Ovine Macrophage Identity and Plasticity: Novel Insights into CSF-Driven Polarization and Species-Specific Responses

Yanina Paola Hecker^1*Montserrat Coronado¹Hurtado-Morillas Clara¹David Arranz-Solís¹Roberto Sanchez Sanchez¹Angel L Corbi²Luis Miguel Ortega-Mora¹

• ¹Complutense University of Madrid, Madrid, Spain
• ²Myeloid Cell Laboratory, Centro de Investigaciones Biologicas Margarita Salas, Madrid, Spain

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 host.