Cytokine Induced Phenotypic and Epigenetic Signatures Are Key to Establishing Specific Macrophage Phenotypes

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 CD14hi CD163hi CD206int FOLR2-expressing M-CSF MΦ and CD14lo CD163lo CD206hi 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.

Immune regulation at the fetal‐maternal interface with focus on decidual macrophages

“…14), making CD163 a potential marker of homeostatic macrophages. This is supported by studies evaluating the effects of M-CSF versus GM-CSF and the additional effects of IL-10, IL-4 and IFN-γ (Ambarus et al, 2012;Kittan et al, 2013). Importantly, CD163 is also expressed on many tissue macrophages that predominantly have homeostatic functions (Van den Heuvel et al, 1999).…”

“…Although it was originally shown to be upregulated by IL-4 and downregulated by IFN-γ (Stein et al, 1992), studies show that GM-CSF macrophages express higher CD206 levels than M-CSF macrophages (Brocheriou et al, 2011;Kittan et al, 2013).…”

“…CD206 was the first described marker that distinguished between macrophages stimulated by IL-4, which induced its expression, or IFN-γ, which decreased its expression (Stein et al, 1992). More recently, also GM-CSF has been shown to strongly upregulate CD206 expression (Brocheriou et al, 2011;Kittan et al, 2013). CD206 has been shown to mediate anti-inflammatory responses in DCs by promoting production of IL-10, downregulation of costimulatory molecules and production of the Th2-associated chemokines CCL17 and CCL22 (Chieppa et al, 2003).…”

“…The most common experimental design for generating polarized macrophages is the differentiation in the presence of M-CSF or GM-CSF and the subsequent activation with cytokines (or TLR ligands) (Martinez et al, 2006;Jaguin et al, 2013;Kittan et al, 2013). However, in papers I and II, macrophages were cultured in the presence of macrophage growth factors as well as polarizing factors (cytokines or hormones) from the first day of culture (Fig.…”

Colony-Stimulating Factor-1 Receptor in the Polarization of Macrophages: A Target for Turning Bad to Good Ones?

“…In spite of this, to refer to GM-CSF and CSF-1 as the cytokines responsible for the prototypical M1 or M2 phenotype in not based on solid evidence [7]. Another important point is that CSF-1-and GM-CSF-differentiated macrophages may react similarly to a further polarization induced by IFNγ or IL-4+IL-13 [8,52]. In this respect, it has been recently shown that the most robust M2 activation phenotype is induced by IL-4/ IL-10/TGFβ following differentiation with CSF-1 [53].…”