TNF-α Regulated Endometrial Stroma Secretome Promotes Trophoblast Invasion.

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However, the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations, therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC), we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-α. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion, we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation.

Novel 3D in vitro models to evaluate trophoblast migration and invasion.

PROBLEM: Embryo implantation depends on the interactions between the developing embryo and the maternal endometrium. Signals originating from the decidua play a critical role in the process of implantation and trophoblast invasion; however, the molecular mechanisms mediating this interaction are poorly understood. The objective of this study was to develop in vitro models that would mimic the processes of attachment, migration, and early invasion of the trophoblast. METHODS OF STUDY: First trimester trophoblast cells (Sw.71 cells) were cultured in low attachment plates to form blastocyst-like spheroids (BLS). Epithelial-mesenchymal transition (EMT) characterization during BLS formation was determined by RT-PCR and Western Blot. The two 3D in vitro culture models consist of (a) trophoblast migration: BLS cultured in suspension (b) trophoblast invasion: human endometrium stromal cells (HESC) plated in the bottom of a 96-well plate, covered by Matrigel and BLS transferred on top. Matrigel was used to mimic the human endometrial extracellular matrix. RESULTS: Using 3D cell culture systems and real-time imaging, we are able to determine the impact of endometrial factors on trophoblast cell function. Endometrial stromal cells promote blastocyst-like spheroid migration of trophoblast cells and invasion of the extracellular matrix. CONCLUSION: We report the characterization of 3D in vitro models to evaluate the interaction between endometrial cells and trophoblast during the process of migration and invasion. The models are useful tools in order to further study the molecular mechanism of embryo-maternal uterine cells interactions.

Trophoblast induces monocyte differentiation into CD14+/CD16+ macrophages.

PROBLEM: During early pregnancy, macrophages and trophoblast come into close contact during placenta development, and regulated cross talk between these cellular compartments is crucial for maintaining a healthy pregnancy. As trophoblast cells constitutively secrete many chemokines and cytokines, we hypothesize that trophoblast-secreted factors can differentiate monocytes into a decidual phenotype. In this study, we describe a unique macrophage phenotype, following monocytes' exposure to trophoblast-soluble factors. METHOD OF STUDY: Peripheral blood monocytes were treated with or without conditioned media (CM) from first trimester trophoblast cells. Phenotypic changes and phagocytic capacity were determined by flow cytometry. Cytokine and chemokine production was determined by multiplex analysis. RESULTS: Monocytes exposed to trophoblast factors undergo morphologic changes characterized by a gain in size and complexity and acquire a unique phenotype characterized by gain of CD14 surface expression as well as CD16. The presence of CD14+/CD16+ macrophages was confirmed in normal decidua. These cells secrete higher levels of IL-1b, IL-10, and IP-10 and have increased capacity for phagocytosis. CONCLUSION: We demonstrate that trophoblast-secreted factors can induce monocyte differentiation into a unique macrophage phenotype. These findings suggest that the microenvironment of the placenta can modulate the phenotype of macrophages present at the decidua.

Effect of culture conditions on the phenotype of THP-1 monocyte cell line.

PROBLEM: Macrophage function has many implications in a variety of diseases. Understanding their biology becomes imperative when trying to elucidate immune cell interactions with their environment, and in vitro cell lines allow researchers to manipulate these interactions. A common cell line used is THP-1, a promyeloid cell line suggestive to outside factors, and therefore sensitive to culture conditions. In this study, we describe how culture conditions can alter THP-1 morphology and in turn affect their response to differentiation stimuli. METHOD OF STUDY: THP-1 cells were cultured in two conditions and treated with phorbol 12-myristate 13-acetate (PMA) or MCSF. CD14 surface expression was determined by flow cytometry and cytokine/chemokine production determined by multiplex analysis. RESULTS: Culture conditions of THP-1 affect their response to PMA. Highly confluent THP-1 cells differentiate into a heterogeneous population responsive to PMA as seen by an increase in CD14 expression. However, these cells, cultured in low confluence, remain as a homogenous population and do not gain CD14. Additionally, there are major differences in the constitutive cytokine profile. CONCLUSION: We demonstrate that the culture conditions of THP-1 cells can alter their response PMA. This suggests that culture techniques may account for the discrepancy in the literature of both basal THP-1 phenotype and their response to PMA.

An in vitro model for the study of human implantation.

