Intra-amniotic infection, the invasion of microbes into the amniotic cavity resulting in inflammation, is a clinical condition that can lead to adverse pregnancy outcomes for the mother and fetus as well as severe long-term neonatal morbidities. Despite much research focused on the consequences of intra-amniotic infection, there remains little knowledge about the innate immune cells that respond to invading microbes. We performed RNA-seq of sorted amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection to determine the transcriptomic differences between these innate immune cells. Further, we sought to identify specific transcriptomic pathways that were significantly altered by the maternal or fetal origin of amniotic fluid neutrophils and monocytes/macrophages, the presence of a severe fetal inflammatory response, and pregnancy outcome (i.e., preterm or term delivery). We show that significant transcriptomic differences exist between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection, indicating the distinct roles these cells play. The transcriptome of amniotic fluid immune cells varies based on their maternal or fetal origin, and the significant transcriptomic differences between fetal and maternal monocytes/macrophages imply that those of fetal origin exhibit impaired functions. Notably, transcriptomic changes in amniotic fluid monocytes/macrophages suggest that these immune cells collaborate with neutrophils in the trafficking of fetal leukocytes throughout the umbilical cord (i.e., funisitis). Finally, amniotic fluid neutrophils and monocytes/macrophages from preterm deliveries display enhanced transcriptional activity compared to those from term deliveries, highlighting the protective role of these cells during this vulnerable period. Collectively, these findings demonstrate the underlying complexity of local innate immune responses in women with intra-amniotic infection and provide new insights into the functions of neutrophils and monocytes/macrophages in the amniotic cavity.
The Author(s). Published by S. Karger AG, Basel.

Background Monocytes, after neutrophils, are the most abundant white blood cells found in the amniotic cavity of women with intra-amniotic inflammation/infection. However, the origin of such cells has not been fully investigated. Herein, we determined (1) the origin of amniotic fluid monocytes/macrophages from women with intra-amniotic inflammation/infection, (2) the relationship between the origin of amniotic fluid monocytes/macrophages and preterm or term delivery and (3) the localization of monocytes/macrophages in the placental tissues. Methods Amniotic fluid samples (n = 16) were collected from women with suspected intra-amniotic inflammation or infection. Amniotic fluid monocytes/macrophages were purified by fluorescence-activated cell sorting, and DNA fingerprinting was performed. Blinded placental histopathological evaluations were conducted. Immunohistochemistry was performed to detect CD14+ monocytes/macrophages in the placental tissues. Results DNA fingerprinting revealed that (1) 56.25% (9/16) of amniotic fluid samples had mostly fetal monocytes/macrophages, (2) 37.5% (6/16) had predominantly maternal monocytes/macrophages and (3) one sample (6.25% [1/16]) had a mixture of fetal and maternal monocytes/macrophages. (4) Most samples with predominantly fetal monocytes/macrophages were from women who delivered early preterm neonates (77.8% [7/9]), whereas all samples with mostly maternal monocytes/macrophages or a mixture of both were from women who delivered term or late preterm neonates (100% [7/7]). (5) Most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (85.7% [12/14]). (6) Women who had mostly fetal monocytes/macrophages in amniotic fluid had abundant CD14+ cells in the umbilical cord and chorionic plate, whereas women with mostly maternal amniotic fluid monocytes/macrophages had abundant CD14+ cells in the chorioamniotic membranes. Conclusion Amniotic fluid monocytes/macrophages can be of either fetal or maternal origin, or a mixture of both, in women with intra-amniotic inflammation or infection. These immune cells could be derived from the fetal and maternal vasculature of the placenta.

