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The Journal of Immunology, 2004, 173: 4286-4296.
Copyright © 2004 by The American Association of Immunologists

Divergent Trophoblast Responses to Bacterial Products Mediated by TLRs

Vikki M. Abrahams*, Paulomi Bole-Aldo*, Yeon Mee Kim{ddagger}, Shawn L. Straszewski-Chavez{dagger}, Tinnakorn Chaiworapongsa§, Roberto Romero and Gil Mor1,*

Departments of * Obstetrics and Gynecology and {dagger} Molecular, Cellular and Developmental Biology, Yale University School of Medicine, New Haven, CT 06520; Departments of {ddagger} Pathology and § Obstetrics and Gynecology, Wayne State University, Detroit, MI 48202; and Perinatology Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intrauterine infections have been associated with pregnancy complications that are also linked with increased trophoblast apoptosis. TLRs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. We hypothesize that intrauterine infections may cause the excessive trophoblast cell apoptosis observed in abnormal pregnancies and that TLR may provide a mechanism of pathogenesis. In this study we describe the expression and function of TLR-2 and TLR-4 in first trimester trophoblast cells. Although ligation of TLR4 induced cytokine production by trophoblast cells, TLR-2 activation induced apoptosis. TLR-2 mediated apoptosis was dependent upon the Fas-associated death domain, the inactivation of the X-linked inhibitor of apoptosis, and the activation of caspases 8, 9, and 3. These results suggest that certain intrauterine infections may directly induce trophoblast cell death through TLR-2. Our findings provide a novel mechanism of pathogenesis for certain pregnancy complications in which there is engagement of the innate immune system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human endometrium becomes an immunologically unique site during pregnancy that must accept the semiallogenic fetus and placenta, while maintaining host defense against an array of microbial pathogens. It is thought that the trophoblast at the maternal-fetal interface also actively participates in preventing allorecognition (1, 2, 3), and in the control of pathogens that may compromise the fetal wellbeing (4). Indeed, intrauterine infections have been associated with pregnancy complications, such as preterm labor and delivery, intrauterine growth restriction (IUGR), 2 and preeclampsia (5, 6, 7, 8, 9). The association between preterm labor and intrauterine infections is now well established and infections have been reported as responsible for up to 40% of cases (10). Furthermore, 80% of preterm deliveries at <30 wk of gestation have evidence of infection (11) and there is evidence that these infections precede the development of preterm labor and delivery (6). Infection, therefore, represents an important and frequent mechanism of disease, yet, the precise mechanisms by which complications of pregnancy arise remain undefined.

The innate immune system responds to infection through a system of pattern recognition receptors (PRR) which recognize conserved sequences known as pathogen-associated molecular patterns (PAMPs), such as bacterial lipoproteins, LPS, and dsRNA (12). These PAMPs are expressed on the surface of, and are unique to, microorganisms. PAMPs, therefore, represent infectious non-self. One of the main families of PRR are TLRs. Originally discovered in Drosophila, the Toll gene was found to be critical for dorsoventricular polarization during embryonic development (13), but later was also found to have immunoprotective properties in the adult fly (14, 15). To date, 11 mammalian Toll homologues have been identified and designated, TLR 1–11 (16, 17). Although extracellularly each TLR is distinct in its specificity, all TLRs contain a leucine-rich repeat extracellular domain and share a common intracellular domain which is homologous to the IL-1R type-1 (IL-1R) intracellular signaling domain, called the Toll/IL-1R homology region (18). Following ligation, TLR signal through the adapter molecule, MyD88, to activate the NF-{kappa}B pathway, which results in an immune response characterized by the production of cytokines, antimicrobial products, and the regulation of costimulatory molecules (12, 16).

Studies in term placenta have demonstrated the expression of TLR-2 and TLR-4 (19, 20). TLR-2 recognizes bacterial lipoproteins, peptidoglycan (PDG) and lipoteichoic acid (LTA) (21, 22, 23), while TLR-4 recognizes Gram-negative bacterial LPS (24). Explants from term placenta have been shown to produce IL-6 and IL-8 following ligation of TLR-2 or TLR-4 (19). We, therefore, hypothesize that intrauterine infections during pregnancy may have a direct effect upon trophoblast cells through TLRs. Because infection may affect placental development and function, and subsequently lead to complications of pregnancy such as preterm labor and delivery, IUGR, and preeclampsia (6, 25, 26), the aim of this study was to characterize the expression and function of TLR-2 and TLR-4 in first trimester trophoblast cells. In this study, we report for the first time that first trimester trophoblast cells express both TLR-2 and TLR-4 and that ligation of TLR-2 by bacterial products mediates activation of the apoptotic pathway in trophoblast cells. In contrast, ligation of TLR-4 results in the production of cytokines by first trimester trophoblast cells. These results suggest a novel and direct mechanism by which intrauterine infections may promote an increase in trophoblast cell apoptosis which might lead to a complicated pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient samples

First trimester placentas (n = 5) were obtained from elective terminations of normal pregnancies performed at Yale-New Haven Hospital (New Haven, CT). All patients signed consent forms and the use of patient samples was approved under Yale University’s Human Investigations Committees.

