Proteomic and morphologic characterization of ovine macrophage differentiation and polarization

Macrophages play crucial roles in inflammation and tissue homeostasis, exhibiting phenotypic and functional plasticity that enables them to initiate, sustain, or resolve inflammation. To support the evidence-based selection of biomedically relevant preclinical models, this study comprehensively characterized ovine macrophage differentiation and polarization by integrating morphological assessments with in-depth proteomic profiling of cellular lysates and secretomes. Monocytes isolated from peripheral blood were differentiated into macrophages using GM-CSF (GMØ) or M-CSF (MMØ), then polarized into M1 and M2 phenotypes. Mass spectrometry identified 4804 proteins in cell lysates and 901 in secretomes, including 42 CD antigens, enabling the establishment of CD marker profiles for monocytes and the distinct macrophage differentiation and polarization states. Proteomic analyses revealed significant upregulation of inflammatory markers in M1 and elevated tissue repair markers in M2 macrophages. Enrichment analysis confirmed activation of antimicrobial and matrix-degrading pathways in M1 macrophages, and regenerative, proteolysis-inhibiting functions in M2 macrophages. In conclusion, this study addresses the limitations of currently available immunological tools by providing an antibody-independent alternative for the classification of ovine macrophages, facilitating more precise phenotypic characterization and functional insight in ovine immunology research.