Cross-Tissue Conserved and Specific Features of Fetal Pig Macrophages and Their Remodeling in Fetal Growth Restriction at Single-Cell Resolution

Macrophages are highly heterogeneous immune cells with context-dependent functions, yet their transcriptional, functional, and metabolic diversity across prenatal tissues of the whole body, sexes and stages remains elusive. During fetal development, macrophages play essential roles in tissue formation and maintaining maternal-fetal immune balance, but their specific phenotypic and functional changes in the context of fetal growth restriction remain relatively underexplored. Herein, we constructed a single-cell cross-tissue macrophage atlas comprising 85,473 cells from 54 tissues of late-gestation fetal pigs. Analysis identified 15 macrophage subtypes, among which two Hofbauer cell subtypes were distinguished by their distinct transcriptional and functional characteristics between the amniotic and chorioallantoic membranes: the HBC GPNMB+ subtype upregulated glycolytic enzymes and extracellular matrix regulators such as LDHA and GPNMB, whereas the HBC PEG10+ subtype highly expressed immunomodulators and developmental regulators including A2M and PEG10. Two dichotomous subtypes, LYVE1highMHCIIlow and LYVE1lowMHCIIhigh, were systematically delineated across 27 tissues, and a core transcriptional signature distinguishing them was defined in both pig and human. Transcriptional profiling of metabolic pathways identified oxidative phosphorylation and the tricarboxylic acid cycle as core pathways across tissues, alongside tissue-specific adaptations such as glycolytic enrichment in amniotic macrophages. Cross-stage comparisons indicated that fetal macrophages predominantly maintained developmental programs, whereas adult macrophages globally displayed elevated immune activity. Cross-sex and cross-species analyses revealed a high degree of similarity in fetal macrophage transcriptomic profiles. In growth-restricted fetuses, chorioallantoic membrane Hofbauer cells exhibited profound remodeling marked by enhanced inflammation, hypoxia response, and vascular dysfunction signaling, alongside altered intercellular communication. Collectively, this work provides a comprehensive cross-tissue resource for understanding macrophage heterogeneity in fetal pigs and offers insights into the potential mechanisms underlying fetal growth restriction.