 3A).
240
Uninfected BeWo cells had similar levels of TNF even after each of treatments (Fig.
241
3B). When BeWo cells were infected with TgChBrUD1, TNF levels were reduced, even
242
after azithromycin treatment when compared to uninfected and azithromycin-treated
243
cells respectively, similar to what was observed in cells that were infected with this
244
strain but remained untreated. In contrast, TgChBrUD2-infected BeWo cells did not
245
show significant changes in TNF production after any treatment conditions, including
246
untreated cells, compared to controls (Fig.3B). TgChBrUD2-infected cells when treated
247
with combination of sulfadiazine and pyrimethamine showed higher TNF level if
248
compared to untreated control. An increased level of TNF in TgChBrUD2-infected cells
249
was also observed with the combination of sulfadiazine and pyrimethamine when
250
compared to azithromycin (P < 0.05) (Fig. 3B).
251
Azithromycin and spiramycin treatments in uninfected BeWo cells showed higher MIF
252
production than untreated control (Fig 3C). TgChBrUD1-infection increased MIF, the
253
same was observed after infection and any drug treatment when compared to control.
254
An increased level of MIF in TgChBrUD2-infected cells and in TgChBrUD2-infected
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256
0.05) (Fig. 3C). Additionally, spiramycin treatment in TgChBrUD2 infected cells
257
increased MIF level when compared to TgChBrUD2 infected and untreated cells. On
258
the other hand, sulfadiazine and pyrimethamine treatment in TgChBrUD2 infected cells
259
reduced MIF production (Fig. 3C).
260
All treatments were able to reduced IL-6 production when compared to respective
261
control, regardless T. gondii infection (P < 0.05) (Fig. 3D).
262
IL-10 production was higher in uninfected and untreated BeWo cells compared to
263
treated cells, regardless of the drug treatment (P<0.05) (Fig. 3E). However, when
264
infected BeWo cells were treated with antibiotics, the levels of IL-10 significantly
265
increased, regardless of the T. gondii strain and drug treatment used compared to each
266
uninfected control (P<0.05) (Fig. 3E). On the other hand, TgChBrUD1-infected BeWo
267
cells treated with spiramycin reduced significantly IL-10 release if compared to only
268
infected cells (Fig. 3E).
269
Finally, TGF-β1 production remained unchanged for all experimental conditions (Fig.
270
3F).
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ACCEPTED MANUSCRIPT 4. Discussion
273
Congenitally acquired toxoplasmosis has been associated with neonatal mortality and
274
morbidity. Clinical symptoms observed at different degrees depend on the virulence of
275
the parasite strain, the maternal immune response and the gestational age when infection
276
occurs (Carlier, et al., 2012). Although the conventional therapy used to treat
277
congenitally acquired toxoplasmosis has led to favorable outcomes, it can also cause
278
several adverse effects (Montoya and Remington, 2008). In the present study, we
279
evaluated the efficiency of using an alternative antibiotic (azithromycin) compared to
280
conventional antibiotics (spiramycin and sulfadiazine/pyrimethamine) in human
281
trophoblastic cells to control infection caused by Brazilian strains of T. gondii.
282
Azithromycin and spiramycin affect BeWo viability at concentration of 600 and 800
283
µg/mL, respectively, when the stock solution was prepared in sterile water (Franco, et
284
al., 2011). Herein it was demonstrated that concentrations ranging from 50 to 200
285
µg/mL of azithromycin, spiramycin or sulfadiazine/pyrimethamine, when diluted in
286
DMSO, were not cytotoxic to BeWo cells. These results were due to the high solubility
287
of these antibiotics in this vehicle. Thus, it is possible to use BeWo cells as an in vitro
288
model for evaluating the effectiveness to use these drugs to treat T. gondii infection.
289
In our study, the moderate TgChBrUD1 strain showed higher infection index and
290
intracellular proliferation when compared to the virulent TgChBrUD2 strain. In a
291
previous in vivo study, we observed that C. callosus females infected by TgChBrUD2
292
presented higher parasitism in many types of tissues, when compared to TgChBrUD1-
293
infected females (Franco, et al., 2014). However, it was detected higher mortality index
294
in females infected by TgChBrUD1. Thus, even TgChBrUD1 has induced lower
295
parasitism in these animals, this strain promoted higher mortality (Franco, et al., 2014).
