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Updated: Jul 11, 2025
Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
Characterization of polarization states of canine monocyte derived macrophages
Qingkang Lyu1,2, Edwin J A Veldhuizen1, Irene S Ludwig1
1Department Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Insights
Canine monocytes can be polarized into M1 or M2 macrophages, exhibiting distinct morphology and surface markers. M2 macrophages show enhanced phagocytic capacity, paving the way for canine immunology research.
Area of Science:
- Immunology
- Cell Biology
- Veterinary Science
Background:
- Macrophage polarization is crucial for immune responses and understanding immune-related diseases.
- Knowledge of canine macrophage polarization is limited, hindering research in veterinary immunology.
- This study addresses the gap in understanding canine macrophage subsets.
Purpose of the Study:
- To polarize canine monocytes into M1 and M2 macrophage subsets.
- To characterize the morphology, surface markers, gene expression, and function of these subsets.
- To establish a foundation for studying canine immune-related diseases using polarized macrophages.
Main Methods:
- Canine monocytes were differentiated into M0, M1 (GM-CSF/IFN-γ + LPS), and M2 (M-CSF + IL-4) macrophages.
- Characterization involved morphology assessment, surface marker analysis (flow cytometry), RNA sequencing, and phagocytosis assays.
- Comparative analysis was performed across the three subsets.
Main Results:
- M1 macrophages displayed a rounder, amoeboid shape, while M2 macrophages were smaller and spindle-like.
- Distinct surface marker profiles were observed: M1 (CD40, CD80, CD86, MHC II) and M2 (CD206, CD209, CD163).
- M2 macrophages demonstrated superior phagocytic capacity for latex beads compared to M0 and M1 cells.
Conclusions:
- Canine M1 and M2 macrophages exhibit distinct characteristics comparable to other species.
- Polarized canine macrophages offer valuable tools for investigating immune-related diseases in dogs.
- Further research into gene expression complexity may reveal novel polarization markers.
Abstract:
Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune‑related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.
