In Utero Transplantation of FVIII-Expressing Human Placental Cells Upregulates Gene Pathways Associated with Immune Tolerance, Altering the Response to Postnatal FVIII Infusion

We have previously reported that in utero transplantation (IUTx) of sheep fetuses (n=14) with human placental cells (PLC) transduced with a lentiviral vector encoding mcoET3, an expression/secretion-optimized, bioengineered fVIII transgene (PLC-mcoET3) increased plasma FVIII activity levels by 57%, 42%, and 35% at 1, 2, and 3 years post-IUTx, respectively, without the development of FVIII/ET3 inhibitors. We also demonstrated that immune tolerance to the cell/gene product was maintained after postnatal administration of PLC-mcoET3 (cells producing 20 IU/kg/24h were administered i.v. for 3 consecutive weeks). However, when IUTx-treated animals received weekly i.v. infusions of purified ET3 protein (20IU/kg) for 5 weeks, all recipients developed a robust ET3-specific IgG response that appeared at week 3 of infusion at titers ranging from 1:70 to 1:857 and inhibitory antibodies that ranged from 3-36 BU. Here, we investigated differences in the immune responses of animals that received IUTx with PLC-mcoET3 and were boosted postnatally with PLC-mcoET3 (IUTx-PLC-mcoET3) vs. ET3 protein (IUTx-ET3) to define the pathways by which the immune system differentially responds to protein vs. cell-secreted ET3. A sheep-specific multiplex gene expression analysis with 165 genes involved in immune cell signaling pathways (NanoString) was used to evaluate mRNA isolated from peripheral blood mononuclear cells collected at Weeks (W) 0, 1, and 5 of postnatal infusions. Significant fold-change expression in these mRNA targets was determined using NanoString nSolver 4.0 software. Animals in the IUTx-PLC-mcoET3 group (known to be devoid of inhibitors to ET3 post-boosting) showed that immunoregulation and immune tolerance gene clusters were among the top three clusters that increased expression from W0 to W5 (adj. p-value<0.01). Differential expression of genes in pathways involved in Th1, Th2, and Th17 responses was also found, at differing levels, in the IUTx-PLC group, suggesting a balance between immunity and tolerance was maintained. Surprisingly, the IUTx-ET3 group, which developed inhibitory antibodies after ET3 boosting, also showed significantly increased expression of immune tolerance genes, and downregulation of Th1 and Th17 cell signaling, when evaluated by direct global significance score. Nevertheless, 66% of these animals had a significant upregulation of Th2 cell signaling by W1 vs W0. To determine if the increase in expression of immune tolerance genes was due to the IUTx treatment, we also evaluated a group of aged-matched, non-transplanted sheep that received ET3 protein under the same dose and schedule. Results from Gene Set Analysis (GSA) demonstrated significant upregulation of genes involved in interferon signaling, class I MHC antigen processing, and Th17 signaling in these animals, suggesting the potential involvement of Th17 cells in the immune response in this group. In conclusion, IUTx with PLC-mcoET3 induces the upregulation of genes associated with immune tolerance, providing an explanation for the long-lasting elevation in plasma FVIII levels in these animals in the absence of inhibitors. Nevertheless, despite the continued expression of tolerogenic genes, administration of purified ET3 protein to these IUTx recipients induced upregulation of Th2 signaling, a pathway that was not observed in animals that only received ET3 protein, demonstrating that the mechanism by which tolerance is broken in IUTx recipients differs from that by which an immune response to ET3 occurs in animals with no prior exposure. Of note is that animals that develop inhibitors by the Th17 pathway had considerably higher inhibitor titers than the IUTx recipients that responded to ET3 infusion by the Th2 pathway. These studies underscore the need for a more complete understanding of the mechanisms by which immune tolerance to FVIII develops during ontogeny. Disclosures Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

Effects of polycyclic aromatic hydrocarbons on the proliferation and differentiation of placental cells

Background The purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons (PAHs) other than bisphenol A (BPA) and BPA substitutes on placental cells. Methods HTR-8/SVneo cells were treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, which is used as a substitute for BPA-free products. After confirming the dose response for each reagent using the prepared cells, the cells were incubated for 24, 48, and 72 h. Cell viability was confirmed using the XTT assay. Each experiment was performed with the minimum number of samples (n = 3) required for statistical analysis. The results were analyzed using t-tests; p < 0.05 was considered statistically significant. Results After treatment with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, the absorbance measured using the XTT assay decreased significantly with increasing concentration. The absorbance decreased significantly over time following treatment with each endocrine disruptor at the concentration confirmed by the dose–response analysis. Conclusions This study showed that anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4-(9-fluorenylidene)diphenol—a BPA substitute—affect cell viability and necrosis in the placental cell line. The study indicates the serious effects of PAHs that negatively affect pregnancy but were previously unknown. Further, this study would serve as a reference for the identification of harmful PAHs during pregnancy prognosis in women who are more susceptible to PAH exposure.

