Regulatory macrophages (Mregs) represent a subset of anti-inflammatory macrophages. In general, macrophages are a very dynamic and plastic cell type and can be divided into two main groups: classically activated macrophages (M1) and alternatively activated macrophages (M2).^[1] M2 group can further be divided into sub-groups M2a, M2b, M2c, and M2d.^[2] Typically the M2 cells have anti-inflammatory and regulatory properties and produce many different anti-inflammatory cytokines such as IL-4, IL-33, IL-10, IL-1RA, and TGF-β.^[3][4] M2 cells can also secrete angiogenic and chemotactic factors.^[5] These cells can be distinguished based on the different expression levels of various surface proteins and the secretion of different effector molecules.^[4]

M2a, mainly known as alternatively activated macrophages, are macrophages associated with tissue healing due to the production of components of extracellular matrix. M2a cells are induced by IL-4 and IL-13.^[2] M2b, generally referred to as regulatory macrophages (Mregs), are characterized by secreting large amounts of IL-10 and small amounts of IL-12.^[6][7] M2c, also known as deactivated macrophages, secrete large amounts of IL-10 and TGF-β. M2c are induced by glucocorticoids and TGF-β.^[8] M2d are pro-angiogenic cells that secrete IL-10, TGF-β, and vascular endothelial growth factor and are induced by IL-6 and A2 adenosine receptor agonist (A2R).^[4][9]

Mreg origin and induction

Mregs can arise following innate or adaptive immune responses. Mregs were first described after FcγR ligation by IgG complexes in the occurrence of pathogen-associated molecular patterns (e. g. lipopolysaccharide or lipoteichoic acid) acting through Toll-like receptors.^[10] Coculture of macrophages with regulatory T cells (Tregs) caused differentiation of macrophages toward Mreg phenotype.^[11] Similar effect provoked interaction of macrophages and B1 B cells.^[12] Mregs can even arise following stress responses. Activation of the hypothalamic-pituitary-adrenal axis leads to production of glucocorticoids that cause decreased production of IL-12 by macrophages.^[13]

Many cell types including monocytes, M1, and M2 can in a specific microenvironment differentiate to Mregs.^[7] Induction of Mregs is strongly linked with the interaction of Fc receptors located on the surface of Mregs with Fc fragments of antibodies.^[14] It has been shown that anti-TNF monoclonal antibodies interacting with Fcγ receptor of Mregs induce differentiation of Mregs through activation of STAT3 signaling pathway.^[15][16] Some pathogens can promote the transformation of cells into Mregs as an immune evasion mechanism.^[7][17] Two signals are needed for Mregs inducement. The first signal is stimulation by M-CSF, GM-CSF, PGE2, adenosine, glucocorticoid, or apoptotic cells.^[9][18] The second signal can be stimulation with cytokines or toll-like receptor ligands. The first signal promotes the differentiation of monocytes to macrophages and the second signal promotes immunosuppressive functions.^[8] In vitro, M-CSF, IFNγ, and LPS are used for the inducement of Mregs.^[7]

Other cells such as eosinophils and innate lymphoid cells type 2 (ILC2) can promote M2 polarization by cytokine secretion. IL-9 can function as a growth factor for ILC-2 and thereby assist in the induction of Mregs. Another cytokine that helps the induction of Mregs is IL-35 which is produced by Tregs.^[7]

Characterization and determination of Mregs

Surprisingly, Mregs resemble classically activated macrophages more than alternatively activated macrophages, due to higher biochemical similarity.^[19] The difference between M1 macrophages and Mregs is, inter alia, that Mregs secrete high levels of IL-10 and simultaneously low levels of IL-12. Out of all macrophages, Mregs show the highest expression of MHC II molecules and co-stimulatory molecules (CD80/CD86), which differentiates them from the alternatively activated macrophages, which show a very low expression of these molecules. Mregs also differ from alternatively activated macrophages by producing high levels of nitric oxide and low arginase activity.^[7][16][19] Lastly, they differ in the expression of FIIZ1 (Resistin-like molecule alpha1) and YM1 which are differentiation markers present on alternatively activated macrophages.^[4] Mregs are recognized by the expression of PD-L1, CD206, CD80/CD86, HLA-DR, and DHRS9 (dehydrogenase/reductase 9).^[4][20] DHRS9 has been recognized as a stable marker for Mregs in humans.^[20]

Biochemical and functional characterization of Mregs

The physiological role of Mregs is to dampen the immune response and immunopathology. Unlike classically activated macrophages, Mregs produce low levels of IL-12, which is important because IL-12 induces differentiation of naïve helper T cells to Th1 cells which produce high levels of IFNγ. Mregs do not contribute to the production of extracellular matrix because they express low levels of arginase.^[12][4]

Mregs show up-regulation of IL-10, TGFβ, PGE2, iNOS, IDO, and down-regulation of IL-1β, IL-6, IL-12, and TNF-α.^[21] By secreting TGF-β they help with the induction of Tregs and by producing IL-10 they contribute to the induction of tolerance and regulatory cell types. Mregs can directly inhibit the proliferation of activated T cells. It has been shown that Mregs co-cultured with T cells have a negative effect on the T-cellular ability to secrete IL-2 and IFN-γ. ^[22] Mregs can also inhibit the arginase activity of alternatively activated macrophages, the proliferation of fibroblasts, and can promote angiogenesis.^[23] The use of Mregs is widely studied as a potential cell-based immunosuppressive therapy after organ transplantation. Mregs could potentially solve the problems (susceptibility to infectious diseases and cancer diseases) associated with the current post-transplant therapy. Since Mregs are still producing nitric oxide they may be more suitable than current treatments, when appropriately stimulated.^[22]

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