296
Probably this phenomenon is due to the exacerbated inflammatory response against
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ACCEPTED MANUSCRIPT TgChBrUD1, promoting high mortality. In addition, surprising results were also found
298
by our group when comparing BeWo cells infected by T. gondii clonal high virulent
299
strain (RH) or by clonal moderate virulent ones (ME49) (Angeloni, et al., 2009). It was
300
observed a lower apoptosis index in BeWo cells infected by T. gondii high virulent RH
301
strain when compared to type II moderate ME49 strain. Therefore, our findings about
302
low or high infection index in BeWo cells during TgChBrUD2 or TgChBrUD1
303
infection, respectively, could be associated with several immune mediators produced by
304
these BeWo cells and future studies are necessary to address this hypothesis.
305
Treatment with azithromycin reduced the infectivity and proliferation of both T. gondii
306
strains included in this study, whereas treatment with spiramycin only decreased the
307
infection index of BeWo cells infected with the TgChBrUD1 strain. Interestingly,
308
treatment with the combination sulfadiazine/pyrimethamine affected only the
309
intracellular proliferation of TgChBrUD2 and not its rate of infectivity. A previous
310
study demonstrated that T. gondii strains might be resistant to sulfadiazine (Meneceur,
311
et al., 2008). Likewise, the Brazilian strains that were assessed in the present study are
312
likely resistant to conventional treatment. The reduction of parasitism that followed
313
treatment with azithromycin was caused by the direct action of this drug on the parasites
314
themselves, as this antibiotic inhibits protein synthesis by binding to the 50S subunit of
315
the pathogen ribosome (Steel, et al., 2012). Both in vitro and in vivo assays have
316
demonstrated that azithromycin has potent activity against clonal strains of T. gondii
317
and can decrease the replication of the parasite in BeWo cells (Franco, et al., 2011).
318
Furthermore, vertical transmission by clonal strains of T. gondii could also be controlled
319
with azithromycin treatment in a murine model (Costa, et al., 2009).
320
The balance between pro- and anti-inflammatory cytokines is important during the
321
gestational period, and the inflammatory responses that are triggered by T. gondii
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ACCEPTED MANUSCRIPT infection could damage the developing fetus (Challis, et al., 2009). In the present study,
323
it was demonstrated that production of the pro-inflammatory cytokine IL-12 in BeWo
324
cells infected with TgChBrUD1 increased after treatment with either azithromycin or
325
spiramycin. This cytokine was also increased in TgChBrUD2-infected cells that were
326
treated with spiramycin. In contrast, TNF production was more closely related to the
327
parasite strain than to the drug treatment, since only TgChBrUD1-infected cells
328
exhibited decreased TNF production, and no changes were observed in cells that were
329
infected with the TgChBrUD2 strain. In addition, infection with both Brazilian strains
330
increased MIF expression.TNF, IL-12 and MIF have central roles in the immune
331
response against T. gondii since these cytokines can active signaling pathways that
332
induce the production of proinflammatory cytokines and are important in controlling the
333
parasite infection (Flores, et al., 2008, Pifer and Yarovinsky, 2011).Thus, reduction of
334
the parasitism after azithromycin treatment observed in the present study seems not to
335
be related with these cytokines and the increased of their levels may be associated with
336
mechanisms of the parasite evasion to the host immune system via the production of
337
virulence factors. Further studies are necessary to confirm this hypothesis. IL-10 and
338
TGF-β1 play important roles during the development of a semi-allogeneic fetus;
339
however, both cytokines have the potential to increase susceptibility to intracellular
340
pathogen infections (Challis, et al., 2009). In our previous study, we demonstrated that
341
T. gondii could invade and replicate within BeWo cells, especially when these cells
342
were stimulated with IL-10 and TGF-β1 (Barbosa, et al., 2008). Thus, the increased
343
production of IL-10 in BeWo cells infected by TgChBrUD1 or TgChBrUD2 strains
344
observed in this study could be related to mechanisms that parasite used to evade hosts
345
cells.