Bacterial and fungal infections are a major cause of morbidity and mortality in neutropenic patients. Donor-derived neutrophil transfusions have been used for prophylaxis or treatment for infection in neutropenic patients. However, the short half-life and the limited availability of large numbers of donor-derived neutrophils for transfusion remain a significant hurdle in the implementation of neutrophil transfusion therapy. Here, we investigate the in vitro and in vivo activity of neutrophils generated from human induced pluripotent stem cells (iPSC), a potentially unlimited resource to produce neutrophils for transfusion. Phenotypic analysis of iPSC-derived neutrophils reveal reactive oxygen species production at similar or slightly higher than normal peripheral blood neutrophils, but have an ∼50%-70% reduced Escherichia coli phagocytosis and phorbol 12-myristate 13-acetate induced formation of neutrophil extracellular traps (NET). Signaling of granulocytic precursors identified impaired AKT activation, but not ERK or STAT3, in agonist-stimulated iPSC-derived neutrophils. Expression of a constitutively activated AKT in iPSC-derived neutrophils restores most phagocytic activity and NET formation. In a model of bacterial induced peritonitis in immunodeficient mice, iPSC-derived neutrophils, with or without corrected AKT activation, migrate similarly to the peritoneal fluid as peripheral blood neutrophils, whereas the expression of activated AKT significantly improves their phagocytic activity in vivo. Stem Cells Translational Medicine 2019;8:557-567.
© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Neutrophils are the most abundant white blood cells found in the amniotic cavity of women with intraamniotic infection and/or inflammation. The current belief is that these neutrophils are of fetal origin. However, abundant neutrophils have been found in the amniotic fluid of women with a severe acute maternal inflammatory response but without a severe fetal inflammatory response in the placenta, suggesting that these innate immune cells can also be of maternal origin or a mixture of both fetal and maternal neutrophils.
We sought to investigate the origin of amniotic fluid neutrophils from women with intraamniotic infection and/or inflammation and to correlate these findings with acute histologic maternal and fetal inflammatory responses in the placenta.
Amniotic fluid was collected from 15 women with suspected intraamniotic infection and/or inflammation (positive microbiological cultures and/or interleukin-6 concentrations ≥2.6 ng/mL). Amniotic fluid neutrophils were purified by fluorescence-activated cell sorting, DNA was extracted, and DNA fingerprinting was performed. DNA fingerprinting was also performed in the umbilical cord and maternal blood DNA. Fluorescence in situ hybridization was assayed in women with male neonates. Blinded placental histopathological evaluations were conducted.
First, DNA fingerprinting revealed that 43% (6/14) of women who underwent a single amniocentesis had mostly fetal neutrophils in the amniotic fluid. Second, DNA fingerprinting showed that 36% (5/14) of the women who underwent a single amniocentesis had predominantly maternal neutrophils in the amniotic fluid. Third, DNA fingerprinting indicated that 21% (3/14) of the women who underwent a single amniocentesis had an evident mixture of fetal and maternal neutrophils in the amniotic fluid. Fourth, DNA fingerprinting revealed that a woman who underwent 2 amniocenteses (patient 15) had fetal neutrophils first, and as infection progressed, abundant maternal neutrophils invaded the amniotic cavity. Fifth, fluorescence in situ hybridization confirmed DNA fingerprinting results by showing that both fetal and maternal neutrophils were present in the amniotic fluid. Sixth, most of the women who had predominantly amniotic fluid neutrophils of fetal origin at the time of collection delivered extremely preterm neonates (71% [5/7]). Seventh, all of the women who had predominantly amniotic fluid neutrophils of maternal origin at the time of collection delivered term or late preterm neonates (100% [6/6]). Eighth, 2 of the women who had an evident mixture of fetal and maternal neutrophils in the amniotic fluid at the time of collection delivered extremely preterm neonates (67% [2/3]), and the third woman delivered a term neonate (33% [1/3]). Finally, most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (87% [13/15]).
Amniotic fluid neutrophils can be either predominantly of fetal or maternal origin, or a mixture of both fetal and maternal origin, in women with intraamniotic infection and/or inflammation. The findings herein provide evidence that both fetal and maternal neutrophils can invade the amniotic cavity, suggesting that both the fetus and the mother participate in the host defense mechanisms against intraamniotic infection.
Copyright © 2017 Elsevier Inc. All rights reserved.

Glioblastoma is the most common and most malignant primary brain tumor with a median survival of 15 months. Moschamine is an indole alkaloid that has a serotoninergic and cyclooxygenase inhibitory effect. In this study, we sought to determine whether moschamine could exert cytotoxic and cytostatic effects on glioma cells in vitro. Moschamine was tested for toxicity in zebrafish. We investigated the effect of moschamine on U251MG and T98G glioblastoma cell lines. Viability and proliferation of the cells were examined with trypan blue exclusion assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the xCELLigence system. Apoptosis (annexin-propidium iodide), cell cycle, and CD24/CD44/CD56/CD15 expression were tested with flow cytometry. Treatment with moschamine significantly reduced cell viability in both cell lines tested. Induction of cell death and cell cycle arrest was confirmed with flow cytometry in both cell lines. After treatment with moschamine, there was a dose-dependent decrease in CD24 and CD44 expression, whereas there was no change in CD56 and CD15 expression in T98G cell line. The zebrafish mortality on the fifth post-fertilization day was zero even for 1 mM of moschamine concentration. The treatment of glioblastoma cell lines with moschamine may represent a novel strategy for targeting glioblastoma.