Reagents and Abs

LPS isolated from Escherichia coli (0111:B4) and camptothecin (CPT) were purchased from Sigma-Aldrich (St. Louis, MO). PDG isolated from Staphylococcus aureus, and LTA isolated from Bacillus subtilis and S. aureus were purchased from Invivogen Life Technologies (San Diego, CA). The caspase-8 inhibitor (Z-IETD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) were obtained from BD Pharmingen (San Diego, CA). Immunohistochemical staining for human TLR-2 was performed using the mouse IgG2a mAb, clone TLR2.1 (Alexis Biochemicals, San Diego, CA) and for human TLR-4 using the rabbit polyclonal Ab, clone H-80 (Santa Cruz Biotechnology, Santa Cruz, CA). This was confirmed by Western blot analysis using the same Abs. The mouse anti-X-linked inhibitor of apoptosis (XIAP) mAb was obtained from BD Transduction Laboratories (San Diego, CA). The mouse mAb for {beta}-actin was purchased from Sigma-Aldrich. Specific signals were detected using either a peroxidase-conjugated horse anti-mouse, or a peroxidase-conjugated goat anti-rabbit secondary Ab (Vector Laboratories, Burlingame, CA).

Isolation and culture of trophoblast cells from first trimester placenta

Primary trophoblast cells from first trimester placentas (n = 5) were prepared as described previously (2). Briefly, tissue specimens were washed with cold HBSS (Invitrogen Life Technologies) to remove excess blood. Cells were scraped from the membranes, transferred to trypsin-EDTA (Invitrogen Life Technologies, Carlsbad, CA) digestion buffer and incubated at 37°C for 10 min with shaking. An equal volume of DMEM medium (Invitrogen Life Technologies) containing 10% FBS was added to inactivate the trypsin. This mixture was vortexed for 20 s, allowed to sediment, and the supernatant was collected. This was repeated twice and the collected supernatant was centrifuged at 1500 rpm for 10 min. Contaminating RBC were removed by resuspending the cellular pellet with HBSS, layering this over the same volume of Lymphocyte Separation Media (ICN Biomedicals, Aurora, OH), and centrifuging at 2000 rpm for 25 min. The cellular interface containing the trophoblast cells was collected and resuspended in DMEM supplemented with 10% normal human serum (Gemini Bio-Products, Woodland, CA) and cultured at 37°C/5% CO2 for no more than three passages. Purity of the trophoblast cells was >98% as determined by immunostaining for cytokeratin-7 (Sigma-Aldrich) (27).

Culture of cell lines

All cells were maintained at 37°C/5% CO2. Two first trimester trophoblast cell lines were used in these studies. The human cytotrophoblast cell line, 3A, which was transformed by SV40 ts30 (28) was purchased from American Type Culture Collection (Manassas, VA). The human extravillous trophoblast cell line, HTR8 (referred to from hereon as H8) (29), was a kind gift from Dr. C. Graham (Queens University, Kingston, Ontario, Canada). The monocytic cell line, THP-1, was a kind gift from Dr. P. Guyre (Dartmouth Medical School, Lebanon, NH). Cell lines were cultured in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS (HyClone, South Logan, UT), 10 mM HEPES, 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 100 nm of penicillin/streptomycin (Invitrogen Life Technologies).

Transfection of trophoblast cells with the dominant-negative Fas-associated death domain (FADD-DN)

H8 and 3A cells were transiently transfected with an expression plasmid containing the AU1-tagged FADD-DN which was a kind gift from Dr. M. Aguero (National Institute of Dental and Craniofacial Research/National Institutes of Health, Bethesda, MD) and has been previously described (30). pcDNA 3.1 was used as a vector control. Briefly, 1 x 106 cells were seeded into a 60-mm dish and cultured overnight until 80–90% confluent. Cells were then transfected for 18 h with 8 µg of DNA using Lipofectamine 2000 (Invitrogen Life Technologies) in a 1:2 ratio. Following transfection, cells were allowed to recover in growth media for 24 h before a treatment experiment was performed.

Immunohistochemistry

The cellular localization of TLR-2 and TLR-4 expression by trophoblast cells from first trimester placenta was performed as previously described (2). In short, placental samples were fixed with 4% paraformaldehyde and then paraffin embedded. Sections of placenta (5 µm) or paraformaldehyde-fixed trophoblast cells, previously adhered to glass slides, were blocked with 10% horse or goat serum in PBS for 1 h at room temperature. Following three washes with PBS, samples were incubated overnight at 4°C with either the anti-TLR-2 mAb or the anti-TLR-4 Ab. Mouse IgG1 or rabbit serum served as negative controls. After three washes with PBS, specific staining was detected by incubating with either a peroxidase-conjugated horse anti-mouse Ab (1/1000 dilution) or a peroxidase-conjugated goat anti-rabbit Ab (1/1000 dilution) for 1 h followed by a 5-min incubation with diaminobenzidine substrate (Vector Laboratories). Cells and tissue sections were then counterstained with hematoxylin (Sigma-Aldrich) before dehydration with ethanol and Histosolve (Shandon, Pittsburgh, PA). Slides were then mounted with Permount (Fisher Scientific, Pittsburgh, PA) and visualized by light microscopy.

