Beaver, Jacob T.

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.