Cytotoxic effects of Shiga toxin-2 on human extravillous trophoblast cell lines

in Reproduction
Correspondence should be addressed to F Sacerdoti; Email: flasacerdoti@gmail.com

Shiga toxin (Stx2) producing Escherichia coli infections during early gestation may impair placentation through a Stx2 damage of extravillous trophoblast (EVT) cells. We have previously demonstrated that Stx2 injected in rats in the early stage of pregnancy causes spontaneous abortion by a direct cytotoxic effect in the highly perfused feto-uteroplacental unit. The main aim was to evaluate the effects of Stx2 on EVT in order to understand the possible adverse effects that the toxin may have on trophoblast cells during early pregnancy. Swan 71 and HTR-8 cell lines were used as human EVT models. The presence of Stx2 receptor, globotriaosylceramide (Gb3), on Swan 71 and HTR-8 cells was evaluated by thin layer chromatography. The effects of Stx2 on cell viability were evaluated by neutral red uptake, migration by wound-healing assay and invasion was determined by the ‘transwell chamber’ assay. Metalloproteinase activity (MMP-2) was evaluated by zymography and tubulogenesis was analyzed by counting the total tube length and the number of branch formation. We have demonstrated that Swan 71 expresses high levels of Gb3 compared to HTR-8 cells. Stx2 decreased significantly Swan 71 viability in a dose-dependent manner after 72 h of toxin exposure. Furthermore, Stx2 impaired migration, invasion and tube-like formation of Swan 71 cells and decreased the MMP-2 activity. These cytotoxic effects were partially prevented by aminoguanidine, an inducible nitric oxide synthase inhibitor. These studies demonstrate that the function and viability of EVT cells may be altered by Stx2 and suggest that NO overexpression may be involved in the detrimental effects.

 

    Society for Reproduction and Fertility

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    Gb3 expression on trophoblast cells and Stx2 cytotoxic effects. (A) Neutral glycolipids extracted from HTR-8, Swan 71 and Vero cells were subjected to TLC and then visualized with orcinol staining. (B) Gb3 glycolipid bands from 1 × 106 cells were quantified by densitometric analysis and compared to standard Gb3 (1 µg/lane, Matreya). (C) Cells were exposed to different concentrations of Stx2 (1 × 10−7–1 µg/mL) in growth-arrested conditions for 24 and 72 h. Then, cells were incubated with neutral red for an additional 1 h. Results are expressed as percentage of cell viability where 100% represents control cells without toxin treatment. Data are shown as means ± s.d. from at least five independent experiments performed in triplicate. *P < 0.05 for Swan 71 (72 h) vs Swan 71 (24), **P < 0.01 for Swan 71 (72 h) vs HRT-8 (24 h) and HTR-8 (72 h).

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    Stx2 inhibits trophoblast cell migration. (A) Cell migration was evaluated in Swan 71 cells by the wound-healing assay in cells grown in 24-well plates and incubated for 24 h with Stx2 (0.001–1 µg/mL) before wound formation. Images of the wound healing were captured at 0 h and 24 h using a light microscope (×200) (B) Cell migration was calculated and expressed as percentages of cell coverage to the initial cell-free zone. Values are presented as means ± s.d. Experiments were repeated three times. Significant differences (*P < 0.05) were found compared to the control.

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    Stx2 inhibits trophoblast cell invasion and MMP-2 activity. Swan 71 cells were grown on transwell insert (8 µm pore size) coated with 0.5 mg/mL Matrigel (Sigma) and incubated for 24 h with Stx2 (0.01 and 0.1 µg/mL). Cells that invaded the lower side of the insert were fixed with methanol and stained with DAPI. (A) Representative photographs of invading cells in each experimental condition (×200). (B) Stx2 at 0.1 µg/mL significantly decreased Swan 71 cell invasion compared to control. MMP-2 activity was evaluated by zymography assay in Stx2-treated and non-treated Swan 71 cells incubated with Stx2 for 24 h. (C) a representative zymography is shown. (D) The densitometric analysis of MMP-2 band intensity showed a significant decrease of MMP-2 activity on Stx2 treated compared to control. Values are presented as means ± s.d. Each experiment was repeated three times. *P < 0.05.

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    Stx2 inhibits tube-like formation of trophoblast cells. Swan 71 cell monolayers treated with Stx2 were seed in 96-well plates coated with Matrigel. Pictures were taken of ten random fields 6 h after tube-like formation (×100). (A) Representative images of tube-like formation. (B) Total tube length and (C) number of branches were quantified in control and Stx2-treated cells. Stx2 significantly decreases each one of the parameters involved in tube-like formation compared to control. Values are presented as means ± s.d. Each experiment was repeated three times. *P < 0.05, **P < 0.01.