Western blot analysis

For analysis of intracellular proteins, cells were lysed using 1% Nonidet P-40 and 0.1% SDS in the presence of protease inhibitors (Roche, Indianapolis, IN). Protein concentrations were calculated by bicinchoninic acid assay (Pierce, Rockford, IL). Proteins were then diluted with gel loading buffer to 20 µg and boiled for 5 min. Proteins were resolved under reducing conditions on either 10 or 12% SDS-PAGE gels and then transferred onto polyvinylidene fluoride paper (PerkinElmer, Boston, MA). Membranes were blocked at room temperature for 1 h with 5% fat-free powdered milk (FFPM) in PBS/0.05% Tween 20 (PBS-T). Following three washes for 10 min each with PBS-T, membranes were incubated overnight at 4°C with primary Ab in PBS-T/1% FFPM. Following this incubation, membranes were washed three times as before and then incubated at room temperature for 1 h with the appropriate secondary Ab conjugated to peroxidase (Vector Laboratories) in PBS-T/1% FFPM. Following three washes for 10 min each with PBS-T and three washes for 10 min each with distilled water, the peroxidase-conjugated Ab was detected by ECL (PerkinElmer). All experiments were repeated at least three times. {beta}-actin was used as internal control, in addition to Ponseau Red, to validate the amount of protein loaded onto the gels.

RT-PCR

Total RNA was isolated from the first trimester trophoblast cells lines and THP-1 cells using the RNeasy kit from Qiagen (Valencia, CA). Reverse transcription was performed on 5 µg of total RNA using the First Strand cDNA Synthesis kit from Amersham Biosciences (Buckinghamshire, U.K.) according to the manufacturer’s directions. The primers used for amplification of human TLR-1 have been described previously (31) and have the following sequences: 5'-TTTGAAAATTGTGGGCACCTTACTG-3', 5'-AAGCAACATTGAGTTCTTGCAAAGC-3'. Thirty cycles of PCR were performed at 95°C for 15 s, 58°C for 20 s, and 72°C for 30 s. The size of the product was 350 bp. The primers used for amplification of human TLR-6 have also been described previously (32) and have the following sequences: 5'-AGAACTCACCAGAGGTCCAACC-3', 5'-GAAGGCATATCCTTCGTCATGAG-3'. Thirty cycles of PCR were performed at 95°C for 30 s, 65°C for 1 min, and 72°C for 1 min. The size of the product was 500 bp.

Cell viability assay

The effects of the TLR agonists on trophoblast cell viability was determined using the CellTiter 96 viability assay (Promega, Madison, WI), as previously described (33). First trimester trophoblast cells were plated in wells of a 96-well plate at 1 x 104 cells per well in growth media and cultured until 70% confluent after which the media was then replaced with the reduced serum medium, Opti-MEM (Invitrogen Life Technologies), and cultured for another 4 h before treatment. Following treatment, the CellTiter substrate, [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium] (MTS), was added to all wells and following a 1–4 h incubation at 37°C, ODs were read at 490 nm. All samples were assayed in triplicate and cell viability was presented as a percentage relative to the untreated control (medium).

Caspase activity assay

The effects of TLR ligation on caspase activity was determined using the Caspase-Glo assay (Promega). Briefly, 10 µg of whole cell lysates were incubated at room temperature in the dark for 1 h with either the caspase 3, 8, or 9 substrate. Following incubation, luminescence was measured using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). The amount of luminescence detected as relative light units was proportional to caspase activity. All samples were assayed in triplicate.

Flow cytometry studies

Apoptosis was monitored by flow cytometry as described previously (33). Following treatment, adherent trophoblast cells (1 x 106) were harvested with 0.05% trypsin-EDTA (Invitrogen Life Technologies). Cells were then washed twice with cold PBS and centrifuged at 1500 rpm for 5 min at 4°C. The cell pellet was then resuspended in 1 ml of cold PBS and incubated on ice for 20 min with propidium iodide (Sigma-Aldrich) at 1 µg/ml and Hoechst 33342 dye (Molecular Probes, Eugene, OR) at 5 µg/ml. Unstained cells served as a negative control. Samples were then analyzed using a FACS Vantage (BD Biosciences, San Diego, CA) with 488 nm/UV dual excitation. Propidium iodide staining was detected in the FL-2 channel and Hoechst staining was detected in the SSc-W channel. Data was analyzed using CellQuest software (BD Biosciences).

Cytokine array studies

The effect of TLR ligation on trophoblast cytokine production was determined using a Human Cytokine Array 3.1 (RayBiotech, Atlanta, GA) according to the manufacturer’s instructions. Briefly, 100 µg of protein from whole cell lysates were incubated with the array membrane. Following incubation with primary biotin-conjugated Abs and HRP-conjugated streptavidin, detection of signals was performed by ECL (PerkinElmer). The intensity of the signals was quantified by densitometry using a digital imaging analysis system and 1D Image Analysis Software (Kodak Scientific Imaging, Melville, NY). The signal intensities were normalized against the positive controls on each array membrane. Fold differences were then calculated against the untreated control experiment.

