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Updated: May 6, 2026
Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis
Published on: November 28, 2015
Cytokine induced phenotypic and epigenetic signatures are key to establishing specific macrophage phenotypes
Nicolai A Kittan1, Ronald M Allen, Abhay Dhaliwal
1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Abstract:
Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14(hi) CD163(hi) CD206(int) FOLR2-expressing M-CSF MΦ and CD14(lo) CD163(lo) CD206(hi) GM-CSF MΦ but show that both macrophage populations reacted similarly to further polarization with IFN-γ or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.
Insights
This study differentiates macrophages (MΦ) using GM-CSF or M-CSF and reveals distinct phenotypes. Epigenetic regulators like MLL are identified as key drivers of MΦ polarization and activation states.
Area of Science:
- Immunology
- Cell Biology
- Epigenetics
Background:
- Macrophages (MΦ) are crucial immune cells with pro-inflammatory (M1) and regulatory (M2) phenotypes.
- Differentiation protocols using M-CSF or GM-CSF alone can induce M1- or M2-like states.
- Epigenetic modifications by chromatin modification enzymes (CME) are known to influence myeloid differentiation.
Purpose of the Study:
- To investigate the phenotypic differences of human MΦ differentiated with GM-CSF versus M-CSF.
- To analyze the role of CME in MΦ polarization towards M1 or M2 states.
- To identify novel markers and regulatory mechanisms of MΦ activation.
Main Methods:
- Differentiated human monocytes into MΦ using GM-CSF or M-CSF.
- Polarized MΦ towards M1 (IFN-γ) or M2 (IL-4+IL-13) phenotypes.
- Analyzed cell surface markers (CD14, CD163, CD206, FOLR2) and CME expression patterns.
- Investigated the role of MLL in regulating CXCL10 expression using an MLL-Menin inhibitor.
Main Results:
- GM-CSF MΦ exhibited distinct phenotypes (CD14lo, CD163lo, CD206hi) compared to M-CSF MΦ (CD14hi, CD163hi, CD206int, FOLR2+).
- Both MΦ types responded similarly to IFN-γ or IL-4+IL-13 polarization, with characteristic M1/M2 gene expression changes.
- M1 MΦ upregulated histone methyltransferase MLL and demethylase KDM6B, while M2 MΦ showed increased DNA methyltransferases and histone deacetylases.
- MLL was identified as a regulator of CXCL10 expression in M1 MΦ, inhibitable by MLL-Menin inhibitor.
- High mannose receptor (CD206) expression is a distinct feature of GM-CSF MΦ.
Conclusions:
- GM-CSF and M-CSF induce distinct MΦ phenotypes with unique surface marker profiles.
- MΦ polarization is regulated by specific epigenetic mechanisms involving CME.
- MLL plays a novel role as a marker for classical M1 activation and regulates inflammatory gene expression.
- Findings offer insights into distinguishing MΦ activation states and potential therapeutic targets.
