Schistosome Egg Antigens Elicit a Proinflammatory Response byTrophoblast Cells of the Human Placenta

Emily A. McDonald,a,b Jonathan D. Kurtis,a,b Luz Acosta,a,c Fusun Gundogan,d Surendra Sharma,e Sunthorn Pond-Tor,a Hai-wei Wu,a,f

Jennifer F. Friedmana,f

Center for International Health Research, Rhode Island Hospital, Brown University Medical School, Providence, Rhode Island, USAa; Department of Pathology andLaboratory Medicine, Rhode Island Hospital, Brown University Medical School, Providence, Rhode Island, USAb; Department of Immunology, Research Institute of TropicalMedicine, Manila, The Philippinesc; Department of Pathology and Laboratory Medicine, Women and Infants Hospital, Brown University Medical School, Providence,Rhode Island, USAd; Department of Pediatrics, Women and Infants Hospital, Brown University Medical School, Providence, Rhode Island, USAe; Department of Pediatrics,Rhode Island Hospital, Brown University Medical School, Providence, Rhode Island, USAf

Schistosomiasis affects nearly 40 million women of reproductive age. Many of these women are infected while pregnant and lac-tating. Several studies have demonstrated transplacental trafficking of schistosome antigens; however, little is known regardinghow these antigens affect the developing fetus and placenta. To evaluate the impact of schistosomiasis on trophoblasts of thehuman placenta, we isolated primary trophoblast cells from healthy placentas delivered at term. These trophoblasts were placedin culture and treated with Schistosoma japonicum soluble egg antigens (SEA) or plasma from S. japonicum-infected pregnantwomen. Outcomes measured included cytokine production and activation of signal transduction pathways. Treatment of pri-mary human trophoblast cells with SEA resulted in upregulation of the proinflammatory cytokines interleukin 6 (IL-6) and IL-8and the chemokine macrophage inflammatory protein 1� (MIP-1�). Cytokine production in response to SEA was dose depen-dent and reminiscent of production in response to other proinflammatory stimuli, such as Toll-like receptor 2 (TLR2) and TLR4agonists. In addition, the signaling pathways extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal protein kinase(JNK), p38, and NF-�B were all activated by SEA in primary trophoblasts. These effects appeared to be mediated through bothcarbohydrate and protein epitopes of SEA. Finally, primary trophoblasts cocultured with plasma from S. japonicum-infectedpregnant women produced increased levels of IL-8 compared to trophoblasts cocultured with plasma from uninfected pregnantwomen. We report here a direct impact of SEA on primary human trophoblast cells, which are critical for many aspects of ahealthy pregnancy. Our data indicate that schistosome antigens can activate proinflammatory responses in trophoblasts, whichmight compromise maternal-fetal health in pregnancies complicated by schistosomiasis.

Schistosomiasis, caused by three principal species of dioecioustrematodes (flatworms), currently affects over 250 million in-

dividuals, results in 1.53 million disability-adjusted life years(DALYs) lost per annum (1), and contributes to poor health andeconomic stagnation in areas in which schistosomiasis is endemic(2). Forty million women of child-bearing age are currently in-fected with schistosomes, yet the impact of schistosomiasis on thehealth of human pregnancy remains understudied. Strong evi-dence exists in rodent models for a host of deleterious outcomesfrom schistosome infection during pregnancy (3, 4). In humans,there is a relatively strong association between schistosome infec-tion and an increased risk of maternal anemia (5, 6). In addition,observational studies have revealed 4 to 18% lower birth weightsfor babies born to infected mothers (7, 8), although clinical trialsinvolving midgestational treatment for schistosomiasis have notdemonstrated improved pregnancy outcomes (9). Schistosomiasisjaponica is associated with multiple proinflammatory response-mediated morbidities in nonpregnant individuals (10, 11), andinflammation is known to result in deleterious birth outcomes,including prematurity, intrauterine growth restriction (IUGR),and low birth weight (LBW) (12–16).

We recently extended these findings to pregnancy, demon-strating that schistosomiasis is associated with elevated proin-flammatory cytokines in human maternal, placental, and cordblood, as well as an increased risk for the development of acutesubchorionitis at the maternal-fetal interface (17). Importantly,those women who displayed the highest levels of inflammatory

markers (tumor necrosis factor alpha [TNF-�] and interleukin 1�[IL-1�] production) also had offspring with lower birth weights,underscoring the potentially detrimental effects of significant lev-els of inflammatory cytokines at the maternal-fetal interface (17).

Despite the importance of molecular events at the maternal-fetal interface in establishing and maintaining pregnancy, the re-sponse of the placenta to schistosomiasis has not been examined.In this study, we examined the effects of schistosome Schistosomajaponicum soluble egg antigens (SEA) on trophoblast cells of thehuman placenta. In addition, we evaluated cytokine productionby term trophoblast cells after 24 h of culture with maternalplasma collected at 32 weeks’ gestation from women with S. ja-ponicum infection or uninfected controls. We report that SEAactivates multiple signaling pathways in trophoblasts, resulting inmarked upregulation of proinflammatory cytokines.