Statistical analysis

Data are expressed as mean ± SD. Statistical significance (p < 0.05) was determined using one-way ANOVA with the Bonferonni correction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
First trimester trophoblast cells express TLRs

The first objective of this study was to determine the expression pattern of TLR-2 and TLR-4 in first trimester placental villous and extravillous tissues. As shown in Fig. 1A, positive immunoreactivity was observed for TLR-2 and TLR-4 in first trimester placental villi (i and iii) and extravillous tissues (ii and iv). Extravillous trophoblast cells displayed strong positive immunoreactivity for both TLR-2 (ii) and TLR-4 (iv). Interestingly, in the villous tissues, expression of TLR-2 (i) and TLR-4 (iii) was restricted to the villous cytotrophoblast cells, while the syncythiotrophoblast cells were negative for both receptors. To further characterize these observations, the expression of TLR-2 and TLR-4 in first trimester trophoblast primary cultures and cell lines was evaluated (Fig. 1B). Similarly, as observed in the tissue sections, first trimester trophoblast cells showed positive immunoreactivity for both TLR-2 (i and iii) and TLR-4 (ii and iv). The staining on the cell cultures was localized both intracellularly, as well as on the cell surface and displayed a polarized pattern.



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  		FIGURE 1. First trimester trophoblast cell expresses TLRs. A, Tissue sections (5 µm) of paraffin-embedded first trimester placental tissue were stained for TLR-2 using a mouse IgG2a mAb (clone TLR2.1) and a peroxidase-conjugated horse anti-mouse Ab, and for TLR-4 using the rabbit polyclonal (clone H-20) and a peroxidase-conjugated goat anti-rabbit Ab (magnification x20). Note the strong positive immunoreactivity of the extravillous trophoblast cells for TLR-2 (ii) and TLR-4 (iv). Villous cytotrophoblast cells also showed immunoreactivity for TLR-2 (i) and TLR-4 (iii). Control cells showed no staining (v and vi). B, First trimester trophoblast cells (3A: i and iii; H8: ii and iv) were stained for TLR-2 using a mouse IgG2a mAb (clone TLR2.1) and a peroxidase-conjugated horse anti-mouse Ab, and for TLR-4 using the rabbit polyclonal (clone H-20) and a peroxidase-conjugated goat anti-rabbit Ab (magnification x60). Note the strong positive immunoreactivity of the trophoblast cells for TLR-2 (i and ii) and TLR-4 (iii and iv) which is localized to both the cytoplasm and plasma membrane. Control cells (v and vi) showed no staining. C, Cell lysates of primary first trimester trophoblast cultures isolated from 6, 8, and 10 wk (W) placental villi and the first trimester trophoblast cell lines (H8 and 3A) were analyzed for TLR-2 (90 kDa), TLR-4 (88 kDa), and MyD88 (35 kDa) expression by Western blot. THP-1 and U937 cell lysates served as positive controls for both TLR proteins. Jurkat and U937 cell lysates served as positive control for MyD88. All cells tested were positive for TLR-2, TLR-4, and MyD88. D, TLR-1 and TLR-6 mRNA expression was evaluated in first trimester trophoblast cells by RT-PCR. Left panel, TLR-1 (350 bp); right panel, TLR-6 (500 bp). (M) marker, (1) THP-1 cells which served as a positive control, (2) 3A cells, and (3) H8 cells.

 
To confirm the specificity of these findings, the expression of TLR-2 and TLR-4 by trophoblast cells was evaluated by Western blot analysis. As shown in Fig. 1C, primary cultures of first trimester trophoblast isolated from placental villi, as well as the first trimester trophoblast cell lines, 3A and H8, expressed a 90-kDa protein corresponding to TLR-2, and the 88-kDa TLR-4 protein. In addition, all cells expressed the 35-kDa TLR signaling adapter protein, MyD88 (Fig. 1C), suggesting that TLR-2 and TLR-4 expressed by first trimester trophoblast cells have the potential to signal and, therefore, be functional.

Because TLR-2 has been shown to co-operate with either TLR-1 or TLR-6 for ligand recognition (34, 35, 36, 37), the expression of these receptors was determined in first trimester trophoblast cells. As shown in Fig. 1D, first trimester trophoblast cells expressed the 350-bp product for TLR-1, but failed to express the 500-bp product for TLR-6.