The Preeclamptic Environment Promotes the Activation of Transcription Factor Kappa B by P53/RSK1 Complex in a HTR8/SVneo Trophoblastic Cell Line

Preeclampsia is a pregnancy disorder associated with shallow placentation, forcing placental cells to live in hypoxic conditions. This activates the transcription factor kappa B (NFκB) in maternal and placental cells. Although the role of NFκB in preeclampsia is well documented, its mechanism of activation in trophoblastic cells has been never studied. This study investigates the mechanism of NFκB activation in a first trimester trophoblastic cell line (HTR8/SVneo) stimulated by a medium containing serum from preeclamptic (PE) or normotensive (C) women in hypoxic (2% O2) or normoxic (8% O2) conditions. The results indicate that in HTR8/SVneo cells, the most widely studied NFκB pathways, i.e., canonical, non-canonical and atypical, are downregulated in environment PE 2% O2 in comparison to C 8% O2. Therefore, other pathways may be responsible for NFκB activation. One such pathway depends on the activation of NFκB by the p53/RSK1 complex through its phosphorylation at Serine 536 (pNFκB Ser536). The data generated by our study show that inhibition of the p53/RSK1 pathway by p53-targeted siRNA results in a depletion of pNFκB Ser536 in the nucleus, but only in cells incubated with PE serum at 2% O2. Thus, the p53/RSK1 complex might play a critical role in the activation of NFκB in trophoblastic cells and preeclamptic placentas.

Identification of Adrenomedullin-Induced S-Nitrosylated Proteins in JEG-3 Placental Cells

Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is important during placentation. Dysregulation of the process leads to pregnancy complications. S-nitrosylation of proteins is associated with cell invasion in many cell types. Adrenomedullin (ADM), a polypeptide expressed abundantly in the first-trimester placentas, induces EVCT invasion by upregulation of protein S-nitrosylation. This study aimed to identify the S-nitrosylated proteins induced by ADM in the JEG-3 placental cells. By using affinity chromatography followed by mass spectrometric analysis, tubulin, enolase, eukaryotic translation initiation factor 4A1, actin, annexin II (ANX II), and glyceraldehyde 3-phosphate dehydrogenaseprotein-1 were found to be S-nitrosylated by ADM. In vitro treatment with ADM or S-Nitrosoglutathione (GSNO) significantly increased the ANX II surface expression, but not its total expression in the JEG-3 cells. Translocation of ANX II to cell surface has been reported to act as a cell surface receptor to plasmin, plasminogen, and tissue plasminogen activator (tPA), thereby stimulating cell invasion and migration. However, in this study, ADM-induced surface expression of ANX II in the JEG-3 cells was not associated with changes in the secretory and membrane-bound tPA activities. Future studies are required to understand the roles of surface expression of S-nitrosylated ANX II on trophoblast functions. To conclude, this study provided evidences that ADM regulated the nitric oxide signaling pathway and modulated trophoblast invasion.

Hofbauer Cells Spread Listeria monocytogenes among Placental Cells and Undergo Pro-Inflammatory Reprogramming while Retaining Production of Tolerogenic Factors

Infection of the placental/fetal unit by the facultative intracellular pathogen Listeria monocytogenes results in severe pregnancy complications. Hofbauer cells (HBCs) are fetal macrophages that play homeostatic anti-inflammatory functions in healthy placentas.

From Coxiella burnetii Infection to Pregnancy Complications: Key Role of the Immune Response of Placental Cells

The infection of pregnant animals and women by Coxiella burnetii, an intracellular bacterium, compromises both maternal health and foetal development. The placenta is targeted by C. burnetii, as demonstrated by bacteriological and histological evidence. It now appears that placental strains of C. burnetii are highly virulent compared to reference strains and that placental injury involves different types of placental cells. Trophoblasts, the major placental cells, are largely infected by C. burnetii and may represent a replicating niche for the bacteria. The placenta also contains numerous immune cells, including macrophages, dendritic cells, and mast cells. Placental macrophages are infected and activated by C. burnetii in an unusual way of M1 polarisation associated with bacterial elimination. Placental mast cells eliminate bacteria through a mechanism including the release of extracellular actin filaments and antimicrobial peptides. In contrast, C. burnetii impairs the maturation of decidual dendritic cells, favouring bacterial pathogenicity. Our aim is to review C. burnetii infections of human placentas, paying special attention to both the action and function of the different cell types, immune cells, and trophoblasts targeted by C. burnetii in relation to foetal injury.