Received 25 October 2012 Returned for modification 13 November 2012Accepted 11 December 2012

Published ahead of print 17 December 2012

Editor: J. F. Urban, Jr.

Address correspondence Emily A. McDonald, emcdonald1@lifespan.org.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01149-12

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MATERIALS AND METHODSCell isolation and culture. Placental samples were collected in accordancewith protocols approved by the institutional review boards at Women andInfants and Rhode Island Hospitals. Cytotrophoblasts (CTBs) were iso-lated from normal, healthy placentas (i.e., from gestations free of anyidentified complications) delivered via elective cesarean section at term asdescribed previously (18). Briefly, villous tissue was dissected away fromthe basal plate and major blood vessels and subjected to enzymatic diges-tion with DNase I (type IV; Sigma Chemical, St. Louis, MO) and trypsin(Invitrogen, Carlsbad, CA). The resulting single-cell suspension was sizefractionated by application to a Percoll density gradient. To ensure a ho-mogenous population of CTBs, the cells underwent negative selectionusing an antibody recognizing human leukocyte antigen A (HLA-A), -B,and -C (W6/32; eBioscience, San Diego, CA) with a magnetically labeledsecondary antibody (Miltenyi Biotec GmBH, Bergisch Gladbach, Ger-many), and the cells were found to be �98% pure by cytokeratin 7 stain-ing (data not shown). Purified cells were cultured in Iscove’s modifiedDulbecco’s medium with 10% fetal bovine serum (FBS), 1% L-glutamine,1% penicillin-streptomycin-amphotericin B and allowed to form syncytiain vitro by incubation in a humidified chamber for 96 h prior to stimula-tion with SEA. All experiments were conducted on cells that were firstallowed to differentiate to better approximate the syncytiotrophoblastlayer found in direct contact with maternal blood in the human placenta.

Positive (lipopolysaccharide [LPS] and zymosan; Sigma Chemical)and negative (Sj68, a highly purified recombinant schistosome proteincomprising amino acids [aa] 20 to 176 of GenBank accession no.CAX72484.1) controls were added for the final 24 h of a 5-day cultureperiod. Cells used in the studies of signaling pathways were cultured for 4days in the presence of 10% fetal bovine serum (FBS) and then switched toserum-free medium. Pharmacological inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) (UO126; Sigma Chemical), p38 mitogen-activated protein (MAP) kinase (SB202190; Sigma), Jun N-terminal pro-tein kinase (JNK) (SP600125; Sigma), and NF-�B (JSH-23; Calbiochem,Merck KGaA, Darmstandt, Germany) were added (10 �M) 1 h prior tothe addition of SEA (25 �g/ml for 24 h). To ensure cell viability afteraddition of the inhibitor, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assays (Sigma) were performed(data not shown).

Antigen preparation. Schistosome eggs were collected from rabbitlivers infected with Schistosoma japonicum. Soluble egg antigen (SEA) wasprepared under endotoxin-free conditions according to standard proce-dures (19). In brief, 7 to 8 weeks after Schistosoma japonicum cercarialexposure, infected rabbits (�2,500 cercariae/rabbit) were perfused, andtheir livers were collected and rinsed with LPS-free phosphate-bufferedsaline (PBS). Liver homogenate was filtered, washed, and centrifuged overa Percoll-0.25 M sucrose gradient. The purified eggs were washed andhomogenized via mortar and pestle in PBS for 20 min. The homogenatewas ultracentrifuged with the resulting supernatant and frozen at �80°C.Preparations were evaluated for contaminating endotoxin using an FDAstandard Limulus amebocyte lysate (LAL) assay (Acila GmbH). Endo-toxin levels for all SEA preparations used were �6 IU/mg protein, whichis at least 1,000-fold lower than levels that have been shown to influencehuman trophoblast cells (5).

We subjected schistosome SEA to a variety of treatments in order toevaluate the relative contribution of carbohydrate and peptideepitopes to cytokine stimulation. The disruption of terminal saccha-ride rings was achieved by treatment with sodium m-periodate (20mM) in sodium acetate buffer (100 mM) for 45 min at 25°C in thedark. Aldehydes produced by this reaction were reduced to primaryalcohols by the addition of sodium borohydride (50 mM) for 30 min at25°C in the dark. Mock-treated SEA was diluted in sodium acetatebuffer without the addition of sodium m-periodate and with the addi-tion of water instead of sodium borohydride. Finally, all samples weredialyzed against PBS prior to use.