TLR-2, but not TLR-4, reduces trophoblast cell viability

Once the expression of TLR-2, TLR-4, and MyD88 by first trimester trophoblast cells had been established, the biological function of these receptors in trophoblast cells was evaluated. As shown in Fig. 2A, when trophoblast cells were incubated in the presence of the TLR-2 agonist, PDG, a significant decrease in cell viability was observed, as determined by the CellTiter 96 assay. Treatment of cells with the TLR-4 agonist, LPS, did not reduce trophoblast cell viability. However, after 48 h of treatment with LPS, there was a slight but significant increase in cell viability (p < 0.001), which was not evident after 72 and 96 h of treatment. In contrast, the effect of PDG on cell viability occurred in a time-dependent manner, with a reduction in trophoblast viability of 25.93 ± 2.31% after 48 h (p < 0.001), 33.75 ± 0.65% after 72 h (p < 0.05), and 72.33 ± 4.79% following 96 h (p < 0.001) of treatment. Although the reduction in trophoblast cell viability induced by PDG was dose-dependent (Fig. 2B), no such effect could be detected following treatment with LPS at varying concentrations (Fig. 2C). In a separate experiment, PDG (80 µg/ml) reduced trophoblast cell viability by 74%, however, in the presence of a blocking anti-TLR-2 mAb (50 µg/ml), PDG reduced cell viability by 66% (p < 0.05 relative to PDG alone; data not shown).



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  		FIGURE 2. TLR-2, but not TLR-4, reduces trophoblast viability. A, First trimester trophoblast cells (3A: 5 x 104) were incubated with either no treatment (medium) as a negative control, CPT at 4 µM as a positive control, PDG at 50 µg/ml, or LPS at 50 µg/ml for 48, 72, and 96 h. Cell viability was then determined using the CellTiter 96 assay. Bar chart shows percentage cell viability relative to the medium control. Treatment with PDG significantly reduced trophoblast cell viability (*, p < 0.05; **, p < 0.001) in a time-dependent manner. LPS failed to reduce trophoblast cell viability, however, after 48 h of treatment a significant increase in trophoblast cell viability was observed (**, p < 0.001). This figure is representative of at least three independent experiments. B, The first trimester trophoblast cells, H8 and 3A (5 x 104), were incubated with PDG at either 0, 10, 20, 40, 80, or 100 µg/ml for 96 h. Cell viability was then determined using the CellTiter 96 assay. Note the induction of trophoblast cell death by PDG in a dose-dependent manner. This figure is representative of at least three independent experiments. C, The first trimester trophoblast cells, H8 and 3A (5 x 104), were incubated with LPS at either 0.1, 1, 10, 50, or 100 µg/ml for 96 h. Cells were incubated with either no treatment (medium) as a negative control, or CPT at 4 µM as a positive control. Cell viability was then determined using the CellTiter 96 assay. Only CPT significantly reduced trophoblast cell viability (**, p < 0.001). This figure is representative of at least three independent experiments.

 
TLR-2, but not TLR-4, mediates apoptosis in first trimester trophoblast cells

The next objective was to determine whether the decrease in trophoblast viability through TLR-2 was the result of an induction in apoptosis. Therefore, first trimester trophoblast cells were incubated with either no treatment (NT), PDG, or LPS (80 µg/ml). Following a 48 h treatment, the cells were double stained with propidium iodide and Hoechst 33342 dye and the number of apoptotic cells were analyzed by flow cytometry. Treatment with PDG resulted in a 61.72% increase in the number of apoptotic trophoblast cells compared with the untreated control, while no such increase in apoptotic trophoblast cells was observed following treatment with LPS (Fig. 3).



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  		FIGURE 3. PDG induces apoptosis in first trimester trophoblast cells. First trimester trophoblast cells (H8) were incubated for 48 h with either no treatment, PDG (80 µg/ml), or LPS (80 µg/ml). Following incubation, trophoblast cells were harvested by rapid trypsinization and then double stained with propidium iodide (1 µg/ml) and Hoechst 33342 dye (5 µg/ml). Fluorescence intensities were analyzed by flow cytometry as described in Materials and Methods. Data are representative of three independent experiments.

 
TLR-2 activates the caspase apoptotic pathway

Because, in the majority of cases, induction of apoptosis is mediated through activation of caspases, the effect of TLR-2 activation by PDG on caspase activity was studied. Trophoblast cells were treated with PDG for 48 h, after which activity of the caspases was determined using the Caspase-Glo assay. As shown in Fig. 4A, treatment of trophoblast cells with PDG resulted in a significant increase in caspase-3, caspase-8, and caspase-9 activity (p < 0.001). Moreover, treatment of trophoblast cells with PDG in the presence of either a caspase-8 or caspase-9 inhibitor resulted in the attenuation of caspase-3 activity (Fig. 4B). The effect of another TLR-2 ligand on trophoblast cell caspase activity was also tested. Like PDG, LTA significantly induced caspase activity in first trimester trophoblast cells (data not shown).