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ACCEPTED MANUSCRIPT IL-6 is expressed throughout pregnancy and plays a pivotal role in embryo implantation,
347
as this cytokine facilitates the growth and invasion of the trophoblast during pregnancy
348
(Sharma, et al., 2016). Furthermore, IL-6 plays an important role during T. gondii
349
infection, as it was demonstrated that BeWo secreted IL-6 even when infected by this
350
parasite (Barbosa, et al., 2015). Previous studies also demonstrated that IL-6 production
351
was significantly reduced after sulfadiazine and macrolides treatment(Bottari, et al.,
352
2015). Thus, our findings are in agreement with these previous studies, since the
353
treatments tested downmodulated IL-6 production, regardless infection.
354
Macrolides have anti-inflammatory and immunomodulatory actions. They impair the
355
activation of pro-inflammatory signaling pathways, such as NF-κB, and they are also
356
able to inhibit the phosphorylation of extracellular signal-regulated kinase (ERK), both
357
of which are important for the expression of pro-inflammatory cytokines (Srivastava, et
358
al., 2011). Our findings showed that azithromycin could inhibit the expression of pro-
359
inflammatory cytokines. Additionally, our results demonstrated that azithromycin could
360
directly affect the parasite itself, reducing the parasitism in BeWo cells.
361
Strain diversity is an evolutionary strategy that can impact biological characteristics that
362
are related to biomedical parameters, such as virulence and drug resistance. The results
363
of several previous studies have indicated that diverse T. gondii strains exist in
364
particular countries, as Brazil (Lopes, et al., 2016, Pena, et al., 2008). However, these
365
studies have primarily focused on genotyping these strains. Experimental models to
366
study specific interactions between the hosts and the parasites of recombinant T. gondii
367
strains have not yet been established, and many details of these interactions need further
368
clarification. In this context, the present study showed that BeWo cells are more
369
susceptible to infection with the TgChBrUD1 T. gondii strain and that this could be
370
related to the decreased production of TNF that is caused by this strain. Additionally,
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ACCEPTED MANUSCRIPT azithromycin treatment was more effective to reduce the infection indexes and
372
intracellular replication of both Brazilian T. gondii strains in trophoblastic cells versus
373
conventional therapy. Therefore, azithromycin might be considered as an appropriate
374
alternative treatment for cases in which congenitally acquired toxoplasmosis is caused
375
by recombinant strains of T. gondii.
376 377
Conflicts of Interest
378
All authors have no conflict of interest to declare.
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Acknowledgments
381
This work was supported by Brazilian Research Agencies (FAPEMIG, CNPq and
382
CAPES).
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ACCEPTED MANUSCRIPT Figure legends
385
Fig. 1. Viability of BeWo cells after treatment with increasing concentrations of
386
azithromycin (AZ) (A), spiramycin (SPI) (B) or the combination of sulfadiazine and
387
pyrimethamine (SDZ/PYR) (C). Cells were cultured in 96-well-plates (5x104 cells/200
388
µL/well) and after 24 h in culture they were treated with varying concentrations of the
389
drugs. Untreated cells were assessed as controls (100% viability). The numbers of
390
viable cells were analyzed using the MTT assay. Data are expressed as mean values ±
391
SEM of the percentages of viable cells compared to controls and are representative of
392
two independent experiments that were conducted in triplicate. *Comparison between
393
treated cells and control (P<0.05).
394
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Fig. 2. T. gondii infection rate (A) and intracellular replication (B) in BeWo cells,
396
following post-treatment with azithromycin (AZ), spiramycin (SPI) or a
397
sulfadiazine/pyrimethamine combination (SDZ/PYR) and T. gondii infection rate (C)
398
and intracellular replication (D) in BeWo cells infected with pre-treated parasites, Cells
399
were cultured in 24-well plates (5x104 cells/200 µL/well) and infected after 24h in
400
culture with either the TgChBrUD1 or TgChBrUD2 strain of T. gondii (ratio of 3
401
parasites per 1 cell). The infected cells were treated with either 100 µg/mL AZ or 100
402
µg/mL SPI or 100/4 µg/mL SDZ/PYR. After 24h, the cells were fixed and stained by
403
toluidine blue, and the percentage of infected cells and number of intracellular parasites
404
per 200 infected cells were analyzed. Data are expressed as mean values ± SEM and are
405
representative of three independent experiments that were conducted in triplicate.