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    Aminoguanidine (AG) partially prevents Stx2 effects on trophoblast cells. AG (100 µM) partially prevented the cytotoxic effects of Stx2 (0.1 µg/mL) on (A) migration, (B) invasion and (C) total tube length and (D) number of branches of Swan 71 cells. Data are shown as means ± s.d. from at least three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001.

References

AlejandraRNataliaSAliciaED 2018 The blocking of aquaporin-3 (AQP3) impairs extravillous trophoblast cell migration. Biochemical and Biophysical Research Communications 499 227232. (https://doi.org/10.1016/j.bbrc.2018.03.133)

Al-HijjiJAndolfELauriniRBatraS 2003 Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium. Reproductive Biology and Endocrinology 1 51. (https://doi.org/10.1186/1477-7827-1-51)

AsagiriKNakatsukaMKonishiHNoguchiSTakataMHabaraTKudoT 2003 Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts. Journal of Obstetrics and Gynaecology Research 29 4955. (https://doi.org/10.1046/j.1341-8076.2003.00066.x)

AthanassakisIAifantisIRanellaAGiouremouKVassiliadisS 1999 Inhibition of nitric oxide production rescues LPS-induced fetal abortion in mice. Nitric Oxide 3 216224. (https://doi.org/10.1006/niox.1999.0224)

BauwensABetzJMeisenIKemperBKarchHMuthingJ 2013 Facing glycosphingolipid-Shiga toxin interaction: dire straits for endothelial cells of the human vasculature. Cellular and Molecular Life Sciences 70 425457. (https://doi.org/10.1007/s00018-012-1060-z)

BerganJDyve LingelemABSimmRSkotlandTSandvigK 2012 Shiga toxins. Toxicon 60 10851107. (https://doi.org/10.1016/j.toxicon.2012.07.016)

BilbanMTauberSHaslingerPPollheimerJSalehLPehambergerHWagnerOKnoflerM 2010 Trophoblast invasion: assessment of cellular models using gene expression signatures. Placenta 31 989996. (https://doi.org/10.1016/j.placenta.2010.08.011)

BlighEGDyerWJ 1959 A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37 911917. (https://doi.org/10.1139/o59-099)

BrosensIPijnenborgRVercruysseLRomeroR 2011 The ‘Great Obstetrical Syndromes’ are associated with disorders of deep placentation. American Journal of Obstetrics and Gynecology 204 193201. (https://doi.org/10.1016/j.ajog.2010.08.009)

BurdetJZottaEFranchiAMIbarraC 2009 Intraperitoneal administration of Shiga toxin type 2 in rats in the late stage of pregnancy produces premature delivery of dead fetuses. Placenta 30 491496. (https://doi.org/10.1016/j.placenta.2009.03.012)

BurdetJZottaECellaMFranchiAMIbarraC 2010 Role of nitric oxide in Shiga toxin-2-induced premature delivery of dead fetuses in rats. PLoS One 5 e15127. (https://doi.org/10.1371/journal.pone.0015127)

CartwrightJEWhitleyGS 2017 Strategies for investigating the maternal-fetal interface in the first trimester of pregnancy: what can we learn about pathology? Placenta 60 145149. (https://doi.org/10.1016/j.placenta.2017.05.003)

CreydtVPSilbersteinCZottaEIbarraC 2006 Cytotoxic effect of Shiga toxin-2 holotoxin and its B subunit on human renal tubular epithelial cells. Microbes and Infection 8 410419. (https://doi.org/10.1016/j.micinf.2005.07.005)

ForstermannUSessaWC 2012 Nitric oxide synthases: regulation and function. European Heart Journal 33 829837. (https://doi.org/10.1093/eurheartj/ehr304)

GiakoumelouSWheelhouseNCuschieriKEntricanGHowieSEHorneAW 2016 The role of infection in miscarriage. Human Reproduction Update 22 116133. (https://doi.org/10.1093/humupd/dmv041)

GibbsRS 2001 The relationship between infections and adverse pregnancy outcomes: an overview. Annals of Periodontology 6 153163. (https://doi.org/10.1902/annals.2001.6.1.153)

GrahamCHHawleyTSHawleyRGMacdougallJRKerbelRSKhooNLalaPK 1993 Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Experimental Cell Research 206 204211. (https://doi.org/10.1006/excr.1993.1139)