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  		FIGURE 4. Ligation of TLR-2, but not TLR-4, activates the caspase pathway in first trimester trophoblast cells. A, First trimester trophoblast cells (H8 and 3A) were incubated with either medium or PDG (80 µg/ml) for 48 h, after which cell lysates were prepared and capsase-3 (i), caspase-8 (ii), and caspase-9 (iii) activities were determined using the Caspase-Glo assay. Bar charts show caspase activity in relative light units (RLU). PDG significantly increased trophoblast cell caspase-3, caspase-8, and capsase-9 activity. *, p < 0.001 relative to the control. B, First trimester trophoblast cells (H8 and 3A) were incubated with or without PDG (80 µg/ml) in the presence or absence of either a caspase-8 inhibitor (Z-IETD-FMK) or a caspase-9 inhibitor (Z-LEHD-FMK) each at 20 µM. After a 48 h incubation, cell lysates were prepared and capsase-3 activity was determined using the Caspase-Glo assay. Bar charts show caspase activity in relative light units (RLU). The presence of either the caspase-8 or the capsase-9 inhibitor blocked the activity of caspase-3 induced by PDG alone. *, p < 0.001 relative to the control. This is representative of three independent experiments. C, First trimester trophoblast cells (H8) were treated for 48 h with either medium (NT), PDG (80 µg/ml), or LPS (80 µg/ml) after which cells were lysed and caspase-3 and XIAP activation analyzed by Western blot. Following treatment with PDG, trophoblast cells expressed the inactive form of XIAP (30 kDa) and the active forms of caspase-3 (19/17 kDa). No activation of caspase-3 or inactivation of XIAP was observed following treatment with either medium or LPS. {beta}-actin shows that equal amounts of protein loaded to all lanes. This is representative of three independent experiments.

 
TLR-2 activation induces XIAP inactivation

Recently, we have demonstrated that the inhibitor of apoptosis, XIAP, prevents caspase activation in trophoblast cells and, therefore, protects the trophoblast against certain apoptotic stimuli (38). Furthermore, inactivation of XIAP appears to be required for first trimester trophoblast cell apoptosis to occur (38). Therefore, we sought to determine whether TLR-2-mediated trophoblast cell apoptosis occurred as a result of XIAP inactivation. The activation status of XIAP and caspase-3 was evaluated by Western blot analysis. Untreated (NT) trophoblast cells or cells treated with LPS expressed only the active form of XIAP (57 kDa) and failed to express the active cleavage products of caspase-3 (Fig. 4C). However, following treatment of trophoblast cells for 48 h with PDG, the inactive form of XIAP (30 kDa) was expressed which correlated with the presence of the active forms of caspase-3 (19/17 kDa) (Fig. 4C).

TLR-2-mediated apoptosis is FADD dependent

The activation of caspase-8 following TLR-2 ligation suggests that following treatment with PDG, trophoblast apoptosis may occur through an intracellular pathway similar to that observed with other membranal receptors, such as Fas and TNF-{alpha} (39). Furthermore, MyD88 has recently been shown to be able to recruit FADD, a primary activator of caspase-8 (40, 41). To determine whether FADD is important for TLR-2-mediated apoptosis in first trimester trophoblasts, cells were transiently transfected with FADD-DN. As shown in Fig. 5i, expression of the FADD-DN significantly reduced the PDG-induced activity of caspase-8 (p < 0.001), when compared with PDG-treated cells transfected with the vector control. Furthermore, caspase-9 and capsase-3 activity was also significantly reduced by the FADD-DN following treatment with PDG (p < 0.001) (Fig. 5, ii and iii).



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  		FIGURE 5. Expression of FADD-DN blocks TLR-2-mediated activation of the caspase pathway in first trimester trophoblast cells. First trimester trophoblast cells (H8 and 3A) transiently transfected with either FADD-DN or empty vector (pcDNA3.1) were incubated with either medium (NT) or PDG (80 µg/ml) for 48 h, after which cell lysates were prepared and caspase activity was determined using the Caspase-Glo assay. Bar charts show caspase activity in relative light units (RLU). Expression of the FADD-DN significantly reduced the activity of caspase-8 (i), caspase-9 (ii), and caspase-3 (iii) induced by PDG when compared with the PDG-stimulated vector control (*, p < 0.001). These data are representative of three independent experiments.

 
Ligation of TLR-2 and TLR-4 results in a differential cytokine profile expressed by first trimester trophoblast cells

The results thus far suggest that ligation of TLR-2 induces trophoblast cell apoptosis, while ligation of TLR-4 promotes their survival. To confirm these findings as well as the functionality of TLR-4, trophoblast cells were either untreated or treated with PDG or LPS, after which cytokine expression was evaluated using the human cytokine array kit. Treatment of cells with LPS induced the up-regulation of both pro- and anti-inflammatory cytokines, while all cytokine levels, except those for IL-6 and IL-8, were either unchanged or down-regulated following treatment with PDG (Table I).


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  		Table I. Cytokine production by first trimester trophoblast cells following ligation of TLR-2 or TLR-4a

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During pregnancy the placenta is not only exposed to the maternal immune system, but also to microorganisms. In this study, we demonstrate for the first time that trophoblast cells have a specific mechanism by which they can recognize and respond to invading pathogens. So far, we have characterized the expression and function of TLR-2 and TLR-4 in first trimester trophoblast cells. Interestingly, the response mounted by trophoblast cells following activation of these receptors is divergent. Although TLR-4 activation results in a more classical response, characterized by the induction of cytokine production, activation of TLR-2 results in the induction of trophoblast cell death.

