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Products Predisposing to Preterm LaborInflammation and Sensitization to Bacterial Viral Infection of the Placenta Leads to Fetal

Enrique Oyarzun, Roberto Romero and Gil MorKoga, Sabine M. Lang, Carmen Booth, Alejandro Manzur, Ingrid Cardenas, Robert E. Means, Paulomi Aldo, Kaori

http://www.jimmunol.org/content/185/2/1248doi: 10.4049/jimmunol.1000289June 2010;

2010; 185:1248-1257; Prepublished online 16J Immunol 

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The Journal of Immunology

Viral Infection of the Placenta Leads to Fetal Inflammationand Sensitization to Bacterial Products Predisposing toPreterm Labor

Ingrid Cardenas,*,1 Robert E. Means,†,1 Paulomi Aldo,* Kaori Koga,* Sabine M. Lang,†

Carmen Booth,‡ Alejandro Manzur,x Enrique Oyarzun,x Roberto Romero,{

and Gil Mor*

Pandemics pose a more significant threat to pregnant women than to the nonpregnant population and may have a detrimental effect

on the well being of the fetus. We have developed an animal model to evaluate the consequences of a viral infection characterized by

lack of fetal transmission. The experiments described in this work show that viral infection of the placenta can elicit a fetal in-

flammatory response that, in turn, can cause organ damage and potentially downstream developmental deficiencies. Furthermore,

we demonstrate that viral infection of the placenta may sensitize the pregnant mother to bacterial products and promote preterm

labor. It is critical to take into consideration the fact that during pregnancy it is not only the maternal immune system responding,

but also the fetal/placental unit. Our results further support the immunological role of the placenta and the fetus affecting the global

response of the mother to microbial infections. This is relevant for making decisions associated with treatment and prevention

during pandemics. The Journal of Immunology, 2010, 185: 1248–1257.

Pregnant women are more susceptible to the effects ofmicrobial products (i.e., endotoxins) and were the mostvulnerable subjects during the 1918 pandemic (influenza A

subtype H1N1), with a mortality rate that ranged between 50 and75% (1). Exposure to the virus during pregnancy may also haveovert or subclinical effects that become apparent only over time.Although substantial progress has been made in the understand-

ing of the immunology of pregnancy, many unanswered questionsremain, especially those associatedwith the susceptibility and sever-ity of infectious agents of mothers and unborn children (2), (3).Epidemiological studies have demonstrated an association

between viral infections and preterm labor (4, 5) and fetal con-genital anomalies of the CNS and the cardiovascular system(6–8). Although some viral infections during pregnancy may beasymptomatic (9), approximately one-half of all preterm delive-

ries are associated with histological evidence of inflammation ofthe placenta, termed acute chorioamnionitis (10), or chronic cho-rioamnionitis (10). Despite the high incidence of acute cho-rioamnionitis, only a fraction of fetuses have demonstrableinfection (11). Most viral infections affecting the mother donot cause congenital fetal infection, and only in a small numberof cases is the virus found in the fetuses (12–17), attesting to theunique ability of the placenta to act as a potent barrier with animmune-regulatory function that protects the fetus from systemicinfection (10, 12, 18, 19).Recent observations indicate that rather than acting as a me-

chanical barrier, the placenta functions as a regulator of the traf-ficking between the fetus and the mother (20–22). Fetal andmaternal cells move in two directions (23, 24); similarly, someviruses and bacteria can reach the fetus by transplacental passagewith adverse consequences (25). Although viral infections arecommon during pregnancy (26), transplacental passage and fetalinfection appear to be the exception rather than the rule (27, 28;reviewed in Ref. 29 and subsequent references).There is a paucity of evidence that viral infections lead to

preterm labor (10, 19, 22–24); however, there are several areas ofcontroversy and open questions. For example, what effects dosubclinical viral infections of the decidua and/or placenta duringearly pregnancy have in response to other microorganisms, such asbacteria; and what is the effect of a subclinical viral infection ofthe placenta on the fetus?The trophoblast is an important component of the placenta, and it

is able to recognize and respond to microorganisms and theirproducts through the expression of TLRs (30–32). TLRs are a familyof innate immune receptors that have an essential role in the rec-ognition of pathogen-associatedmolecular patterns (33–35). Troph-oblasts are able to produce cytokines/chemokines and antiviralfactors following TLR-3 ligation in vitro, suggesting the potentiallyactive role of these cells in the control of viral infections (20, 36).Some of these receptors (chemokine and TLRs) may also functionas viral receptors mediating viral recognition and entry into the

*Department of Obstetrics, Gynecology and Reproductive Sciences, †Department ofPathology, and ‡Department of Comparative Medicine, School of Medicine, YaleUniversity, New Haven, CT 06520; xDepartment of Obstetrics and Gynecology,Pontificia Universidad Catolica, Santiago, Chile; and {Perinatology Research Branch,Eunice Kennedy Shriver National Institute of Child Health and Human Development,National Institutes of Health, Department of Health and Human Services, Detroit, MI48201

1I.C. and R.E.M. contributed equally to this work.

Received for publication January 27, 2010. Accepted for publication May 10, 2010.

