Programming of the Endometrial Immune Milieu by Interferon Gamma during Early Pregnancy in Pigs

Release of interferon-gamma (IFNG), a pro-inflammatory type II interferon, by the early conceptus is critical for pregnancy establishment in the pig. Yet, the cellular and molecular mechanisms underpinning conceptus-derived IFNG actions in maternal peripheral immune cells and at the embryo–maternal interface remain unclear. Here we show that pregnancy status up-regulates expression of IRF1, an IFNG target-gene, in peripheral blood mononuclear cells on day 15 of pregnancy in the pig. In a second study, loss-of-function IFNG (IFNG–/–) embryos were generated by using CRISPR/Cas9 gene editing and somatic cell nuclear transfer. Single-nuclei RNA sequencing of endometrium from gilts carrying wild-type (Control) or IFNG–/– conceptuses on day 15 of pregnancy revealed cell-type-specific signatures at the embryo–maternal interface in response to conceptus IFNG. Changes in the transcriptome of epithelial and IFNG receptor-expressing immune cells were evident between Control and IFNG–/– samples, along with a downregulation of IFNG target-genes involved with chemotaxis and immune cell differentiation in IFNG–/– recipient endometria. A notable reduction in monocytes and macrophages was observed in IFNG–/– samples, confirmed by immunohistochemistry for AIF1. Differential gene expression (DEG) analysis revealed 391 DEGs in monocytes, implicating IFNG in macrophage polarization, with evidence suggesting a shift toward an M2 phenotype. The study concludes that conceptus IFNG plays an important role in monocyte recruitment and macrophage polarization at the embryo–maternal interface for regulation of inflammation upon conceptus attachment. Insufficient numbers or skewed IFNG-activated macrophages within the endometrium may contribute to pregnancy failure in the IFNG–/– pig model. Overall design: On day 15 of estrous cycle/pregnancy, recipient (IFNG-/- and IFNG+/+) and bred gilts were euthanized via jugular injection of pentobarbital sodium and phenytoin sodium. The reproductive tracts were excised and transported to the laboratory for immediate processing. Uterine horns were trimmed free of adjacent tissues and processed for sample collection. After flushing and recovery of stage-matched conceptuses to confirm pregnancy, uterine horns were opened longitudinally along the antimesometrial line and endometrial fragments were dissected from the mesometrial side. Tissue samples were snap frozen and stored at -80°C. Prior to flushing, distal parts of each uterine horn near the uterine body were clamped, sectioned transversely, and processed for tissue fixation.