A number of complicated pregnancies have been associated with the presence of intrauterine infections (5, 6, 7, 8, 9), however, the precise mechanism by which a pathogen might impact a pregnancy has, as yet, been undefined. Another key observation in a number of abnormal pregnancies is that during the first trimester, trophoblast cell apoptosis is significantly elevated (42, 43, 44). It is thought that such trophoblast cell death may result in the pathology of conditions such as preeclampsia, IUGR, and preterm labor. The findings of this current study support our hypothesis that intrauterine infections may cause the excessive trophoblast cell apoptosis observed in abnormal pregnancies, and that TLRs may provide the mechanism of pathogenesis. Specifically, our results implicate TLR-2 in providing a direct mechanism for the induction of first trimester trophoblast cell apoptosis in response to certain infections.

Because pregnancy complications such as preeclampsia, IUGR, and preterm labor are thought to be established early in pregnancy (25, 26) and infection may precede implantation (6), this study evaluated the expression and function of TLRs in first trimester trophoblast cells. Studies in term placenta have demonstrated that TLR-1–10 are expressed at the mRNA level (45, 46). At the protein level, TLR-2 and TLR-4 have been detected in term trophoblast cells and their expression is reportedly restricted to the syncytiotrophoblast, intermediate trophoblast, and extravillous trophoblast cell populations (19, 20). In contrast, we report that the extravillous trophoblast and villous cytotrophoblast cells of first trimester placenta express TLR-2 and TLR-4, while the syncytiotrophoblast cells are negative for both receptors. These results suggest that, at least during the first trimester of pregnancy, the placenta may only respond to a microorganism that has breached this outer layer and entered either the decidual or placental villous compartments. These results are in keeping with the premise that a microorganism is only pathogenic once it has penetrated a certain physical barrier and accessed a tissue compartment where PRRs are expressed (12). Such a hypothesis has helped to explain how an immune response can be mounted against pathogenic, but not commensal, bacteria. In further support of this analogy, as well as the restricted expression of TLR by first trimester trophoblast cells, intestinal epithelial cells have been shown to express TLR on their basolateral side so that these cells will only respond to a bacterium that has invaded the basolateral compartment from the apical surface (47). Immunocytochemical staining of first trimester trophoblast cells for TLR-2 and TLR-4 revealed that these receptors, in addition to being expressed on the cell surface, were highly expressed intracellularly. Such cytoplasmic expression may provide rapid mobilization of additional receptors to the cell surface following initial bacterial recognition (48, 49, 50). Alternatively, cytoplasmic expression may serve to facilitate intracellular recognition and responses (51, 52).

Once expression of TLR-2 and TLR-4 by first trimester trophoblast cells had been established, their biological function in these cells was investigated. As shown in this study, first trimester trophoblast cells express MyD88, an adapter protein that allows all TLRs to signal through a common intracellular pathway. Following ligation of a TLR by its ligand, MyD88 is recruited to the intracellular domain of the TLR (53, 54). MyD88, in turn, recruits and activates IL-1R-associated kinase (IRAK) (54). IRAK then dissociates from the receptor complex and becomes associated with TNFR-associated factor-6 (55), resulting in downstream activation of the NF-{kappa}B and MAPK signaling pathways (56). Such TLR signaling results in an inflammatory immune response characterized by the production of cytokines and antimicrobial factors (57). In first trimester trophoblast cells, ligation of TLR-4 by LPS appears to trigger such a classical response, characterized by the up-regulation of both pro- and anti-inflammatory cytokines (Table I). However, ligation of TLR-2 expressed by first trimester trophoblast cells had a very different effect. Treatment of first trimester trophoblast cells with the TLR-2 ligand, PDG, up-regulated only IL-6 and IL-8 expression levels, while all other cytokine levels were down-regulated or unchanged. Moreover, ligation of TLR-2 caused first trimester trophoblast cells to undergo apoptosis, consistent with the recent reports of TLR-2-mediated apoptosis in other cell types (40, 41, 58, 59).

The induction of apoptosis in first trimester trophoblast cells was observed following ligation of TLR-2 with either PDG or LTA. Recognition of these bacterial products by TLR-2 has been reported to require the additional recruitment of TLR-6 or TLR-1 (36, 37). In this study, we have demonstrated that first trimester trophoblast cells respond similarly to both PDG and LTA, possible through a combination of TLR-2 and TLR-1, but not through TLR-6 because trophoblasts do not express this receptor. Furthermore, recent studies have shown that TLR-6 augments, but is not essential, for TLR-2-mediated responses (41, 60). Therefore, our findings suggest that PDG and LTA can induce trophoblast cell apoptosis through either a TLR-2 homodimer or a TLR-1/TLR-2 heterodimer.