This work was in part supported by grants from the National Institutes of Health(NICDH P01HD054713 and 3N01 HD23342) and the Intramural Research Programof the Eunice Kennedy Shriver National Institute of Child Health and Human De-velopment, National Institutes of Health, Department of Health and Human Services.

Address correspondence and reprint requests to Dr. Gil Mor, Department of Obstet-rics, Gynecology and Reproductive Sciences, Reproductive Immunology Unit, YaleUniversity School of Medicine, 333 Cedar Street, LSOG 305A, New Haven, CT06520. E-mail address: gil.mor@yale.edu

The online version of this article contains supplemental material.

Abbreviations used in this paper: DN, dominant negative; dpi, days postinfection; E,embryonic day; FGF-2, fibroblast growth factor-2; FIRS, fetal inflammatory responsesyndrome; GRO-a, growth-related oncogene-a; IP-10, IFN-g–inducible protein-10; KO,knockout;MHV-68,murine g-herpesvirus 68; poly(I:C), polyinosinic:polycytidylic acid;TIR, Toll/IL-1R; VEGF, vascular endothelial growth factor; wt, wild type.

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trophoblast. Signaling through TLRs has been shown to inducemurine g-herpesvirus 68 (MHV-68) reactivation in vivo (37).Herpesviruses are the most common cause of viral-related peri-

natal neurologic injury in the United States (38). However, amongthe eight known human herpesviruses, most reported adversepregnancy and neonatal outcomes are the result of the HSVs(HSV-1 and HSV-2) and CMV (39) and usually occur due toa primary infection of the mother during the first trimester orinfection of the infant during delivery. MHV-68 (murid herpesvi-rus 4 [NC_001826.2]) is a g-herpesvirus of rodents that sharessignificant genomic colinearity with two human pathogens, EBVand Kaposi’s sarcoma-associated herpesvirus (40). As in thesetwo viruses, the effect of MHV-68 in pregnancy is unknown.We developed a novel murine model to evaluate the role of viral

infection in pregnancy and fetal development. Our data suggest thateven in the absence of placental passage of the virus, the fetus couldbe adversely affected by an inflammatory response mounted inresponse to viral invasion of the placenta. Furthermore, we dem-onstrate that a viral infection in early pregnancy sensitizes thepregnant mother to the effects of bacterial products later on ingestation, and specifically, to premature labor. These data suggestthat exposure to early viral infections may program the immuneresponse of mother and fetus. Such observations have importantconsequences for understanding the potential risk of viral infec-tions during pregnancy and the importance of adequate surveillanceto prevent maternal mortality and subclinical fetal injury, leading tolong-term consequences.

Materials and MethodsVirus culture

MHV-68 expressing GFP (provided by R. Sun, University of California, LosAngeles, CA) was passaged in NIH 3T3 cells with DMEM plus 10% FCS.After lysis, supernatant was harvested, filtered (0.45-mm pore), and titeredby 2-fold serial dilutions. To determine virus load in infected mice, frozenhomogenized tissues were minced and subjected to 10-fold serial dilutions,and endpoint titers were determined in NIH 3T3 cells by GFP (41, 42). Asingle virion or DNA copy was sufficient to show a positive result byplaque assay or PCR.

Animal procedures

C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor,ME), and TLR-3 knockout (KO) was provided by R. A. Flavell (Yale Uni-versity, New Haven, CT). Adult mice (8–12 wk of age) with vaginal plugswere infected i.p. at embryonic day (E) 8.5 postconception with either 1 3106 PFU MHV-68 expressing GFP (in 200 ml vol) or DMEM (vehicle).Three or 9 d postinfection (dpi), animals were sacrificed, and organs wereremoved, fixed in 4% paraformaldehyde, and/or stored at 280˚C. Allanimals were maintained in the Yale University School of Medicine An-imal Facility under specific pathogen-free conditions. All experimentswere approved by the Yale Animal Resource Committee.

Reagents and Abs

LPS (Escherichia coli O111:B4) was purchased from Sigma-Aldrich(St. Louis, MO). Lymphocyte separation media was purchased from MPBiomedicals (Solon, OH).

For NK and macrophage detection, biotinylated lectin Dolichos biflorusagglutinin from Sigma-Aldrich and rat anti-mouse F4/80 Ab (eBioscience,San Diego, CA) were used, respectively. Anti–NF-kB p65 mAb waspurchased from Santa Cruz Biotechnology (Santa Cruz, CA). Dominant-negative (DN) TLR1 Toll/IL-1 receptor (TIR) and TLR2TIR, incapable oftranducing a signal after ligand binding, were purchased from InvivoGen(San Diego, CA). Bio-plex Pro custom 18-plex panel (catalogueM500FHB86U;171B6007M) for cytokine detection was purchased fromBio-Rad (Hercules, CA).

Cell lines

Human first trimester trophoblast HTR-8 cells were gifted from C. Graham(Queen’s University, Kingston, Ontario, Canada). Human first trimestertrophoblast 3A cells were stably transfected with DN TLR2 and TLR1

genes, as previously described (22). Following transfection, cells express-ing the TLR1TIR (TLR1-DN) or the TLR2TIR (TLR2-DN) were selectedwith puromycin. Vector alone-transfected cells served as negative control.