In addition, SEA was subjected to protein degradation by incubation

with the serine protease proteinase K (50 �g/ml) for 15 min at 37°C.Reactions were stopped by the addition of phenylmethylsulfonyl fluoride(PMSF) (5 mM). As a negative control, an aliquot of SEA was denaturedby heating to 95°C for 10 min. Modified SEA (25 �g/ml) was used tostimulate primary trophoblast cells as outlined above.

Cytokine assays. Primary trophoblast cells (n 12 trophoblast prep-arations from distinct placentas) were treated with SEA (25 �g/ml) ormedium alone for 24 h. Cytokine assays were performed on culture mediacollected at the end of the treatment period. IL-1�, IL-6, gamma inter-feron (IFN-), tumor necrosis factor alpha (TNF-�), IL-4, Il-5, IL-10,IL-13, IL-12, IL-8, and IL-2 were measured with a bead-based platform(BioPlex; Bio-Rad, Hercules, CA) using a sandwich antibody-based assayas described previously (20). In addition, chemokines, including CCL3(macrophage inflammatory protein 1� [MIP-1�]), CCL18, and CCL2(monocyte chemotactic protein 1 [MCP-1]), were measured using a sim-ilar bead-based approach. As expected, many of these cytokines were notproduced at detectable levels, reflecting the nonimmune origin of thesyncytiotrophoblast.

Signaling assays. Primary trophoblasts were collected and allowed todifferentiate as described above. Cultures were treated with SEA (25 �g/ml) or medium alone for 24 h, 30 min, 15 min, or 5 min. Cells were thenharvested using a cell lysis kit (Bio-Rad) in accordance with the manufac-turer’s instructions. Briefly, cells were washed and then lysed in the pres-ence of protease inhibitors by shaking for 20 min at 4°C. Cellular debriswas cleared by centrifugation at 4,500 � g and 4°C for 20 min. The result-ing whole-cell lysates were quantitated using a standard bicinchoninicacid (BCA) assay (Thermo Scientific, Rockford, IL) to ensure that allsamples were within the working range of the assay. Total and phosphor-ylated protein levels for the signaling molecules ERK1/2, JNK, p38 MAPkinase (MAPK), Akt, and I�B� were assessed using the BioPlex phospho-protein and total protein detection kits (Bio-Rad), according to the man-ufacturer’s instructions, on a bead-based analyzer (BioPlex; Bio-Rad).Data were analyzed as the ratio of phosphorylated to total protein for agiven signaling molecule.

Progesterone assay. Progesterone was measured in the culture mediafrom trophoblasts that had been in culture for 5 days, with SEA exposure(25 �g/ml) for the final 24 h. Hormone levels were measured using aprogesterone enzyme immunoassay (EIA) kit (Cayman Chemical, AnnArbor, MI) according to the manufacturer’s instructions. All sampleswere run in duplicate at a 1:100 dilution.

Human plasma assays. Term trophoblasts (described above) werecultured for 4 days before being cultured for 24 h in serum-free mediawith the addition of 10% plasma collected from pregnant women at 32weeks’ gestation. All plasma samples were collected from women residingin Leyte, the Philippines, an area where schistosomiasis is endemic. Thestudy population was the same as has been described elsewhere (17). Fromthis larger population, we selected 9 women infected with schistosomiasisand 8 uninfected women matched for socioeconomic status (SES), coin-fections (Ascaris lumbricoides, Trichuris trichiura, and hookworm), grav-ida, parity, gestational age, body mass index (BMI), smoking status, andmaternal age (Table 1). Socioeconomic status was calculated from a de-tailed questionnaire that we previously validated in this study population(17) and is reported as a composite score.

Following the 24 h of incubation with maternal plasma, trophoblastculture supernatants were collected and analyzed for cytokine productionas described above.

Statistical analysis. All data are reported as means plus or minus thestandard errors of the mean (SEM). Data analysis was performed usingJMP 10 (SAS Institute, Cary, NC). All data were evaluated using matched-pair Wilcoxon signed-rank analysis, with experiments performed on tro-phoblast preparations from unique placentas (n number of distinctplacentas used for each experiment). Statistical significance was consid-ered to be indicated by a P value of �0.05. SEA manipulation experiments(see Fig. 4) were analyzed using matched-pair analysis, with a one-sided Pvalue of �0.05 considered significant.

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RESULTSSchistosome egg antigens stimulate proinflammatory cytokinerelease from trophoblast cells. In order to examine the directeffect that schistosome infection might exert on the trophoblastcells of the placenta, we treated term trophoblast cells that hadformed syncytia in vitro with schistosome SEA in culture. After 24h of exposure to SEA, the media were collected and analyzed for avariety of cytokines, including interleukin 1� (IL-1�), IL-6,gamma interferon (IFN-), TNF-�, IL-4, IL-5, IL-10, IL-13, IL-12, IL-8, and IL-2. Of these, secretion of IL-6 and IL-8 was in-creased 6.0-fold (P 0.03) and 2.0-fold (P � 0.01), respectively,after 24 h of treatment with SEA (Fig. 1A and B). Both of theseproinflammatory cytokines are known to play important roles atthe maternal-fetal interface, and IL-8 in particular can also act as achemokine to attract immune cells (21, 22).