The proapoptotic effect induced by TLR-2 in first trimester trophoblast cells occurred through the activation of caspases. Caspase-mediated apoptosis can occur through either the extrinsic or the intrinsic pathway (61). The former occurs when a membranal receptor activates caspase-8, which in turn directly activates caspase-3. However, under circumstances where active caspase-8 levels are low, the intrinsic pathway is directed through the mitochondria (62). We have recently demonstrated that XIAP prevents caspase activation in trophoblast cells (38) by preventing both caspase-9 and caspase-3 activation (63, 64). Furthermore, inactivation of XIAP renders trophoblast cells sensitive to apoptotic stimuli (38). In this current study, we have demonstrated that activation of TLR-2 expressed by trophoblast cells results in XIAP inactivation, thus allowing apoptosis to occur. Moreover, the mitochondrial pathway appears to be critical for TLR-2-mediated apoptosis in trophoblast cells, because both caspase-8 and caspase-9 activation are required for the downstream activation of caspase-3. In contrast, TLR-2-mediated apoptosis in macrophages responding to the 19-kDa Mycobacterium tuberculosis protein is dependent upon caspase-8, but not caspase-9, activation (59). An explanation for these differing results may be the cell type studied or the TLR-2 ligand used.

Because caspase-8 activation was observed, we hypothesized that TLR-2 may be functioning similarly to death receptors such as, Fas (CD95), or the type I TNFR (TNFRI) (39). Both Fas and TNFRI use FADD, which recruits and activates procaspase-8 (65, 66), to mediate their apoptotic signal (67). Because MyD88 contains a death domain (68) through which it can directly interact with FADD (40, 41), we postulated that TLR-2-mediated trophoblast cell apoptosis may use this pathway. When first trimester trophoblast cells were transfected with a FADD-DN, TLR-2-mediated apoptosis was inhibited, suggesting that in first trimester trophoblast cells, TLR-2 activation of the apoptotic caspase cascade is occurring through a MyD88/FADD-dependent pathway.

Previous studies have shown that TLR-2-mediated apoptosis is also accompanied by early NF-{kappa}B activation (40, 41). Although it has been suggested that FADD may preclude IRAK from the MyD88-receptor complex (69), NF-{kappa}B activation may still occur. One possibility may be that caspase-8 activation promotes NF-{kappa}B activation (70, 71). Alternatively, IRAK may still have the ability to interact with one of the two MyD88 molecules recruited to the heterodimer TLR complex. Notwithstanding, ours and previous findings suggest that any antiapoptotic signals promoted by NF-{kappa}B (72) may be outweighed by the proapoptotic signals mediated through TLR-2 (40, 41). Consequently, TLR-2 may provide a direct mechanism by which apoptosis can be induced in first trimester trophoblast cells.

Although TLR-4-mediated apoptosis has recently been reported in macrophages (73), we report herein, that TLR-4 ligation by LPS fails to induce apoptosis in first trimester trophoblast cells. TLR-4 differs from TLR-2 in that it can signal via both MyD88-dependent and -independent pathways (74). TLR-4 can associate with the TIR domain-containing adaptor protein inducing IFN-{beta}, which can directly bind TNFR-associated factor-6 and subsequently activate NF-{kappa}B (75). Hence, the additional antiapoptotic signals generated through the NF-{kappa}B pathway (72) may outweigh any proapoptotic signals generated by LPS thus resulting in trophoblast cell survival in vitro. However, the in vivo scenario may be quite different. Animal models of pregnancy complications have been generated by the administration of LPS (76, 77, 78). In this current study, we have demonstrated that LPS, through TLR-4, triggers first trimester trophoblast cells to produce high levels of cytokines, including TNF-{alpha} and IFN-{gamma}. We and others have shown that trophoblast cells are highly sensitive to these cytokines, suggesting that TNF-{alpha} and IFN-{gamma} expression in the placenta may induce trophoblast cell apoptosis (27, 79, 80). Therefore, while LPS does not directly induce trophoblast cell death, its indirect effects may have a significant impact on trophoblast cell survival during pregnancy.

In summary, we have demonstrated that trophoblast cells are able to recognize pathogens through the expression of TLR-2 and TLR-4, a system characteristic of innate immune cells. Interestingly, upon activation of these two receptors, distinct trophoblast cell responses were observed. TLR-4 ligation promoted cytokine production, while ligation of TLR-2 induced apoptosis in first trimester trophoblast cells. These findings suggest that a pathogen, through TLR-2, may directly promote the elevated trophoblast cell death observed in a number of pregnancy complications. TLR-2-mediated trophoblast apoptosis, therefore, provides a novel mechanism of pathogenesis by which certain intrauterine infections may contribute to conditions such as preterm labor, IUGR, and preeclampsia.


   Acknowledgments
 
We thank Drs. Seth Guller and Susan Richmond for tissue procurement and Thomas Taylor for his assistance with the FACS Vantage flow cytometry studies.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Gil Mor, Reproductive Immunology Unit, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, FMB 301, New Haven, CT 06520. E-mail address: Gil.Mor{at}yale.edu Back

2 Abbreviations used in this paper: IUGR, intrauterine growth restriction; PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pattern; XIAP, X-linked inhibitor of apoptosis; FADD-DN, dominant-negative Fas-associated death domain; PDG, peptidoglycan; LTA, lipoteichoic acid; FFPM, fat-free powdered milk; IRAK, IL-1R-associated kinase; CPT, camptothecin; NT, no treatment. Back

Received for publication June 3, 2004. Accepted for publication July 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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