Human trophoblast isolation

First trimester trophoblast cells were isolated and cultured, as described (43).Briefly, after washing, tissues were minced and incubated in PBS, 0.125%trypsin, and 30 U/ml DNase I for 1 h at 37˚C. A 70-mm filtered suspensionwas layered on lymphocyte separation media (MP Biomedicals) and cen-trifuged. The interface layer was collected, washed, and resuspended withMEM. Cells were cultured in MEM with D-valine (Caisson Laboratories,North Logan, UT), 10% human serum, placed in a type IV collagen-coatedplate (BD Biosciences, Franklin Lakes, NJ), and kept at 37˚C/5% CO2.

Mouse embryonic fibroblast cell preparation

Embryos were harvested on days 11.5–13.5, and fibroblast cells wereprepared according to the method described by Bowtell’s laboratory (44–46). This is a standard method used for the preparation of supporting fi-broblast cells for stem cell growth. Mice embryonic fibroblasts werepropagated and maintained according to the 3T3 protocol (47).

Cytokine analysis

Cytokine concentration was determined, as previously described (48–50),using the cytokine multiplex assays from Bio-Rad. Briefly, wells of a 96-well filter plate were loaded with either 50 ml prepared standard solution or50 ml cell-free supernatant and incubated on an orbital shaker at6500 rpmfor 2 h in the dark at room temperature. Wells were then vacuum washedthree times with 100 ml wash buffer. Samples were then incubated with 25ml biotinylated detection Ab at 6500 rpm for 30 min in the dark at roomtemperature. After three washes, 50 ml streptavidin-PE was added to eachwell and incubated for 10 min at 6500 rpm in the dark at room temper-ature. After a final wash, the beads were resuspended in 125 ml assaybuffer for measurement with the LUMINEX 200 (LUMINEX, Austin,TX). The cytokines included in the Multiplex assay were as follows: IL-1b, IL-10, GM-CSF, IFN-g, TNF-a, IL-1a, IL-6, IL-12p40, IL-12p70, G-CSF, KC, MIP-1a, RANTES, MCP-1, and MIP-1b.

Immunohistochemistry

After Ag retrieval with Retrievagen A (pH 6.0; BD Biosciences), macro-phages were detected in paraffin-embeddedmurine uteri with rat anti-mouseF4/80 Ab at 1:20. NK cell detection was performed, as previously reported(31, 51).

For the localization of the p65 subunit of NF-kB, MHV-68–infectedhuman trophoblast cells were fixed and incubated with the mouse anti–NF-kB p65 Ab. Slides were then incubated with Alexa Fluor546 anti-mouse IgG and counterstained with Hoechst 33342 dye (Molecular Probes,Eugene, OR).

Total RNA isolation

Total RNA was extracted using TRIzol, and cDNA was prepared usinga Verso cDNA kit per manufacturer’s protocol (Thermo Scientific, Wal-tham, MA).

Real-time PCR

Real-time PCR was performed in duplicate using SYBR Green (Invitrogen,Carlsbad, CA) in an ABI Prism 7500 (Applied Biosystems, Foster City,CA). cDNA sample (1 ml) was amplified with gene-specific primers usingoptimized PCR cycles. GAPDH was used as an endogenous control forrelative comparison of human TLR-2, TLR-3, and TLR-4. GAPDH ex-pression did not vary with treatments. TLR-2 forward, 59-ATGCCTACT-GGGTGGAGAAC-39; TLR-2 reverse, 59-TGCACCACTCACTCACA-39;TLR-3 forward, 59-GTGCCGTCTATTTGCCA-39; TLR-3 reverse, 59-AG-TCTGTCTCATGATTCTGTTG-39; TLR-4 forward, 59-CAGCTCTTGGT-GGAAGTTGA-39; TLR-4 reverse, 59-GCAAGAAGCATCAGGTGAAA-39; GAPDH forward, 59-GAGTCAACGGATTTGGTCGT-39; GAPDH re-verse, 59-GACAAGCTTCCCGTTCTCAGCC-39.

The TLR-2, TLR-3, and TLR-4 cycle threshold value was analyzed usingthe DD cycle threshold Livak method (52).

ELISA

Serum from wild-type (wt) and TLR3 KO mice was analyzed for the pres-ence of anti-viral IgM or IgG Abs. For coating of ELISA plates, MHV-68GFP stocks were filtered through a 0.45-mm-pore membrane, and virionswere centrifuged through a 5% sucrose cushion (SW28, 20,000 rpm for

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75 min at 4˚C). Virion pellets were resuspended in PBS containing 0.5%Triton X-100 and 0.5% FBS to achieve 50003 concentration. Maxisorbplates (Nunc-immuno plates) (Corning Life Sciences, Corning, NY) werecoated with 91 mg virus protein/well, washed, blocked with 0.3 mg BSA,and incubated with serum from either wt or TLR3 KO with or withoutMHV-68 infection. After washing, goat anti-mouse IgG or IgM, HRP-conjugated Ab (Southern Biotechnology Associates, Birmingham, AL)was added at 1/4000 dilution in PBS/1% BSA for 2 h at room temperature,washed, and detected with tetramethylbenzidine substrate at 405 nm.

Statistical analysis

Data are expressed as mean 6 SE for in vitro study and median 6 first orthird quartiles for in vivo study. Statistical significance (p , 0.05) wasdetermined using either two-tailed unpaired Student t tests or Mann-Whitney U test for nonparametric data. Unless stated otherwise, all experi-ments were performed in duplicate.

