Skountzou, Ioanna

Abstract Placental immunity is dichotomous: tolerance of the semiallogenic fetus is balanced with limiting transmission of maternal pathogens. HCs are the major fetal immune cell at the placenta, but mechanisms responsible for maintaining immune homeostasis while preventing infection require elucidation. We determined the phenotype of human HCs throughout gestation and analyzed stimulation response. Activated HCs were present in early pregnancy and reduced in number by term while maintaining similar phenotypes. Tolerant HC numbers were highest in midgestation, after a relatively intolerant phenotype early in gestation. We saw a significant shift in macrophage polarization as gestation progressed. Transcription of ARG2 exceeded iNOS at all points, reaching 10-fold higher at term. Following treatment with IL4+IL13 or IFNγ+LPS midgestation HCs underwent significant phenotype change and activation. Basal expression of antiviral IFN stimulated genes (ISGs) was lowest at midgestation and was enhanced by IFN-α and IFN-λ1 with a 10-fold stronger response to IFN-α. RIG-I agonism induced HC activation, 10-fold iNOS upregulation and enhanced transcription of IFNs, MDA5, RIG-I, and ISGs. Response to stimulation by IFNγ+LPS, IL4+IL13, IL1β+HAGG was limited to loss of tolerance at term. IL-10 treatment increased numbers of CD163+ HCs, and IFNγ+LPS caused loss of discernible polarization patterns. Basal expression of RIG-I, MDA5 and ISGs was highest in term HCs, but IFN-α did not activate them. RIG-I agonism reduced markers of tolerance. HCs are variable macrophages, with phenotype and immune capacity strongly dependent on gestational age. Understanding placental immunobiology is fundamental to addressing key pregnancy morbidities.

Abstract Fetal placental macrophages (Hofbauer cells (HCs)) appear at 18 days post-conception and are maintained throughout pregnancy. Despite their identification more than a century ago, there are few studies characterizing the evolution of human HC phenotype through gestation. Using high-dimensional flow cytometry, we measured expression of CD68, CD80, CD86, HLA-DR, CD163, CD206, and CD209 on human HCs ex vivo. This panel includes markers previously validated on term human HCs for immune activation (CD80, CD86) and modulation (DC-SIGN, HLA-DR). To best capture the spectrum of HC diversity across gestation, we employed an unbiased approach to analyze processed datasets from HCs isolated from placentae 12–17 weeks gestational age (n=5), 17–24 weeks gestational age (n=7) and at term (n=5) using t-distributed stochastic neighbor embedding analysis (tSNE). HC populations were more diverse early in pregnancy and at term, compared to the second trimester; this is anticipated given the changing microenvironment following initial placental anchoring to eventual parturition. Marker expression heatmaps were generated to assess gestation-dependent changes in HC phenotype; HCs expressing activation markers were most frequent in early gestation, and reduced by term. HCs bearing markers of immune modulation were most frequent at mid-gestation, and least in early gestation. These results demonstrate the power of utilizing computational methods to analyze high-dimensional flow cytometry data collected from an understudied cell type and demonstrate how HCs phenotypically evolve during human pregnancy.

Abstract The importance of fetal placental macrophages (Hofbauer cell [HCs]) is underscored by their appearance 18 d postconception and maintenance through term; however, how human HCs evolve during healthy pregnancy and how microenvironment and ontogeny impact phenotype and function remain unknown. In this study, we comprehensively classify human HCs ex vivo, interrogate phenotypic plasticity, and characterize antiviral immune responses through gestation. Activated HCs were abundant in early pregnancy and decreased by term; molecular signatures emphasize inflammatory phenotypes early in gestation. Frequency of HCs with regulatory phenotypes remained high through term. Furthermore, term HCs exhibited blunted responses to stimulation, indicating reduced plasticity. IFN-λ1 is a key placental IFN that appeared less protective than IFN-α, suggesting a potential weakness in antiviral immunity. Ligand-specific responses were temporally regulated: we noted an absence of inflammatory mediators and reduced antiviral gene transcription following RIG-I activation at term despite all HCs producing inflammatory mediators following IFN-γ plus LPS stimulation. Collectively, we demonstrate sequential, evolving immunity as part of the natural history of HCs through gestation.