Hecker, Yanina P.Coronado, MontserratHurtado Morillas, ClaraArranz Solís, DavidSánchez Sánchez, RobertoCorbí, ÁngelOrtega Mora, Luis Miguel2025-12-192025-12-192025Hecker, Y. P., Coronado, M., Hurtado-Morillas, C., Arranz-Solís, D., Sánchez-Sánchez, R., Corbí, Á., & Ortega-Mora, L. M. (2025). Ovine macrophage identity and plasticity: novel insights into CSF-driven polarization and species-specific responses. Frontiers in immunology, 16, 1680086. https://doi.org/10.3389/fimmu.2025.168008610.3389/fimmu.2025.1680086https://hdl.handle.net/20.500.14352/129496Author contributions YH: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. MC: Data curation, Formal analysis, Resources, Software, Visualization, Writing – review & editing. CH-M: Formal analysis, Software, Visualization, Writing – review & editing. DA-S: Investigation, Methodology, Resources, Visualization, Writing – review & editing. RS-S: Investigation, Methodology, Resources, Visualization, Writing – review & editing. AC: Conceptualization, Formal analysis, Investigation, Resources, Supervision, Visualization, Writing – review & editing. LO-M: Conceptualization, Funding acquisition, Investigation, Resources, Supervision, Visualization, Writing – review & editing.Macrophages (MØs) are pivotal immune cells exhibiting significant plasticity that has been widely studied in human and murine models. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are key regulators of macrophage differentiation from monocytes. In this study, we comprehensively investigated the immunophenotypic, functional, and transcriptomic profiles of ovine MØs differentiated with GM-CSF (GM-oMØs) or M-CSF (M-oMØs) to provide a more nuanced understanding of their activation states. After 7 days, GM-oMØs displayed a smaller, more varied morphology with lower cell yields compared to the larger, uniformly amoeboid M-oMØs. Immunophenotypically, M-oMØs showed significantly higher CD163 expression, consistent with human M-MØs, while CLEC5A was uninformative for differentiation. Transcriptomic analysis, complemented by qPCR and ELISA, revealed clearly distinct profiles, with GM-oMØs exhibiting a pronounced pro-inflammatory phenotype and showing significantly higher expression of 408 genes, mostly associated with interferon and inflammatory response pathways, a feature that aligns with the functional and phenotypic characteristics of human and mouse GM-MØ. Conversely, M-oMØs displayed a regulatory and anti-inflammatory profile, marked by a significantly higher expression of IL-10 and a set of 248 genes involved in cellular homeostasis. Notably, LPS stimulation dramatically shifted the M-oMØ phenotype toward a pro-inflammatory state, unequivocally demonstrating their substantial plasticity, and mirroring human M-CSF-polarized monocytes. Our findings fundamentally challenge the prevailing M1/M2 simplification in ovine macrophage biology and provide a robust foundation for selecting appropriate in vitro macrophage models for future investigations into ovine host defense and disease pathogenesis. This study demonstrated that M-oMØs exhibit greater plasticity, making them more suitable for pathogen-host interaction studies. Unlike GM macrophages, which already have a defined phenotype, M-oMØs more accurately reflect the dynamic immune response induced by a pathogen in the hostengAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article1664-3224https://doi.org/10.3389/fimmu.2025.168008641376638https://pubmed.ncbi.nlm.nih.gov/41376638/open access576.8GM-CSFM-CSFRNAseqimmunophenotypeOvine macrophagesParasitología (Medicina)2401.12 Parasitología Animal