ResultsMaternal infection with MHV-68 does not induce pretermlabor

Systemic administration of polyinosinic:polycytidylic acid [poly(I:C)] to pregnant mice induced preterm labor and delivery, andproduction of proinflammatory cytokines (53, 54). The inflammatoryresponse to the TLR-3 ligand was found in the placenta at E17.5(local response) as well as in the spleen (systemic response) (30,55) characterized by upregulation of IL-6, IL-12p40, MCP-1, MIP-1b, growth-related oncogene-a, and RANTES. These observationsindicated that the placenta was able to recognize and respond to viralproducts. To understand the effects of viral infection in pregnancy,we used MHV-68. C57BL/6 pregnant mice received 1 3 106 PFUMHV-68 i.p. on E8.5 of pregnancy, and were followed up to E17.5.Control animals received media as a placebo. This viral dose haspreviously been shown to infect mice organs and produce a systemicviral response (37).Maternal infection with MHV-68 had no effect on pregnancy

outcome, including litter size, weight, or gestational age at delivery(Fig. 1). We then evaluated whether the absence of viral infectionto the placenta and decidua was responsible for the lack of effectin pregnancy outcome. To test this hypothesis, replicative viralloads (PFU/ml) were determined using a limiting dilution plaqueassay on frozen tissue taken from the placenta and decidua (local),and spleen and lymph node (systemic and classical target organ

for MHV-68) (56) from pregnant mice who had received MHV-68on E8.5 of pregnancy and sacrificed 3 or 9 d after viral adminis-tration (dpi).Three days after viral administration, we observed a high viral

load in the spleen and lymph nodes. Interestingly, viral titers in thedecidua were significantly higher than those in the spleen (Fig. 2A).The placenta was also infected, but overall viral titers were lowerthan those in the spleen.Nine days after viral administration, we observed a substantial

increase in splenic viral titers (higher than 3 logs), a slight decreasein the decidua, and increasing viral titers in the placenta (Fig. 2B).Most notably, no viruses were detected in the fetus of infectedmothers using the plaque assay or by PCR, even 9 d after viraladministration (Fig. 2A and data not shown).These results suggest that the viral load administered to the mice

is able to infect all the organs, including placenta and decidua;however, in contrast to the effect observed with poly(I:C), the viralinfection in the placenta and decidua did not seem to have an effecton pregnancy outcome. To better understand the differences be-tween the two responses [virus versus poly(I:C)], we evaluated thecytokine/chemokine profile in the placenta and spleen of miceinfected with MHV-68. MHV-68 infection did not induce the pro-duction of chemokines and inflammatory cytokines seen with poly(I:C) administration (Fig. 2C, 2D). Moreover, we observed inhi-bition on the production levels of IL-6, MIP-1b, and RANTES inthe placenta of MHV-68–infected mice. These data suggest thatthe change in the cytokine profile may play an important role inthe induction of preterm labor/delivery.

TLR-3 is necessary for the control of early viral infection

The effects of poly(I:C) in pregnancy outcome (and, in particular,preterm labor) are mediated through TLR-3 (30, 35), and thispattern recognition receptor plays an important role in theimmune response to herpesviruses (57). Moreover, some virusessuch as influenza A and Kaposi’s sarcoma-associated herpesvirushave been associated with activation of the TLR-3 pathway inhumans (58, 59). Thus, we evaluated the role of TLR-3 onMHV-68 infection during pregnancy by using TLR-3 KO mice.Similarly, as in the wt mice, administration of MHV-68 had noeffect on the duration of pregnancy (i.e., there was no premature

FIGURE 1. Pregnancy outcome in wt mice infec-

ted with MHV-68. Wt pregnant mice were infected

i.p. with 13 106 PFUMHV-68 or vehicle at E8.5 and

sacrificed at E17.5. A, Pups, uterus, and gestational

sacs from wt treated with vehicle, and B, pups, uterus,

and gestational sac from wt infected with MHV-68,

showing no differences in gross anatomy. C, Fetal

weight at the time of delivery. Note the lack of differ-

ence between the two groups. n = 6 mice per group.

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FIGURE 2. Effect of MHV-68 viral infection in pregnant mice. Viral titers as PFU/ml were determined in wt pregnant mice infected with MHV-68 (1 3106 PFU) 3 d (E11.5; A) and 9 d (E17.5; B) postinfection. Viral titers were observed in lymph nodes, placenta, decidua, and spleen, but were absent in the

fetuses. pp , 0.05, decidua versus spleen. Placenta (C) and spleen (D) cytokine profile was determined in wt pregnant mice treated with poly(I:C) or

MHV-68 4 and 9 d postinfection, respectively. pp , 0.05, MHV-68 versus control; #p , 0.05, poly(I:C) versus MHV-68. E and F, Viral titers as PFU/ml

were determined in TLR-3 KO pregnant mice infected with MHV-68: E, 3 d (E11.5), and F, 9 d (E17.5) postinfection. Note the high levels of viral titers in

lymph nodes, placenta, decidua, and spleen, but absent in the fetuses. Bars show median 6 SEM. n = 6 mice per group.