406
*
407
(P<0.05). #Comparison between TgChBrUD1- and TgChBrUD2-infected cells
408
(P<0.05).
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Comparison between infected/treated cells and control (infected/untreated cells)
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ACCEPTED MANUSCRIPT Photomicrographs of BeWo cells after TgChBrUD1 infection (E), TgChBrUD1
410
infection and azithromycin treatment (F), TgChBrUD2 infection (G) and TgChBrUD2
411
infection and azithromycin treatment (H). Arrows indicate parasitophorous vacuoles.
412
Toluidine blue staining.
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Fig. 3. IL-12 (A), TNF (B), MIF (C), IL-6 (D), IL-10 (E) and TGF-β (F) measurements
415
by ELISA in cell-free supernatants of BeWo cells that were either not infected or
416
infected with the TgChBrUD1 or TgChBrUD2 strains of T. gondii and subsequently
417
treated with either azithromycin (AZ), spiramycin (SPI) or a combination of
418
sulfadiazine and pyrimethamine (SDZ/PYR). Data are expressed as mean values ± SEM
419
and are representative of two independent experiments that were conducted in triplicate.
420
*
421
(uninfected/untreated or uninfected/treated cells) (P<0.05). #Comparison between
422
different treatments in uninfected or infected cells (P<0.05).
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Angeloni, M. B., Silva, N. M., Castro, A. S., Gomes, A. O., Silva, D. A. O., Mineo, J. R., and Ferro, E. A. V., 2009. Apoptosis and S Phase of the Cell Cycle in BeWo Trophoblastic and HeLa Cells are Differentially Modulated by Toxoplasma gondii Strain Types. Placenta 30, 785-791. Barbosa, B. F., Lopes-Maria, J. B., Gomes, A. O., Angeloni, M. B., Castro, A. S., Franco, P. S., Fermino, M. L., Roque-Barreira, M. C., Ietta, F., MartinsFilho, O. A., Silva, D. A. O., Mineo, J. R., and Ferro, E. A. V., 2015. IL10, TGF Beta1, and IFN Gamma Modulate Intracellular Signaling Pathways and Cytokine Production to Control Toxoplasma gondii Infection in BeWo Trophoblast Cells. Biology of Reproduction 92, 82-82. Barbosa, B. F., Silva, D. A. O., Costa, I. N., Mineo, J. R., and Ferro, E. A. V., 2008. BeWo trophoblast cell susceptibility to Toxoplasma gondii is increased by interferon-γ, interleukin-10 and transforming growth factor-β1. Clinical & Experimental Immunology 151, 536-545. Bottari, N. B., Baldissera, M. D., Tonin, A. A., Rech, V. C., Nishihira, V. S. K., Thomé, G. R., Camillo, G., Vogel, F. F., Duarte, M. M. M. F., Schetinger, M. R. C., Morsch, V. M., Tochetto, C., Fighera, R., and Da Silva, A. S., 2015. Effects of sulfamethoxazole-trimethoprim associated to resveratrol on its free form and complexed with 2-hydroxypropyl-β-cyclodextrin on cytokines levels of mice infected by Toxoplasma gondii. Microbial Pathogenesis 87, 40-44. Carlier, Y., Truyens, C., Deloron, P., and Peyron, F., 2012. Congenital parasitic infections: A review. Acta Tropica 121, 55-70. Carneiro, A. C. A. V., Andrade, G. M., Costa, J. G. L., Pinheiro, B. V., Vasconcelos-Santos, D. V., Ferreira, A. M., Su, C., Januário, J. N., and Vitor, R. W. A., 2013. Genetic Characterization of Toxoplasma gondii Revealed Highly