Of the other cytokines measured, most were undetectable (IL-1�, IFN-, TNF-�, IL-5, IL-13, and IL-12), while others did notdiffer across treatment groups (IL-4, IL-10, and IL-2). Most ofthese (IL-1�, IFN-, TNF-�, IL-12, IL-4, IL-10, and IL-2) havebeen reported to be expressed by human trophoblasts (23–26),although in a number of different treatment/culture paradigms aswell as cytokine measurement techniques. The diversity amongthese reports, ours included, likely explains the divergence in cy-tokine production by trophoblast cells in culture. Interestingly,the anti-inflammatory cytokine IL-10 was not significantly alteredfollowing SEA treatment (data not shown). This is particularlyimportant given that chronic schistosome infection is typicallyassociated with a systemic anti-inflammatory Th2-type (high IL-10) response in other tissues (27).

In addition to assessing the levels of cytokines in the culturemedia following SEA treatment, we also evaluated the secretion ofselect chemokines, including MCP-1 and MIP-1�. Of these,MIP-1� was significantly upregulated (2.8-fold, P 0.03) by pri-mary trophoblasts exposed to SEA for 24 h in vitro compared tothe corresponding control cells that received media alone(Fig. 1C). These data suggest that, in addition to augmenting theproinflammatory cytokine response, trophoblasts also respond toSEA by releasing chemokines, which might ultimately influencethe immune cell milieu at the maternal-fetal interface.

Cytokine production in trophoblast cells is SEA dose depen-dent. Previous dose-ranging experiments performed in our labo-ratory using SEA and placental explant cultures showed an opti-mal dose of SEA to be 25 �g/ml (data not shown). To determinethe optimal SEA concentration for stimulation of purified tropho-blasts, we stimulated isolated trophoblast cells that had been cul-tured for 4 days with various levels of SEA (2.5, 10, 25, and 50�g/ml). With as little as 10 �g/ml SEA, evaluation of the proin-flammatory cytokines IL-6, IL-8, and MIP-1� showed significantupregulation, which continued to rise as the level of SEA increased(Fig. 2A, C, and E). These dose response data are comparable tothose reported for studies of SEA stimulation of professional im-mune cells (28–31) and informed our selection of an SEA dose of25 �g/ml for 24 h.

Importantly, the trophoblast responses detected in our exper-iments are specific to SEA, as exposure of trophoblast cells to anirrelevant schistosome protein, Sj68 (25 �g/ml for 24 h), did notelicit any cytokine response (Fig. 2B and D). This result is in con-trast to that observed for the positive controls of lipopolysaccha-ride (LPS), a TLR4 agonist and known stimulant of proinflamma-tory cytokines, and zymosan (TLR2 agonist) (Fig. 2B and D). Ofnote, despite being recognized stimulants of proinflammatory cy-tokines in isolated term trophoblast cells, both LPS and zymosan(32) showed high variability between placental preparations intheir stimulation potential, whereas SEA, at lower doses, is muchmore consistent in its proinflammatory stimulation.

Trophoblast cells respond to SEA through a variety of signal-ing pathways. SEA has been shown to activate members of theMAPK signaling cascades in other cell types (33–35). In order todetermine intracellular signaling pathways that are stimulated bySEA in trophoblast cells, we performed a bead-based assay mea-suring the activated (i.e., phosphorylated) and total forms of thesignaling molecules: Akt, c-Jun N-terminal kinase (JNK), extra-cellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-acti-vated protein kinase (p38 MAPK), and nuclear factor of � lightpolypeptide gene enhancer in B-cells inhibitor � (I�B�). Within15 min of SEA stimulation, all three MAPK signaling pathwaysevaluated (JNK, ERK1/2, and p38 MAPK) were activated abovethe media-only control, with a significant elevation in ERK1/2 as

TABLE 1 Demographic data for selected plasma samples from pregnant women collected at 32 weeks of gestation, matched on a number ofpotential confounders

Potential confoundera In Schistosoma japonicum-infected samples (n 9)In Schistosoma japonicum-uninfected samples(n 8)

Coinfections (n)Ascaris lumbricoides 8 7Trichuris trichiura 8 8Hookworm 7 6

SES (mean [95% CI]) 12.90 (10.03, 15.77) 14.22 (10.90, 17.53)

Smoking status (n)Yes 0 0No 9 8

Gestational age (wk) (mean [95% CI]) 38.67 (36.18–41.16) 38.88 (37.93–39.82)BMI (kg/m2) (mean [95% CI]) 22.49 (20.38–24.60) 22.39 (19.02–25.75)Maternal age (yr) (mean [95% CI]) 28.43 (25.11–31.74) 32.64 (27.28–38.00)Parity (mean [95% CI]) 2.56 (0.97–4.15) 3.63 (2.15–5.10)Gravida (mean [95% CI]) 3.78 (2.46–5.10) 4.89 (3.30–6.45)a SES, socioeconomic status; BMI, body mass index; CI, confidence interval.