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labor). However, we observed higher viral titers in all tissues,including the decidua and placenta of TLR-3 KO mice, both at3 and 9 dpi (Fig. 2E, 2F). The fetuses of either wt or TLR-3 KO-infected mothers were not infected, suggesting that TLR-3 is notrequired for the protection of fetuses against viral invasion.We then evaluated whether the viral dose used in this study is

able to mount an adaptive immune response by assessing sero-conversion. IgG anti–MHV-68 were significantly higher in both wtand TLR-3 KO-infected pregnant mice than in noninfected ani-mals. However, when we compared the response between wt andTLR-3 KO, we observed that IgG anti MHV-68 levels were sig-nificantly lower in the TLR-3 KO (Fig. 3). These results confirmthat MHV-68 infections during pregnancy are able to mount a spe-cific adaptive immune response characterized by the presence ofanti–MHV-68 IgG. The presence of higher viral titers and lowlevels of anti–MHV-68 IgG in the TLR-3 KO mice indicated thatTLR-3 expression is required to elicit a potent antiviral Ab re-sponse against this dsDNA virus.

Effect of MHV-68 infection on the placenta

Because we observed viral infection of the placenta and decidua inmothers, the next objective was to determine the effect of MHV-68infection on the placental and decidual pathology. Thus, utero-placental units were collected at 9 dpi, and H&E staining wasperformed. All histological samples were analyzed in a blindedmanner by an independent animal pathologist (C.B.). Sites ofedema were observed only in the decidua of infected mice; necrosisand inflammation foci were observed in the labyrinth of infectedmice (Fig. 4A). Significant pathologic changes present within thelabyrinth of TLR3 KO-treated mice included an overall tissuehyperesinophilia, nuclear pyknosis, cellular fragmentation, andmultifocal loss of tissue of architecture (necrosis) in the labyrinth(Fig. 4B).We then evaluated changes of the number and distribution of NK

(lectin-positive) cells and macrophages (F4/80 positive). NK cellswere observed in decidua of control as well as infected mice. Nochange in the location of these cells was observed as a result ofthe infection (data not shown). Macrophages are mainly localizedin the myometrium and decidua of the pregnant mice (Fig. 4C,arrows). Fewmacrophages are also found in the placenta. No differ-ences in the distribution and number of macrophages were foundin the decidua and placenta from infected and control groups.In addition, we observed an increase in collagen deposition in the

perivascular spaces of infected animals, predominantly in the lab-yrinth layer, as compared with control mice (Fig. 4D). The presence

of collagen in the perivascular areas suggests that an active repairprocess was taking place in the placenta of infected mice. Similarchanges were observed in TLR-3 KO mice (Fig. 4D).

Fetal response to placental infection

Although we observed high viral titers in the placenta, no virus wasdetected in any of the fetuses, as determined by the limiting dilutionplaque assay and confirmed by PCR. To determine whether the lackof fetal infection was due to inability of the virus to infect fetalcells, mouse embryonic fibroblast cells were isolated and infectedwith MHV-68 in vitro with a similar dose as that used for tropho-blast cells (see below). Eighty percent of embryonic fibroblastswere infected by MHV-68 in less than 12 h, as shown by GFP-positive signal; however, the viral infection induced a lytic effect(data not shown). These results suggest that the placenta is func-tioning as an immunological barrier, capturing the virus and pre-venting it from reaching the fetus. To determine whether theinfection of the placenta could have an effect on the developingfetus, we next assessed fetal morphology from mice infected withMHV-68 during pregnancy versus those receiving a placebo.Analysis of the fetuses revealed that viral infection of the mother

has a transient effect on development. Three dpi, fetuses of infectedmothers were smaller and had a lower weight (in both wt and TLR-3KO mice), although this effect was more evident in the TLR-3 KOgroup (Supplemental Fig. 1A). Furthermore, we observed a delay inthe process of differentiation of the eye, tails, and limbs (Supple-mental Fig. 1B). However, after 9 d, the differences between thefetuses from infected and noninfected mice from both wt andTLR-3 KO were no longer detectable (Supplemental Fig. 1C).These important observations are evidence of the remarkable plas-ticity of the developing fetus.Because we observed an early effect on fetal development, we

then evaluated the integrity of fetal organs and tissues using mi-croscopic sections. Despite the absence of viruses in the fetuses, wenoted severe pathological changes in the fetal tissues of infectedmothers from both wt and TLR-3 KO.We observed hydrocephalus,defined as an increase in the subarachnoid space, in the brains of allfetuses from infected mothers (Fig. 4E). We did not see anychanges in the lateral ventricles, nor did we detect abnormal im-mune infiltration or white matter damage.In the thoracic cavity, the pathological changeswere characterized

by the presence of hemorrhage inside the lungs and pericardium inall treated animals compared with the controls (Fig. 4F). However,there was no damage in the abdominal cavity or the limbs.We then evaluated the cytokine profile in fetuses from infected

and control mothers. Interestingly, 9 dpi, we observed a significantincrease in the levels of fetal proinflammatory cytokines (Fig. 4G),including high levels of IFN-g and TNF-a. The presence of thesetwo cytokines may explain some of the morphological changesobserved in these fetuses.Collectively, these data suggest that although there is no demon-

strable fetal viral infection, the presence of an active inflammatoryresponse in the placenta and decidua can have a direct effect onfetal development.