Diverse Genotypes for Isolates from Newborns with Congenital Toxoplasmosis in Southeastern Brazil. Journal of Clinical Microbiology 51, 901-907. Challis, J. R., Lockwood, C. J., Myatt, L., Norman, J. E., Strauss, J. F., and Petraglia, F., 2009. Inflammation and Pregnancy. Reproductive Sciences 16, 206-215. Costa, I. N., Angeloni, M. B., Santana, L. A., Barbosa, B. F., Silva, M. C. P., Rodrigues, A. A., Rostkowsa, C., Magalhães, P. M., Pena, J. D. O., Silva, D. A. O., Mineo, J. R., and Ferro, E. A. V., 2009. Azithromycin Inhibits Vertical Transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae). Placenta 30, 884-890. Dupont, C. D., Christian, D. A., and Hunter, C. A., 2012. Immune response and immunopathology during toxoplasmosis. Seminars in Immunopathology 34, 793-813. Ferguson, D. J. P., Bowker, C., Jeffery, K. J. M., Chamberlain, P., and Squier, W., 2012. Congenital Toxoplasmosis: Continued Parasite Proliferation in the Fetal Brain Despite Maternal Immunological Control in Other Tissues. Clinical Infectious Diseases 56, 204-208. Flores, M., Saavedra, R., Bautista, R., Viedma, R., Tenorio, E. P., Leng, L., Sanchez, Y., Juarez, I., Satoskar, A. A., Shenoy, A. S., Terrazas, L. I., Bucala, R., Barbi, J., Satoskar, A. R., and Rodriguez-Sosa, M., 2008. Macrophage migration inhibitory factor (MIF) is critical for the host resistance against Toxoplasma gondii. The FASEB Journal 22, 3661-3671.
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EP
12.
AC C
472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517
40
*
SDZ/PYR ( µg/mL)
1,000/40
800/32
60
600/24
80
400/16
0
200/8
400
200
100
40
50
60
100/4
80
M AN U
100
50/1
B
Control
TE D Cell viability (%)
SC
AZ (µg/mL)
400
RI PT 200
100
50
Control
0
Control
EP
Cell viability (%)
Figure 1
Cell viability (%)
AC C
ACCEPTED MANUSCRIPT A 100 *
50
*
20
SPI (µg/mL)
C
100
*
*
*
20
0
ACCEPTED MANUSCRIPT
Figure 2 Post-treatment of T. gondii-infect Bewo trophoblast cells B
60 #
#
*
40
Intracellular replication
Infection index (%)
#
* *
20
0 AZ
SPI
TgChBrUD2 #
#
1,000 #
*
*
500
0
SDZ/PYR
Control
AZ
*
*
SPI
SDZ/PYR
SC
Control
TgChBrUD1
1,500
RI PT
A
D
60 #
#
40
#
*
0 Control
SPI
1,000
G
# #
750
#
*
500
* *
*
250
0 Control
SDZ/PYR
F
EP
E
AZ
TE D
*
20
AC C
Infection index (%)
#
Intracellular replication
C
M AN U
Pre-treatment of T. gondii tachyzoites
H
AZ
SPI
SDZ/PYR
ACCEPTED MANUSCRIPT
Figure 3
A
B
#
50
Medium AZ SPI SDZ/PYR
20
RI PT
#
25
#
*
*
#
* §
*
10
*
0
0 TgChBrUD2
#
#
*
25
*
*
0
E
*
AC C
#
600
#
#
400
# #
200
TgChBrUD2
TgChBrUD1
TgChBrUD2
20
0 Control
F 15000
#
#
TgChBrUD1
40
TgChBrUD2
#
#
IL-6 (pg/ml)
TgChBrUD1
§
*
EP
Control
*
TE D
IL-10 (pg/mL)
# #
TGF-β (pg/mL)
D
50
800
Control
Produção de MIF (pg/ml)
C
TgChBrUD1
M AN U
Control
#
*
SC
#
#
§
TNF (pg/mL)
IL-12 (pg/mL)
*
10000
# #
#
*
* *
# *
*
5000
*
0
0 Control
TgChBrUD1
TgChBrUD2
Control
TgChBrUD1
TgChBrUD2
ACCEPTED MANUSCRIPT Highlights
• Azithromycin reduced infection by Brazilian T. gondii genotypes in BeWo cells; • Azithromycin was more effective than classical antibiotic therapy;
AC C
EP
TE D
M AN U
SC
RI PT
• Azithromycin is an appropriate alternative therapy for congenital toxoplasmosis.