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early as 5 min after treatment (Fig. 3A to C). As expected, theseresponses were transient, as all three signaling molecules returnedto control levels within 30 min of SEA stimulation.

In addition, I�B� was activated in a transient manner, with asignificant increase in phosphorylation 15 min post-SEA expo-sure. Taken together, these results suggest that SEA acts through avariety of signaling molecules to influence the biology of the tro-phoblast cell. In contrast, Akt was not significantly altered bytreatment with SEA (data not shown), demonstrating the speci-ficity of the MAPK stimulation seen after SEA exposure.

We next used pharmacological inhibitors to specifically blockthe activity of each signaling pathway (UO126, ERK1/2;SB202190, p38 MAPK; SP600125, JNK; JSH23, NF-�B) and eval-uated the trophoblast response to SEA. In most cases, pretreat-ment with an inhibitor of a specific signaling pathway prior to

24-h SEA exposure resulted in an intermediate level of cytokineproduction compared to the level in either the vehicle controlalone or vehicle control plus SEA (Fig. 4A, B, and E through H),with the notable exception of SB202190, the inhibitor of the p38MAPK pathway. Pretreatment with SB202190 lowered the levelsof IL-6 and IL-8 production compared to pretreatment with di-methyl sulfoxide (DMSO) (vehicle), but not to the point of statis-tical significance (Fig. 4C and D). Of note, inhibition of no singlepathway resulted in complete abolishment of the proinflamma-tory response to SEA, suggesting that multiple pathways might be

FIG 1 The proinflammatory cytokines IL-6 and IL-8 and the chemotacticcytokine MIP-1� are upregulated by SEA in trophoblast cells. Trophoblastcells isolated from healthy pregnancies at term formed syncytia in vitro for 4days before being treated with SEA (25 �g/ml) for 24 h. Media from all treat-ment conditions were collected and measured for cytokine expression. (A)IL-6 production is upregulated 6.0-fold in trophoblast cells exposed to SEA(n 12, P 0.03). (B) IL-8 production is increased 2.0-fold after SEA treat-ment (n 12, P � 0.01). (C) MIP-1� production is increased 2.8-fold follow-ing SEA treatment in trophoblasts (n 12 individual trophoblast preparationsfrom distinct placentas, P 0.03).

FIG 2 Trophoblast cells respond to SEA in a dose-dependent manner. Tro-phoblast cells isolated from healthy pregnancies at term were allowed to formsyncytia in vitro for 4 days before being treated with SEA for 24 h. Media fromall treatment conditions were collected and measured for cytokine expression.(A) IL-6 expression is increased with higher doses SEA (n 7, P 0.03). (B)IL-6 production in trophoblasts responds to SEA in a specific manner (n 6,P 0.03). (C) IL-8 production by trophoblasts increases with increasing dosesof SEA exposure (n 7, P 0.02). (D) SEA stimulates IL-8 production introphoblasts at a level on par with the TLR4 and TLR2 agonists LPS andzymosan, respectively (n 6, P 0.03). (E) The chemokine MIP-1� is alsodose responsive to SEA (n 7 individual trophoblast preparations for distinctplacentas, P 0.02). nt, no treatment.

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activated simultaneously to lead to IL-6 and IL-8 production bytrophoblasts in response to SEA.

SEA activates trophoblasts via both carbohydrate and pep-tide epitopes. SEA is a heterogeneous mix of potential antigens. Inorder to broadly classify the nature of the relevant epitopes, weevaluated the impact of periodate and protease treatment on SEA-induced trophoblast activation. Sodium meta-periodate oxida-tively cleaves between vicinal diols of sugar residues on glycopro-teins, thus altering carbohydrate-associated antigenicity (36),while proteinase K, which has broad specificity, destroys peptideepitopes.

Pretreatment of SEA with either periodate or proteinase K at-tenuated the IL-6 and IL-8 responses of stimulated trophoblasts(Fig. 5), suggesting that the syncytiotrophoblast responds to bothcarbohydrate and peptide epitopes in SEA.

Exposure to SEA might enhance hormone production by tro-phoblast cells. Progesterone levels in the culture media were as-sayed following 24-h exposure to SEA (25 �g/ml) in vitro. Al-though not statistically significant, with n 8 placentalpreparations there was a trend for increased levels of progesteronein the culture media from cells that had been exposed to SEA forthe final 24 h of the 5-day culture period (Fig. 6). This is an in-triguing finding that warrants further investigation.