Trophoblast-viral interaction

To understand the implications of these observations in humans,trophoblast cells were isolated from first trimester human placentasand infected with GFP-MHV-68 for either 24 or 48 h. Infection wasmonitored by the presence of GFP. Positive GFP-MHV-68–infectedtrophoblast cells were observed �12 h postinfection and remainedviable up to 6 dpi (Fig. 5A).Next, we determined the cytokine response induced by MHV-68

in trophoblast cells in vitro. Contrary towhat we observedwith poly

FIGURE 3. Seroconversion in wt and TLR-3 KO pregnant mice infected

with MHV-68. Wt and TLR-3 KO mice were infected i.p. with MHV-68

(1 3 106 PFU) or vehicle at E8.5. Serum samples were collected 9 dpi,

and levels of IgG Abs were determined by ELISA. Note the high levels of

IgG anti–MHV-68 Abs in the wt treated group compared with controls. A

significantly lower response was observed in TLR-3 KO-treated mice. n = 6

mice per group. pp , 0.05.

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(I:C) treatment (which induces robust proinflammatory cytokine/chemokine production [30]), MHV-68 has a unique profile char-acterized by inhibition of chemokines and lack of production ofproinflammatory cytokines (Table I). However, we observed a mildincrease in modulatory cytokines, such as IL-6, IL-1b, and theimmune suppressor vascular endothelial growth factor (VEGF)(60) (Table I). Evaluation of the cytokine response by trophoblastisolated from wt mice or TLR-3 KO mice to MHV-68 infectionrevealed a similar profile in terms of the inhibition of chemokines.However, contrary to the wt mice, we did not observe an increase inIL-1b and IL-6, suggesting that TLR-3 expression might be neces-sary for the production of these two cytokines (data not shown).

Differential regulation and role of the TLR/NF-kB pathwayduring MHV-68 infection in trophoblast cells

The generalized inhibition of chemokine expression and the in-creased secreted levels of the immune suppressor VEGF representan important immune-regulatory mechanism by which MHV-68can escape immune surveillance and successfully infect trophoblastcells.Our next objective was to determine the potential mechanism by

which MHV-68 inhibits chemokine production. Because the NF-kBpathway is a major regulator of cytokine and chemokine production,we evaluated the status of p65 (a regulatory subunit of NF-kB) introphoblast cells followingMHV-68 infection. Trophoblast cells arecharacterized by constitutive NF-kB activity and cytokineproduction (20, 30). Therefore, p65 is localized in the nuclei ofthe cells (Fig. 5B). FollowingMHV-68 infection, no nuclear stainingwas observed along with a decrease in cytoplasmic expression (Fig.5B), suggesting that the NF-kB pathway is inhibited in trophoblastcells infected by MHV-68. The viral-induced inhibition of NF-kBcorrelates with the inhibition on chemokine production observed inthe infected trophoblast and placenta.TLR/MyD88 signal has been shown to be important in the

regulation of MHV-68 replication (61). Trophoblast cells expressTLRs and are able to respond to TLR ligands (20, 30); therefore,we evaluated whether viral infection could affect the expression orfunction of TLRs. MHV-68 infection induces TLR-2 andTLR-4 mRNA expression in human first trimester trophoblastcells in a time-dependent manner. In contrast, TLR-3 mRNA lev-els were not affected or were decreased postinfection (Fig. 5C).The significant increase in TLR-2 expression followingMHV-68

infection indicates a potential association between TLR-2 and theviral adaptation to the host. Thus, wt trophoblasts, trophoblasts sta-bly transfected with a TLR-2 DN or TLR-1 DN, were infected withMHV-68, and 48 h postinfection, supernatants from these cultureswere collected and transferred to new cultures of wt first trimestertrophoblast cells. No de novo infection was observed followingtransfer of supernatants obtained from TLR-2 DN trophoblasts,and similarly, TLR1 DN reinfectivity was greatly reduced, suggest-ing that TLR-2 is necessary for viral reactivation, replication, oregress (Supplemental Fig. 2).

MHV-68 infection sensitizes to bacterial LPS

Because we observed that MHV-68 infection leads to an increasein the expression levels of TLR-4 and TLR-2, we next tested thehypothesis that viral infection in early pregnancy could affect theresponse to microbial products. We injected MHV-68 into pregnantwt mice at E8.5 (early pregnancy), followed by LPS injection atE15.5 (late pregnancy). We selected a dose of LPS that has a modesteffect on pregnancy outcome (20 mg/kg) (30). Control mice receivedonly vehicle or only LPS at E15.5. LPS treatment of MHV-68–infected pregnant mice induced preterm labor/delivery in less than24 h in all of the treatedmice (Fig. 6). Anatomical examination of the

mothers showed vaginal bleeding and 100% fetal death in theMHV-68 plus LPS-treated group (Supplemental Fig. 3). LPS admin-istration, without previous viral infection, induced preterm labor/de-livery in 29% of cases, and we did not observe major anatomicalchanges in the mother or the fetus (Supplemental Fig. 4).

