ABSTRACT
Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.
ABSTRACT
Problem: Trophoblast organoids derived from human placental villi provide a powerful 3D model system of placental development, but access to first-trimester tissues is limited due to ethical and legal restrictions. Here we sought to establish a methodology for establishing 3D trophoblast organoids from naïve human pluripotent stem cells (hPSCs), which have an expanded potential for extraembryonic differentiation. Method of Study: We previously demonstrated that naïve hPSCs readily give rise to self-renewing human trophoblast cells (hTSCs) that resemble post-implantation cytotrophoblast (CTB) progenitors and can further differentiate into specialized trophoblast lineages. Here we examined whether hTSCs derived from three distinct sources (naïve hPSCs, human blastocysts, and first-trimester placental tissues) have the potential to self-organize into 3D trophoblast organoids by transfer to Matrigel droplets in the presence of trophoblast organoid medium. The expression of protein markers in the resulting stem cellderived trophoblast organoids (SC-TOs) was examined by immunofluorescence and light-sheet microscopy, while their single cell transcriptome was analyzed using the 10X Genomics platform. We also investigated the X chromosome inactivation (XCI) status of organoids derived from female naïve hPSCs and their ability to differentiate into invasive extravillous trophoblast (EVT) organoids. Finally, we evaluated whether SC-TOs are susceptible to infection by various emerging pathogens (SARS-CoV-2 and Zika virus), as a basis for establishing a stem cell-based model system of placental infections during the first trimester. Results: Trophoblast organoids generated from naïve and primary hTSCs displayed comparable tissue architecture, placental hormone secretion, microRNA expression, and capacity for long-term selfrenewal. In-depth single cell transcriptome profiling revealed that SCTOs encompass a variety of trophoblast identities that closely correspond to CTB progenitor, syncytiotrophoblast (STB) and EVT cell types found in human post-implantation embryos. Interestingly, the cellular composition in trophoblast organoids derived from naïve and primary hTSCs was highly similar, which suggests that trophoblast organoid culture represents a powerful attractor state in which the influence of subtle epigenetic differences between naïve and primary hTSCs is mitigated. These organoid cultures displayed clonal XCI patterns previously described in the human placenta.Upon differentiation into specialized EVT organoids, extensive trophoblast invasion was observed in co-culture assays with human endometrial cells. We further demonstrated that SC-TOs display selective vulnerability to infection by SARS-CoV-2 and Zika virus, which correlated with the expression levels of their respective entry factors. Conclusions: The generation of trophoblast organoids from naïve hPSCs provides an accessible and patient-specific 3D model system of the developing placenta and its susceptibility to emerging pathogens. The ability to genetically manipulate naïve hPSCs prior to differentiation into SC-TOs enables functional interrogation of regulatory factors implicated in placental organogenesis.
ABSTRACT
Problem: The placenta performs various functions of the lung/GI/GU tract for the developing fetus, while also moderating host defenses of the fetus against infections in utero, and likely educates the developing fetal immune system. It thus has long-term impacts on the health of both the woman and the child. Knowledge is limited about the underlying mechanisms that enable the placenta to serve as a protective barrier for the fetus against infection. The long-term goals of my research program are to, 1) elucidate the normal barriers to infection in the placenta and show how dysfunction in barrier function can lead to adverse maternal-fetal outcomes, 2) define how viral infections impact placental biology, and 3) characterize possible functional roles for the newly described microbiota at the maternal-fetal interface. Method of Study: To address the above questions, our research includes the use human placentas, primary human trophoblasts and immune cells derived from term placentas, cultured placental cells, trophoblast organoids, and mousemodels. Results: We found that placentas from women who gave birth prematurely exhibit reduced autophagy activity. Prematurity and reduced autophagy levels were also strongly associated with maternal infection. In a mouse model of pregnancy, we showed that placentas from mice deficient for Atg16L1 were significantly less able to withstand infection, and the deficient mice gave birth prematurely upon an inflammatory stimulus. We have also shown that the autophagy pathway plays a key role in ZIKV vertical transmission from mother to fetus. We demonstrated that hydroxychloroquine (HCQ), an autophagy inhibitor approved for use in pregnant women, can attenuate placental and fetal ZIKV infection and ameliorate adverse placental and fetal outcomes. More recently, we have identified a small molecule inhibitor that targets the NS2B-NS3 protease of ZIKV and inhibits viral replication. It has recently become evident that SARS-CoV-2 infection is also associated with adverse outcomes for pregnant women, including preterm birth, preeclampsia, and fetal growth restriction. We localized SARS-CoV-2 to the placenta and showed that infection alters the Renin Angiotensin System (RAS) that regulates blood pressure, thereby increasing risk for preeclampsia. In new work, we are showing that SARS-CoV-2 non-structural proteins affect autophagy in different ways than in Zika virus. Finally, we have discovered that the maternal fetal interface of the placenta harbors intracellular resident microbes, and functionally demonstrated that they do not induce any inflammatory response or cell death but may promote immune tolerance and support normal pregnancy outcomes. Conclusions: For the past 10 years of my career, I have been working on host microbial interactions at the maternal fetal interface. Our work has led to new insights into viral infections, showing how they co-opt host defenses, and that tolerance may have microbial drivers. We have shown how cellular pathways in the placenta such as autophagy and RAS mechanistically regulate host defenses against pathogens, including ZIKV and SARS-CoV-2. Additionally, our studies provide a foundation for understanding possible 'commensal' microbial- placental interactions and hint at the functional importance of microbes at the fetal maternal interface in maintaining placental health and supporting fetal development.