Factors present in human plasma from infected pregnantwomen cause increased proinflammatory cytokine productionby term trophoblast cells. A subset of plasma samples collected at

32 weeks’ gestation was selected from a larger study performed toexamine the impact of schistosomiasis on pregnancy (17). Pri-mary term trophoblast cells from North American women withhealthy pregnancies were placed in culture and allowed to differ-entiate for 4 days. The media were then replaced with serum-freemedia containing 10% plasma from individual subjects infectedwith schistosomiasis, an uninfected matched control, or 10% FBS(negative control). All media were collected after 24 h and assayedfor IL-6 and IL-8 levels. IL-8 was significantly upregulated in thosecells exposed to plasma from women with an active schistosomeinfection compared to cells exposed to plasma from their unin-fected counterparts (Fig. 7), with IL-6 also showing a trend towardan increased response to plasma from infected women (data notshown). IL-8 and IL-6 levels in the plasma alone ranged fromundetectable to more than 100-fold lower than the lowest levels inany of the trophoblast cultures (data not shown). Therefore, thematernal plasma itself is not a significant contributor of IL-8 orIL-6 production in these trophoblast cultures.

DISCUSSION

Schistosomiasis is responsible for significant morbidity in low-and middle-income countries and significantly contributes to theglobal burden of anemia, undernutrition, and hepatic fibrosis (2).Although praziquantel (PZQ) effectively treats schistosomiasis,PZQ remains an FDA pregnancy class B drug, and thus schisto-some-infected women in some regions where schistosome infec-

FIG 3 SEA stimulates a variety of signaling pathways in trophoblast cells. Trophoblast cells isolated from healthy pregnancies at term formed syncytia in vitro for4 days before being treated with SEA. Whole-cell lysates from all treatment conditions were collected and measured for expression of total and phosphorylatedforms of signaling molecules. ERK1/2 (A), JNK (B), p38 MAPK (C), and I�B� (D) activity in trophoblast cells is upregulated within 15 min of exposure to SEA(n 5 individual trophoblast preparations from distinct placentas).

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tion is endemic, such as the Philippines, are excluded from treat-ment during pregnancy and lactation.

In mouse models, schistosomiasis is associated with strikinglypoor pregnancy outcomes, even at low infection intensity. CBA/Jmice infected with approximately 15 cercariae of Schistosoma

mansoni produce 66% fewer viable litters than uninfected con-trols, a result attributable to increased rates of abortion (20% ver-sus 1%), maternal death (5% versus 0%), and infanticide (42%versus 23%) (3). In addition, pup weight at 2 weeks was signifi-cantly lower in pups born to infected mothers (3). In separatestudies, heavier (40 cercariae/mouse) and chronic infections (4) ofC57BL/6 mice resulted in similar profoundly adverse birth out-comes, including increases in maternal death rate, abortion, andinfanticide from infected pregnancies. Interestingly, these datawere not replicated in a study involving exposure to S. japonicumcercariae 1 to 2 weeks postconception (37). These data suggest thateither (i) different schistosome species (S. mansoni versus S. ja-ponicum) influence the health of the pregnancy differently or (ii)schistosome infection is most detrimental if the exposure and ini-tial response to infection occur prior to successful breeding.

As might be expected given the discordance in placental struc-ture, the effect of schistosomiasis on pregnancy outcomes appearsto be somewhat species specific. Pigs infected with �9,000 cercar-iae of S. japonicum early in gestation (week 4) produce offspringthat appear to be smaller and weaker and fail to thrive, while pigsinfected prior to insemination or late in gestation produce normaloffspring (38). To our knowledge, no studies have examined thespecific effect of schistosome infection on the placenta itself ineither animal models or in humans. Reports suggest that maternal

FIG 4 The ERK1/2 and NF-�B signaling pathways are the dominant media-tors of the proinflammatory cascade initiated by SEA in trophoblasts. Phar-macological inhibition of the JNK, p38, and ERK1/2 MAPK pathways as well asthe NF-�B pathway prior to SEA (25 �g/ml) exposure for 24 h. (A, C, E, and G)IL-6 production after SEA with the addition of either vehicle (DMSO) orspecific inhibitors. (B, D, F, and H) IL-8 production from the same experi-ments. (A and B) Inhibition of the ERK1/2 signaling pathways (n 6). (C andD) Inhibition of the p38 MAPK signaling pathway (n 6). (E and F) Inhibi-tion of the JNK signaling pathway (n 5). (G and H) Inhibition of the NF-�Bsignaling pathway (n 6 individual trophoblast preparations from distinctplacentas). All data are reported as means � SEM. Unique letters denote sta-tistically significant differences at P values of �0.05.