DiscussionWe demonstrated that maternal viral infection can lead to pro-ductive replication in the placenta and a fetal inflammatory re-sponse, even though the virus is not detected in the fetus. Theexperiments described in this work are intended to show that viralinfection of the placenta can elicit a fetal inflammatory response,which in turn can cause organ damage and, potentially, downstreamdevelopmental deficiencies. Furthermore, we demonstrated thata viral infection of the placenta may sensitize to bacterial infectionand promote preterm labor.Pregnant women are exposed to many infectious agents that are

potentially harmful to the fetus. The risk evaluation has been fo-cused on whether there is a maternal viremia or fetal transmission(62). Viral infections that are able to reach the fetus by crossingthe placenta might have a detrimental effect on the pregnancy (63,64). It is well accepted that in those cases infection can lead toembryonic and fetal death, induce miscarriage, or induce majorcongenital anomalies (62, 65). However, even in the absence offetal viral infection, the fetus could be adversely affected by thematernal response to the infection. Examples are infections withHIV, hepatitis B, varizella zoster virus, and parvovirus B19,among others (5, 28, 66, 67). Indeed, viral crossing of the placentamay be the exception rather than the rule.One of the main questions of this study was how amicroorganism,

in this case a virus, might initiate a response that may not lead to pre-term labor, but would alter the immunologic balance at the maternalfetal interface. Poly(I:C) has been used in several studies as a modelfor TLR3activation and shown to be a potent inducer of preterm labor(68). However, the use of MHV-68, which is able to activateTLR-3 (61), did not show the same outcome. Our results indicatethat only a condition characterized by the expression of inflamma-tory cytokines at the maternal-fetal interface will trigger a cascade ofevents leading to the termination of the pregnancy. In contrast, a viralinfection in the placenta that triggers a mild inflammatory responsewill not terminate the pregnancy, but is able to activate the immunesystem not only of the mother, but of the fetus as well.It is critical to take into consideration the fact that during

pregnancy it is not only the maternal immune system responding,but also the fetal/placental unit. Our results further support theimmunological role of the placenta and the fetus affecting theglobal response of the mother to microbial infections. This isrelevant for making decisions associated with treatment and pre-vention during pandemics.An important observation in this study is the fact that even

though there is a high viral titer in the placenta and decidua, novirus was detected in the fetus. This result further confirms our andothers’ studies suggesting that the placenta is an active barrier,able to control an infection and protect the fetus (49, 69–72).However, the inflammatory response originated on the maternalside has a negative impact on the fetus and triggers a fetal in-flammatory condition.Fetal inflammatory response syndrome (FIRS) is a condition in

which, despite an absence of cultivable microorganisms, neonateswith placental infections have very high circulating levels of in-flammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-a (73–75).We observed a similar outcome in our animal model in whichMHV-68 infection of the placenta triggers a fetal inflammatory

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FIGURE 4. Effect of MHV-68 viral infection on the maternal/fetal interface. Morphological changes were observed in the placenta and decidua of

MHV-68–infected pregnant mice associated with the following: A, edema (p) in the D, but absent in the L and S. Upper and lower panel, scale bar, 200 and

300 mm, respectively. B, Necrosis in placenta, marked loss of cellular detail, fragmentation, hypereosinophilia (boxes) in the labyrinth subjacent to the

epithelium (E), and necrosis of scattered giant cells (arrowheads). These changes were more accentuated in the TLR-3 KO compared with wt mice. Scale

bars, 200 mm. C, Immunohistochemistry for F4/80-positive macrophages (brown) localized in the myometrium (MYO) and decidua (DEC) at E11.8 and

E17.5. Black arrows show the edge between myometrium and decidua. Original magnification 320. D, Increase in collagen deposition (arrows) in the

labyrinth layer of MHV-68–infected mice using Trichromic Mason staining (original magnification 320. E, Presence of fetal brain hydrocephalus (black

arrows) in wt and TLR-3 KO MHV-68–infected mice (middle and right panel) compared with normal controls (left panel). Note the width of LVs. Original

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response similar to the one observed in FIRS, even though the viruswas not able to reach the fetus. In the case of human FIRS, thesecytokines have been shown to affect the CNS and the circulatorysystem (76). In this study, we showed that fetal morphologic ab-normalities may be caused by fetal proinflammatory cytokines,such as IL-1, TNF-a, MCP-1, MIP1-b, and IFN-g. Beyondmorphological effects on the fetal brain, the presence of FIRSincreases the future risk for schizophrenia, neurosensorialdeficits, and psychosis induced in the neonatal period (77–79).

Therefore, we propose that an inflammatory response of theplacenta, which alters the cytokine balance in the fetus, mayaffect the normal development of the fetal immune system,leading to anomalous responses during childhood or later in life(77–79). One example of this is the differential responses inchildren to vaccination or the development of allergies (11, 80).Antenatal infections can have a significant impact on later vaccineresponses. We can observe this type of outcome in other conditionsassociated with placental infection, such as malaria. A few studiessuggest that surviving infants with placental malaria may sufferadverse neurodevelopmental sequelae and may have an abnormalresponse to a later infection with the parasite (81). In the majority ofthe cases, the parasite did not reach the fetus, but the inflammatoryprocess in the placenta affected the normal fetal development (82).The differential cytokine response observed between poly(I:C)