PROBLEM: Implantation remains the rate-limiting step for the success of in vitro fertilization. Appropriate models to study the molecular aspects of human implantation are necessary in order to improve fertility. METHODS: First trimester trophoblast cells are differentiated into blastocyst-like spheroids (BLS) by culturing them in low attachment plates. Immortalized human endometrial stromal cells and epithelial cells (ECC-1) were stably transfected with GFP or tdTomato. Co-culture experiments were monitored using Volocity imaging analysis system. RESULTS: This method demonstrates attachment and invasion of BLS, formed by trophoblast cells, into stromal cells, but not to uterine epithelial cells. CONCLUSION: We have developed an in vitro model of uterine implantation. The manipulation of this system allows for dual color monitoring of the cells over time. Additionally, specific compounds can be added to the culture media to test how this may affect implantation and invasion. This model is a helpful tool in understanding the complexity of human implantation.

Enhanced stimulation of anti-ovarian cancer CD8(+) T cells by dendritic cells loaded with nanoparticle encapsulated tumor antigen.

PROBLEM: Dendritic cell (DC)-based cancer therapies are favored approaches to stimulate anti-tumor T-cell responses. Unfortunately, tolerance to tumor antigens is difficult to overcome. Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) are effective reagents in the delivery of drugs and tumor-associated antigens (TAA). In this study, we assessed the capacity of a PLGA NP-based delivery system to augment CD8 T-cell responses to ovarian cancer TAA. METHOD OF STUDY: Human DC were generated from blood monocytes by conventional in vitro differentiation and loaded with either soluble tumor lysate or NP/lysate conjugates (NPL). These antigen-loaded DC were then used to stimulate autologous CD8(+) T cells. Cytokine production and activation markers were evaluated in the CD8(+) T cells. RESULTS: DC loading with NPL increased cytokine production by stimulated CD8 T cells and induced T-cell expression of cell surface co-stimulatory molecules, typical of anti-tumor immune responses. In contrast, delivery of naked tumor lysate antigens preferentially induced a T-cell profile characteristic of tolerization/exhaustion. CONCLUSION: These findings indicate that delivery of TAA in NP enables DC to efficiently activate anti-tumor CD8(+) T cells. PLGA NP encapsulation of tumor-derived lysate protein antigens is an encouraging new preparative methodology for DC-based vaccination meriting clinical testing.

TMS, a chemically modified herbal derivative of resveratrol, induces cell death by targeting Bax.

Breast cancer recurrence after an initial favorable response to treatment is a major concern for patients who receive hormonal therapies. Additional therapies are necessary to extend the time of response, and ideally, these therapies should exhibit minimal toxicity. Our study described herein focuses on a non-toxic pro-apoptotic agent, TMS (2,4,3',5'-tetramethoxystilbene), which belongs to the Resveratrol family of stilbenes. Prior study demonstrated that TMS was more effective than Resveratrol for inducing apoptosis. Additionally, TMS was effective for invoking death of relapsing breast cancer cells. As TMS was effective for reducing tumor burden, we sought to determine the mechanism by which it achieved its effects. Microarray analysis demonstrated that TMS treatment increased tubulin genes as well as stress response and pro-apoptotic genes. Fractionation studies uncovered that TMS treatment causes cleavage of Bax from the p21 form to a truncated p18 form which is associated with the induction of potent apoptosis. Co-localization analysis of immunofluorescent studies showed that Bax moved from the cytosol to the mitochondria. In addition, the pro-apoptotic proteins Noxa and Bim (EL, L, and S) were increased upon TMS treatment. Cell lines reduced for Bax, Bim, and Noxa are compromised for TMS-mediated cell death. Electron microscopy revealed evidence of nuclear condensation, formation of apoptotic bodies and DAPI staining showed evidence of DNA fragmentation. TMS treatment was able to induce both caspase-independent and caspase-dependent death via the intrinsic death pathway.

Local injury of the endometrium induces an inflammatory response that promotes successful implantation.

OBJECTIVE: To study whether an injury-induced inflammation might be the mechanism underlying the favorable effect of endometrial biopsy on the implantation rate in in vitro fertilization (IVF) patients. DESIGN: Controlled clinical study. SETTING: A medical center IVF unit and a research institute. PATIENT(S): Women undergoing IVF who had previous failed treatment cycles. INTERVENTION(S): Endometrial samples were collected from two groups of patients on day 21 of their spontaneous menstrual cycle. The experimental, but not the control group underwent prior biopsy treatment on days 8 or/and 11 to 13 of that same cycle. MAIN OUTCOME MEASURE(S): Abundance of immune cells, cytokines/chemokines level, correlation between these parameters and pregnancy outcome. RESULT(S): A statistically significantly higher amount of macrophages/dendritic cells (HLA-DR+ CD11c+ cells) and elevated proinflammatory cytokines, tumor necrosis factor-α (TNF-α), growth-regulated oncogene-α (GRO-α), interleukin-15 (IL-15), and macrophage inflammatory protein 1B (MIP-1B), were detected in day-21 endometrial samples of the experimental group. A direct stimulatory effect of TNF-α on MIP-1B, GRO-α, and IL-15 messenger RNA (mRNA) expression was demonstrated. A positive correlation was found between the levels of macrophages/dendritic cells, MIP-1B expression, and TNF-α expression and the pregnancy outcome. CONCLUSION(S): A biopsy-induced inflammatory response may facilitate the preparation of the endometrium for implantation. Increased MIP-1B expression could possibly serve for prediction of implantation competence.