FIG 5 The proinflammatory effects of SEA on trophoblast cells involve bothcarbohydrate and protein epitopes. SEA was manipulated in a manner thatdisturbed carbohydrate (periodate) or peptide (proteinase) epitopes prior totreatment of trophoblasts. Heat-denatured SEA was included as a negativecontrol. Secretion of the proinflammatory cytokines IL-6 (A) and IL-8 (B),normally stimulated by SEA exposure, was disturbed with SEA manipulation.mock, SEA subjected to dilution and incubations in parallel to periodate-treated SEA, without the addition of periodate. Matched-pair analysis with aone-sided t test; a P value of �0.05 was considered significant (n 5 individualtrophoblast preparations from distinct placentas).

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schistosomiasis in humans is associated with an increased risk ofmaternal anemia (5, 6), lower birth weight (7, 8), and risk of lowbirth weight (LBW) and preterm delivery (8, 39). In addition, werecently demonstrated that pregnant women infected with S. ja-ponicum had increased proinflammatory cytokine levels in mater-nal peripheral, placental, and cord blood (17).

Two observational studies, as well as a recently completed ran-domized controlled trial (RCT) in Uganda, have addressed therole of schistosomiasis in the pathogenesis of adverse pregnancyoutcomes in humans (7–9). A large (n 592) observational studyin which S. japonicum infection was examined in women in Chinarevealed lower birth weights among firstborn children fromwomen with schistosomiasis than among uninfected women;however, no adjustment was made for potential confounders suchas maternal nutritional status and socioeconomic status. A sepa-rate case-control study of Schistosoma haematobium-infectedpregnant women in Ghana revealed differences in birth weight ofinfants born to infected versus uninfected women, although thiswas significant only in premature deliveries (n 8 S. haemato-bium-infected women) (8). The results from this study were alsolikely influenced by selection bias, with infected women beingrecruited only if they felt poorly enough to present to the hospitalfor care. This possible bias, coupled with the low sample size in thesingle stratum in which effects were demonstrated, makes the re-sults of this study difficult to interpret.

Finally, a recent RCT conducted in Uganda addressed the effi-cacy of PZQ and/or albendazole given during the second or thirdtrimester (9). Treatment with praziquantel was not associatedwith decreased risk of maternal anemia or low birth weight com-pared to placebo. An RCT nearing completion in the Philippines(ClinicalTrials.gov, registered study number NCT00486863) willaddress the efficacy of praziquantel given earlier (12 to 16 weeks’gestation) and in the context of infection with S. japonicum, whichis considered the more virulent species.

In this study, we examined for the first time the impact ofschistosome infection on trophoblast cells of the human placenta.The human placenta is composed of a multitude of cell types, with

the syncytiotrophoblast layer comprising the functional unit re-sponsible for nutrient, waste, and gas exchange between motherand fetus. Although isolated reported studies have demonstrateddirect trafficking of schistosome worms and whole eggs to theplacental compartment in humans, this event appears to be rela-tively rare and is difficult to assess (40). Exposure and transfer ofsoluble worm and egg antigens across the placenta, however, iswell documented and occurs in the majority of human pregnan-cies complicated with schistosome infection (41). We thereforeutilized schistosome SEA in our experiments as a starting point forexamination of the effect that schistosome infection might haveon trophoblast cells. As a follow-up experiment, we also placedtrophoblast cells in culture with plasma from pregnant womeninfected with schistosomiasis.

We previously reported that schistosome infection results in aproinflammatory cytokine response detectable in both maternaland fetal blood at term (17). Herein, we report results that estab-lish that the trophoblast cells of the placenta contribute, in part, tothis proinflammatory response. In addition to direct productionof proinflammatory cytokines, trophoblasts collected at term andallowed to form syncytia in vitro produce increased amounts ofthe chemokine MIP-1� in response to SEA, suggesting that tro-phoblast cells might alter the immune cell repertoire at the mater-nal-fetal interface in response to SEA exposure. Schistosome eggantigen has been previously shown to induce MIP-1� productionby macrophages during granuloma formation in a murine model(42). In this study, we extended this paradigm to the placenta, withimplications for an altered immune cell environment at the ma-ternal-fetal interface.

Trophoblasts have been shown to produce a variety of cyto-kines, depending on the gestational age, differentiation status ofthe trophoblast cells, and placental environment (43–46). The

FIG 6 Progesterone production increases in trophoblasts exposed to SEA invitro. Progesterone levels in the culture media following 24-h SEA exposure(25 �g/ml) were measured by an EIA. Although not statistically significant, atrend toward increased levels of progesterone was observed in those cellstreated with SEA (n 8 individual trophoblast preparations from distinctplacentas, P 0.06).

FIG 7 Plasma from pregnant women with schistosomiasis causes increasedIL-8 production by human trophoblast cells. Term trophoblasts from NorthAmerican control women were cultured for 4 days prior to culture for 24 h inmedia supplemented with plasma samples (10% final concentration). Plasmasamples were collected at 32 weeks’ gestation from women with and withoutschistosomiasis residing in an area of the Philippines where schistosomiasis isendemic. IL-8 production is significantly elevated in trophoblasts that wereexposed to plasma from women infected with schistosomiasis. Data are ex-pressed as the median fold increase in IL-8 levels over the no-plasma controlsfor each trophoblast preparation (n 6 individual trophoblast preparationsfrom distinct placentas) (n 17 plasma samples, P 0.04).