and MHV-68 infection questioned the role of TLRs during a viralinfection. However, our finding demonstrates a unique interactionbetween the virus and TLR expression and function at the placentaand decidua. We confirmed that TLR-3 is necessary for the controlof viral replication, as demonstrated by the presence of higher titersof MHV-68 found in the TLR-3 KO mice. According to this, clin-ical data proved that TLR-3 controls herpesvirus infection, becausechildren with a TLR-3 deficiency are very susceptible to HSV-1–induced encephalitis (83). In contrast, the virus requires TLR-2and TLR-1 expression for its own replication. These findings openthe possibility of using TLR-2 or TLR-1 antagonists as potentialagents for preventing herpes viral replication.Viral infectionmay influence theoutcomeofaconcurrentbacterial

infection (84); however, to date there is no evidence indicatingwhether a viral infection sensitizes to bacterial infection duringpregnancy.We showed thatMHV-68–infected pregnant mice under-go preterm labor following injection of a low dose of LPS, whichhas almost no effect on noninfected mice. These results suggest thata viral infection during pregnancy increases the risk of preterm labor

magnification 310. F, Fetal thoracic cavity from WT and TLR-3 KO pregnant mice infected with MHV-68. Note the areas of hemorrhage in lung right

middle lobe and pericardium. G, Fetal cytokine profile from pregnant mice infected with MHV-68. Fetal lysates were obtained, and cytokines/chemokines

were measured by Luminex. Bars show median 6 SEM. n = 6 mice per group. pp , 0.05. Figures are representative of six animals per group and three

independent experiments. D, decidua; L, labyrinth; LV, lateral ventricle; S, sponginous layer.

FIGURE 5. Primary cultures of human first trimester trophoblast cells

infected with GFP-MHV-68. A, Infection was monitored by the presence of

GFP-labeled MHV-68. Positive GFP-MHV-68–infected trophoblast cells

(white arrows) were observed �12 h postinfection. B, Inhibition of NF-kB

activity in MHV-68–infected trophoblast cells. Expression of p65 was deter-

mined by immunofluorescence. Note the decrease in the number of tropho-

blast cells with nuclear p65 (white dots) following MHV-68 infection. C,

Expression of TLR-2, -3, and -4 by human first trimester trophoblast cells

followingMHV-68 infection. TLR-2, -3, and -4 expressions were determined

by real-time quantitative RT-PCR. Note the significant increase on TLR-2

and -4 expression and decrease in TLR-3 in MHV-68–infected cells com-

pared with the control. n = 3 samples per group. pp , 0.05.

Table I. Cytokine/chemokine profile of poly(I:C)- or MHV-68–infectedhuman trophoblast

Factor Poly(I:C) MHV-68

IL-6 ↑6.3 ↑3.3IL-1B — ↑4.5VEGF — ↑1.2FGF-2 ↑2.0 ↑5.7IL-8 ↑7.2 ↓11.5MCP-1 ↑1.7 ↓162.5RANTES ↑2.3 ↓2.4GRO-a ↑6.5 ↓8.7IL-1a ↑115.8 —GM-CSF ↑261.1 —IL-12p70 ↑2.2 ↓2.0IFN-g ↑6.9 ↑1.5IP-10 ↑225.2 ↓3.3IFN-a ↑2.6 ↓1.5IFN-b ↑60.0 ↑3.0

Cytokine/chemokine profile of poly(I:C)-treated and MHV-68–infected humanprimary trophoblast. Isolated human first trimester trophoblast cells were treatedwith either 25 mg/ml poly(I:C) or MHV-68 at a multiplicity of infection of 1.4 for72 h. Supernatants were collected, and cytokines and chemokines were measured byMultiplex. Fold changes (mean 6 SEM) of cytokine/chemokine secretions with poly(I:C) and MHV-68. n = 3 samples per group. pp , 0.05.

↓, Decrease; ↑, increase; FGF-2, fibroblast growth factor-2; GRO-a, growth-related oncogene-a; IP-10, IFN-g–inducible protein-10.

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or maternal death in response to other microorganisms, such asbacterial infection. In the pandemic of 1918, high rates of pregnancyloss and preterm delivery were reported (1), and during the pan-demic of 1957–1958, an increase in CNS defects and other adverseoutcomes were reported. In the more recent H1N1 influenza virusinfection, 13% of the deaths were pregnant women (3). In all thesecases, a bacterial-associated complication was reported.In conclusion, we demonstrate that even in the absence of fetal

viral infection, the inflammatory response originating in the pla-centa and decidua induces an inflammatory process with potentialdamage in fetal organs. It is therefore essential to evaluate thepresence of maternal viral infections prenatally to prevent long-term adverse outcomes for the child and the mother. Future studiesare needed to develop useful biomarkers for viral infections duringpregnancy even in a subclinical state as a strategy of early detectionand prevention of fetal damage and maternal mortality. Further-more, it is extremely important to take into consideration the pos-sibility of placental infection when determining a response toemerging infectious disease threats.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 6. MHV-68 infection sensitizes to bacterial LPS. Wt mice were

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Corrections

Cardenas, I., R. E. Means, P. Aldo, K. Koga, S. M. Lang, C. Booth, A. Manzur, E. Oyarzun, R. Romero, and G. Mor. 2010. Viral infec-tion of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor. J. Immunol. 185:1248–1257.

The sixth author’s middle initial was omitted. The correct name is Carmen J. Booth.

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