Non-Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

Ovine macrophage identity and plasticity: novel insights into CSF-driven polarization and species-specific responses.

Abstract

Macrophages (MOs) 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 MOs differentiated with GM-CSF (GM-oMOs) or M-CSF (M-oMOs) to provide a more nuanced understanding of their activation states. After 7 days, GM-oMOs displayed a smaller, more varied morphology with lower cell yields compared to the larger, uniformly amoeboid M-oMOs. Immunophenotypically, M-oMOs showed significantly higher CD163 expression, consistent with human M-MOs, while CLEC5A was uninformative for differentiation. Transcriptomic analysis, complemented by qPCR and ELISA, revealed clearly distinct profiles, with GM-oMOs 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-MO. Conversely, M-oMOs 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-oMO 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-oMOs exhibit greater plasticity, making them more suitable for pathogen-host interaction studies. Unlike GM macrophages, which already have a defined phenotype, M-oMOs more accurately reflect the dynamic immune response induced by a pathogen in the host.