HambartsoumianESrivastavaRKSeibelMM 2001 Differential expression and regulation of inducible nitric oxide synthase (iNOS) mRNA in human trophoblasts in vitro. American Journal of Reproductive Immunology 45 7885. (https://doi.org/10.1111/j.8755-8920.2001.450203.x)

HusseinHS 2007 Prevalence and pathogenicity of Shiga toxin-producing Escherichia coli in beef cattle and their products. Journal of Animal Science 85 E63E72. (https://doi.org/10.2527/jas.2006-421)

IbarraCAmaralMMPalermoMS 2013 Advances in pathogenesis and therapy of hemolytic uremic syndrome caused by Shiga toxin-2. IUBMB Life 65 827835. (https://doi.org/10.1002/iub.1206)

KarmaliMAPetricMLimCFlemingPCArbusGSLiorH 1985 The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. Journal of Infectious Diseases 151 775782. (https://doi.org/10.1093/infdis/151.5.775)

KrauseBJHansonMACasanelloP 2011 Role of nitric oxide in placental vascular development and function. Placenta 32 797805. (https://doi.org/10.1016/j.placenta.2011.06.025)

LalaPKChakrabortyC 2003 Factors regulating trophoblast migration and invasiveness: possible derangements contributing to pre-eclampsia and fetal injury. Placenta 24 575587. (https://doi.org/10.1016/S0143-4004(03)00063-8)

Melton-CelsaAR 2014 Shiga toxin (Stx) classification, structure, and function. Microbiology Spectrum 2. (https://doi.org/10.1128/microbiolspec.EHEC-0024-2013)

MuthingJSchweppeCHKarchHFriedrichAW 2009 Shiga toxins, glycosphingolipid diversity, and endothelial cell injury. Thrombosis and Haemostasis 101 252264. (https://doi.org/10.1160/TH08-05-0317)

ObrigTGLouiseCBLingwoodCABoydBBarley-MaloneyLDanielTO 1993 Endothelial heterogeneity in Shiga toxin receptors and responses. Journal of Biological Chemistry 268 1548415488.

PeltierMR 2003 Immunology of term and preterm labor. Reproductive Biology and Endocrinology 1 122. (https://doi.org/10.1186/1477-7827-1-122)

SacerdotiFAmaralMMZottaEFranchiAMIbarraC 2014 Effects of Shiga toxin type 2 on maternal and fetal status in rats in the early stage of pregnancy. BioMed Research International 2014 384645. (https://doi.org/10.1155/2014/384645)

SacerdotiFAmaralMMAisembergJCymeryngCBFranchiAMIbarraC 2015 Involvement of hypoxia and inflammation in early pregnancy loss mediated by Shiga toxin type 2. Placenta 36 674680. (https://doi.org/10.1016/j.placenta.2015.03.005)

SacerdotiFScaliseMLBurdetJAmaralMMFranchiAMIbarraC 2018 Shiga toxin-producing Escherichia coli infections during pregnancy. Microorganisms 6. (https://doi.org/10.3390/microorganisms6040111)

SpragueAHKhalilRA 2009 Inflammatory cytokines in vascular dysfunction and vascular disease. Biochemical Pharmacology 78 539552. (https://doi.org/10.1016/j.bcp.2009.04.029)

Staun-RamEShalevE 2005 Human trophoblast function during the implantation process. Reproductive Biology and Endocrinology 3 56. (https://doi.org/10.1186/1477-7827-3-56)

StrasbergPGreyAWarrenISkomorowskiMA 1989 Simultaneous fractionation of four placental neutral glycosphingolipids with a continuous gradient. Journal of Lipid Research 30 121127.

Straszewski-ChavezSLAbrahamsVMAlveroABAldoPBMaYGullerYRomeroRMorG 2009 The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan. Placenta 30 939948. (https://doi.org/10.1016/j.placenta.2009.08.007)

TangLHeGLiuXXuW 2017 Progress in the understanding of the etiology and predictability of fetal growth restriction. Reproduction 153 R227R240. (https://doi.org/10.1530/REP-16-0287)

TorresAG 2017 Escherichia coli diseases in Latin America-a ‘one Health’ multidisciplinary approach. Pathogens and Disease 75. (https://doi.org/10.1093/femspd/ftx012)

WaddellTCohenALingwoodCA 1990 Induction of verotoxin sensitivity in receptor-deficient cell lines using the receptor glycolipid globotriosylceramide. PNAS 87 78987901. (https://doi.org/10.1073/pnas.87.20.7898)

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