Viral ssRNA induces first trimester trophoblast apoptosis through an inflammatory mechanism.

PROBLEM: Infection during pregnancy represents a significant cause of mobility and mortality. While viruses pose a major threat, little is known about their effect on early pregnancy, or the mechanisms involved. The objective of this study was to characterize the trophoblast response following exposure to viral ssRNA. METHOD OF STUDY: First trimester trophoblast cells were treated with or without viral ssRNA. Cytokine production was measured using multiplex analysis and ELISA. Apoptosis was determined using Hoechst staining, cell viability, and caspase activity assays. RESULTS: Treatment of trophoblasts with viral ssRNA increased their secretion of IL-8, IL-6, and IFNbeta. However, the ssRNA also induced trophoblast apoptosis. To test whether the viral ssRNA-induced inflammatory response was responsible for this induction of apoptosis, conditioned media (CM) from trophoblasts were added to a fresh culture of cells. The CM from viral ssRNA-treated induced higher levels of trophoblast apoptosis than the control CM. Moreover, recombinant IFNbeta induced trophoblast apoptosis. CONCLUSION: We demonstrate that viral ssRNA induces a pro-inflammatory and type I interferon response in the trophoblast and this inflammatory process may indirectly induce trophoblast apoptosis. These results provide a novel mechanism by which certain viral infections might compromise placental integrity and function, and therefore, pregnancy outcome.

Toll-like receptors and pregnancy: trophoblast as modulators of the immune response.

During normal pregnancy, the decidua is populated by a variety of leucocytes; however, cells of the innate immune system seem to dominate this tissue. Their presence suggests that the innate immune system is not indifferent to the fetus and has been associated with a response of the maternal immune system to the 'semi-allograft' fetus. New evidences, however, indicate that these immune cells are critical for decidual and trophoblast development rather than induction of tolerance. We hypothesized that during implantation, an inflammatory environment is necessary for the attachment and invasion of the blastocyst. The existence of an 'inflammatory-mediated embryo implantation' condition is dependent on the proper 'education' of the innate immune system which we propose is mediated by the trophoblast. Here we postulate that trophoblast cells successfully orchestrate their inflammatory environment and regulate immune cells differentiation and activation through Toll-like receptors (TLR). We will describe potential functions of TLR in trophoblast cells, their recognition and response to microorganisms, and their involvement in innate immunity.

TLR6 modulates first trimester trophoblast responses to peptidoglycan.

Intrauterine bacterial infections are a well-established cause of pregnancy complications. One key observation in a number of abnormal pregnancies is that placental apoptosis is significantly elevated. First trimester trophoblast cells are known to express TLR1 and TLR2 and to undergo apoptosis following exposure to Gram-positive bacterial peptidoglycan (PDG). Thus, the objectives of this study were to determine whether PDG-induced pregnancy complications are associated with placental apoptosis and to characterize the cellular mechanisms involved. We have demonstrated, using an animal model, that delivery of PDG to pregnant mice early in gestation resulted in highly elevated placental apoptosis, evidenced by trophoblast M-30 and active caspase 3 immunostaining. Using an in vitro model of human first trimester trophoblasts, apoptosis induced by PDG was found to be mediated by both TLR1 and TLR2 and that this could be blocked by the presence of TLR6. Furthermore, in the presence of TLR6, exposure to PDG resulted in trophoblast NF-kappaB activation and triggered these cells to secrete IL-8 and IL-6. The findings of this study suggest that a Gram-positive bacterial infection, through TLR2 and TLR1, may directly promote the elevated trophoblast cell death and that this may be the underlying mechanism of pregnancy complications, such as preterm delivery. Furthermore, the expression of TLR6 may be a key factor in determining whether the response to PDG would be apoptosis or inflammation.

A novel three-dimensional in vitro system to study trophoblast-endothelium cell interactions.

INTRODUCTION: Pregnancy complications have been linked to improper trophoblast migration and failure of spiral artery transformation. Endothelial cells play an essential role in directing trophoblast migration and transformation, although by an unknown mechanism. We describe a novel in vitro model to evaluate endothelial-trophoblast interaction and signaling in a three-dimensional system. METHOD OF STUDY: Immortalized human endometrial endothelial cell line and first trimester trophoblast cells were co-cultured. Endothelial transformation into vessel-like structures occurred in Matrigel(TM) OpenLab Image Analysis software was used to monitor labeled trophoblast migration and endothelium transformation. Cytokine/chemokine production was determined using Multiplex. RESULTS: Trophoblast migrates toward endothelial cells in Matrigel, aligns on top of the endothelium within 4-8 hr and achieves complete replacement of the endothelium by 72-96 hr. Lipopolysaccharide treatment damages the endothelium and disrupts endothelium-trophoblast interaction. CONCLUSION: We report a novel three-dimensional in vitro and in vivo system of trophoblast-endothelium cell interaction. Significant changes in endothelial cells' phenotype are observed upon differentiation in Matrigel. These changes may be necessary for endothelium to direct trophoblast migration and transformation.

Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy.

PROBLEM: Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells 'educate' monocytes/macrophages to create an adequate environment that promote trophoblast survival. METHOD OF STUDY: CD14(+) monocytes were isolated from peripheral blood using magnetic beads. Co-culture experiments were conducted using a two-chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. RESULTS: Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro-inflammatory cytokines [interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha] and chemokines (growth-related oncogen-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre-exposed to trophoblast cells. CONCLUSION: The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro-inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.

Expression and secretion of antiviral factors by trophoblast cells following stimulation by the TLR-3 agonist, Poly(I : C).

BACKGROUND: During pregnancy, the placenta may become exposed to micro-organisms, such as viruses, which may pose a substantial threat to the embryo/fetus well-being. Recent insight into the immunological capabilities of the trophoblast suggests that the placenta may function as an active barrier by recognizing and responding to pathogens through Toll-like receptors (TLRs). METHODS: The objective of this study was to determine whether the engagement of TLR-3 with viral dsRNA by first-trimester trophoblast could induce the production of factors necessary to generate an antiviral response. Therefore, trophoblast cells were exposed to the TLR-3 agonist, Poly(I : C). RESULTS: We report that following stimulation with Poly(I : C), first-trimester trophoblast cells produce interferon beta (IFNbeta) and secretory leukocyte protease inhibitor (SLPI), as well as the intracellular factors 2',5'-oligoadenylate synthetase (OAS), Myxovirus-resistance A (MxA) and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G). This response is TLR-3 specific because the TLR-4 ligand, lipopolysaccharide (LPS), had no effect on the production of these antimicrobial factors. Furthermore, we describe a positive feedback mechanism in which IFNbeta enhances the antiviral response by promoting the production of OAS, MxA and APOBEC3G. CONCLUSIONS: These findings suggest that trophoblast cells are able to recognize and specifically respond to viral products in a highly regulated fashion and that the placenta may be pivotal in the control of viral infections at the maternal-fetal interface.

A role for TLRs in the regulation of immune cell migration by first trimester trophoblast cells.

Normal pregnancy is characterized by the presence of innate immune cells at the maternal-fetal interface. Originally, it was postulated that the presence of these leukocytes was due to an immune response toward paternal Ags expressed by the invading trophoblasts. Instead, we and others postulate that these innate immune cells are necessary for successful implantation and pregnancy. However, elevated leukocyte infiltration may be an underlying cause of pregnancy complications, such as preterm labor or preeclampsia. Furthermore, such conditions have been attributed to an intrauterine infection. Therefore, we hypothesize that first trimester trophoblast cells, upon recognition of microbes through TLRs, may coordinate an immune response by recruiting cells of the innate immune system to the maternal-fetal interface. In this study, we have demonstrated that human first trimester trophoblast cells constitutively secrete the chemokines growth-related oncogene, growth-related oncogene alpha, IL-8, and MCP-1 and are able to recruit monocytes and NK cells, and to a lesser degree, neutrophils. Following the ligation of TLR-3 by the viral ligand, poly(I:C), or TLR-4 by bacterial LPS, trophoblast secretion of chemokines is significantly increased and this in turn results in elevated monocyte and neutrophil chemotaxis. In addition, TLR-3 stimulation also induces trophoblast cells to secrete RANTES. These results suggest a novel mechanism by which first trimester trophoblast cells may differentially modulate the maternal immune system during normal pregnancy and in the presence of an intrauterine infection. Such altered trophoblast cell responses might contribute to the pathogenesis of certain pregnancy complications.

Is the trophoblast an immune regulator? The role of Toll-like receptors during pregnancy.

In the early 1950s, Medawar recognized for the first time the unique immunology of the maternal/ fetal interface and its potential relevance for transplantation. In his original work, he described the fetal allograft analogy whereby the fetus may be viewed as a semi-allogeneic conceptus that has evaded rejection by the maternal immune system. Although numerous hypotheses have been proposed to prove this observation, none have demonstrated that the maternal immune system is antagonist to the invading trophoblast. In the present article, we have reviewed recent studies demonstrating the expression and function of Toll-like receptors (TLRs) in trophoblast cells, and, based on these data, we propose an alternative view for maternal/fetal immune interactions.