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

Abstract Authors: Programming of the Uterine Immune Milieu by Interferon Gamma during Early Pregnancy in Pigs
 
Mariana Sponchiado¹; Weihong Gu¹; Darling M. C. Madrid¹; Rachel Carroll¹; Kimberly M. Davenport²; Caroline G. Lucas¹; Riley M. Sullivan¹; Kevin D. Wells¹; John P. Driver¹; Wesley C. Warren¹; Randall S. Prather¹; Rodney D. Geisert¹
 
1. Division of Animal Sciences, University of Missouri, Columbia, MO, USA
2. Department of Animal Sciences, Washington State University, Pullman, WA, USA

Abstract Text: Attachment and/or invasion of the allogenic conceptus to the bearing uterus is a paradoxical phenomenon that requires well-orchestrated adaptations by the maternal immune system in response to the conceptus and its signals. In the pig, which has an epitheliochorial type of placentation, the conceptus secretes interferon-gamma (IFNG), a pro-inflammatory type II interferon, during early attachment to the uterine surface. Ablation of conceptus IFNG is associated with altered endometrial regulation of inflammation, attachment of conceptuses, and organization of the extracellular matrix in the endometrium resulting in degeneration of the conceptuses on day 17 of pregnancy in pigs (Johns et al., 2021; BOR 105:1577-1590). This study investigated the role of conceptus–derived IFNG in controlling proinflammatory endometrial cellular and molecular responses to avoid pregnancy loss. Loss-of-function IFNG (IFNG^–/–) embryos were generated by using CRISPR/Cas9 gene editing and somatic cell nuclear transfer. Endometrial samples were collected along the mesometrial side from gilts carrying wild-type (Control; n=4) or IFNG^–/– (n=3) conceptuses on day 15 of pregnancy, and subjected to single-nuclei RNA sequencing (snRNAseq; 10x Genomics). A dimensionality reduction analysis identified 19 nuclei clusters including epithelial (luminal, glandular and ciliated), stromal, endothelial, perivascular, lymphatic, and immune cells. Changes in distribution of epithelial and IFNG receptor-expressing immune cells were evident between Control and IFNG^–/– endometria. A notable reduction in monocytes and macrophages was observed in IFNG^–/– samples, confirmed by immunohistochemistry for allograft inflammatory factor 1 (AIF1), a marker for monocytes and macrophages. AIF1-positive cells were visualized in endometrial blood vessels, stroma, glands and sub-epithelial cellular compartments. Downstream target genes of IFNG signaling, such as C-X-C motif chemokine ligand 9 (CXCL9) and 10 (CXCL10), which are involved with chemotaxis and immune cell differentiation, were downregulated in IFNG^–/– compared to Control endometria. In addition, higher expression of monocyte chemotactic protein 1 (CCL2) in perivascular cells from Control samples indicates enhanced recruitment of monocytes within the endometrium in response to IFNG. Analysis of differentially expressed genes (DEGs) revealed 391 DEGs in monocytes, implicating IFNG in monocyte activation and macrophage polarization, with evidence suggesting a shift toward an M2 (anti-inflammatory) phenotype in Control endometria. The study concludes that conceptus-derived IFNG plays an essential role in monocyte recruitment and macrophage polarization at the embryo–maternal interface for the regulation of inflammation upon conceptus attachment. Insufficient numbers or skewed macrophage polarization within the endometrium may contribute to pregnancy failure in the IFNG^–/– pig model.
Support: USDA-NIFA (2017-67015-26451 and 2020-67015-33402).