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data described herein are similar to the effects seen in trophoblastcells in response to other inflammatory agents, such as lipopoly-saccharide (LPS) (47); however, trophoblast cells also secrete cy-tokines throughout a routine gestation. In the course of normalpregnancy, IL-10 is upregulated, and it is thought to play an im-portant role in the maintenance of immune tolerance toward thedeveloping fetus (48). It is thus somewhat surprising that we didnot observe an increase in IL-10 secretion by trophoblast cellswhen they were exposed to SEA. This finding, however, is consis-tent with the proinflammatory Th1 signature that we observedboth in the trophoblasts themselves (Fig. 1) and in maternal andnewborn compartments following exposure to SEA (17).

The recapitulation of SEA-induced trophoblast activation byplasma from pregnant women with schistosomiasis furtherstrengthens evidence for the association between schistosomiasisand poor pregnancy outcomes. Our data suggest that the levels ofcirculating schistosome-associated antigens in maternal plasmaare sufficient to directly activate trophoblasts in vitro. Despitematching on measured potential confounders (geohelminths, so-cioeconomic status, etc.), we recognize that residual, unmeasuredconfounders related to both S. japonicum infection and tropho-blast activation might also partly explain this relationship.

Schistosome egg antigens have been shown to stimulate boththe ERK1/2 and p38 MAPK signaling pathways in dendritic cells(33, 34) and macrophages (35). Classical TLR activators, such asLPS (TLR4), Pam3cys (TLR2), and flagellin (TLR5), also activatedthese pathways to various degrees in dendritic cells (34), lendingcredence to the possibility of SEA working through a TLR-medi-ated pathway. Despite these findings, the ligand-receptor pairsand consequent signaling cascades initiated by SEA have not beenreported in cells that are not classic immune cells (e.g., trophoblastcells). In this study, we demonstrated that SEA can robustly acti-vate the ERK1/2 and p38 MAPK pathways, as well as the JNKMAPK pathway in trophoblast cells. In addition, the NF-�B path-way is activated in trophoblasts in response to SEA. These data areconsistent with the proinflammatory response seen following SEAexposure of trophoblasts.

The schistosome egg antigen represents an admixture of manydifferent proteins, including cytoplasmic and secretory products.Some of the most abundant products secreted from schistosomeeggs have biological activity from both protein and carbohydrateepitopes (49), and antibody responses directed against SEA com-prise both antiglycan and antipeptide responses (50). In addition,at least one of the major glycoproteins secreted by S. mansoni eggs,interleukin-4-inducing principle from schistosome eggs (IPSE/alpha-1), is known to stimulate cytokine responses by basophils,albeit prominently skewed toward a Th2 environment (51). Ourmanipulation of SEA, directed at either the glycan or peptideepitopes, resulted in reduced proinflammatory responses in tro-phoblast cells. Although these data suggest that both sugars andproteins in SEA play a role in initiating a proinflammatory re-sponse within trophoblast cells, they by no means preclude otherpossibilities, such as the importance of multiple carbohydratestructures from the same molecule initiating a proinflammatoryresponse. More detailed analysis of the role of glycans and/or pro-teins in this response is warranted. In addition, neither manipu-lation abrogated the cytokine response to the level of untreatedcells, suggesting that both components of SEA might activate thesyncytiotrophoblast. These data are in keeping with the multitudeof signaling pathways that we have observed to be initiated by SEA

in trophoblast cells. Studies designed to specifically identify theligand-receptor pairs responsible for SEA-induced trophoblastactivation are currently ongoing.

In conclusion, we have demonstrated that antigens releasedfrom schistosome eggs and present in maternal blood activatehuman trophoblast cells to secrete specific proinflammatory cyto-kines and chemokines. Additional data suggest that SEA mightinfluence other basic functions of the trophoblast cells, such ashormone production. However, the changes in progesterone pro-duction we observed did not reach statistical significance and thusshould be interpreted with caution. We do show, however, thatSEA activates a variety of signaling pathways in the trophoblastcell, including members of the MAPK family and the NF-�B path-way. These effects are mediated through both carbohydrate andprotein epitopes. To our knowledge, this is the first report of adirect effect of SEA on cells of the placenta, and it represents animportant initial characterization of how schistosomiasis mightinfluence the placenta and subsequent health of the pregnancy.

ACKNOWLEDGMENTS

This work was supported by grants 1F32AI093043-01A1 (to E.A.M.) and4U01AI066050-06 (to J.F.F.) from the National Institute of Allergy andInfectious Diseases at the National Institutes of Health.

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