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Orekhov et al. Vessel Plus 2019;3:10 I http://dx.doi.org/10.20517/2574-1209.2019.04 Page 5 of 20
[29]
macrophages induced by LPS .

According to the modern nomenclature, at least five M2-like macrophage subsets can be distinguished.
Three of those induced by IL-4 (IL-13), immune complexes or IL-10 [termed M(IL4), M(Ic), and M(IL-10)
respectively] exhibit the most anti-inflammatory properties and correspond roughly to M2a, M2b, and M2c
subsets of the old classification. In mice, IL-4-acivated macrophages are characterized by high expression of
STAT-6, but also express STAT-1, SOCS2, and IRF-4, while M(IL-10) macrophages produce STAT-3, SOCS3,
nuclear factor, interleukin 3-regulated (NFIL3), and strawberry notch homolog 2 (Drosophila) (SBNO2)
transcription factors. While SOCs are needed to block the pro-inflammatory activation of macrophages,
[30]
both NFIL3 and SBNO2 contribute to the downstream anti-inflammatory effects of IL-10 .
In humans, IL-4-activated macrophages produce IRF-4, SOCS1, and GATA3 essential for the establishment
of the IL-4-dependent anti-inflammatory transcriptional program. In humans, M(IL10) cells highly produce
SOCS3 while TGF-β and glucocorticoids-induced macrophages (roughly corresponding to M2c phenotype)
express SMAD2, DNA-binding protein inhibitor ID3, and regulator of G-protein signaling 1 transcriptional
regulators. These proteins are required for the activation of the multistep TGF-β-specific transcription
program. Glucocorticoid-dependent stimulation of the surface expression of the TGF-β receptor II is
[31]
necessary for TGF-β-mediated signaling . Glucocorticoid hormones are also involved in the induction of
TGF-β receptor II in human M(GC) macrophages. By contrast, expression of this receptor is absent in IL-4-
[31]
induced human macrophages .

IRF/STAT SIGNALING
The IRF/STAT-regulated pathways are key mediators of M1/M2 macrophage activation. In response to Th1
cytokines and inflammatory stimuli, stimulation of STAT-1/STAT-2 and IRF-5 primes differentiation to M1
cells, while STAT-3/STAT-6 and IRF-3/IRF-4 critically contribute to the formation of the M2 phenotype.

Role of IRF/STAT signaling in M1 differentiation
Generally, GM-CSF drives the commitment of the myeloid cell lineage to the bone marrow but also supports
monocyte transformation to M1 macrophages. The GM-CSF receptor exists as heterodimer consisted of
[32]
α- and β-subunits, which can form a homohexamer as a functional ternary complex . Activation of the
GM-CSF receptor leads to Janus kinase 2 (Jak2)-mediated stimulation of STAT-5 and Erk/Akt-dependent
[33]
pathway, and nuclear translocation of transcription factors IRF5 and NF-κB .

In monocytes, stimulation with GM-CSF upregulates the antigen-presenting function, phagocytosis, anti-
microbial activity, and production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and growth
factors (M-CSF, GM-CSF). A global transcriptome analysis of GM-CSF-induced macrophages revealed up-
regulation of 340 genes responsible mainly for antigen presentation, lipid metabolism, and innate immune
signaling including macrophage-specific surface markers/receptors such as CD14, CD163, C5R1, CSF3R,
[34]
GDF15, and FcγR1A .
STAT-1 and STAT-2
IFN-γ binding to its receptor leads to the recruitment of Jak1/2 and formation of the functionally active
[35]
STAT-1 dimer , which then binds to the interferon-γ activated sequence (GAS) in the promoter of its target
[36]
genes, such as IL-12 and inducible NO-synthase (iNOS) and stimulates their expression . LPS binding to
TLR4 induces the subsequent activation of NF-κB, which drives transcription of a whole set of inflammatory
[37]
genes . LPS also stimulates production of Type 1 IFN, which through autocrine binding to its receptor
[38]
IFNAR leads to the Jak1/Tyk2-mediated activation of STAT-1 and STAT-2 . Activated STAT-1 and STAT-
2 form, in turn, a complex with IRF-9. The assembly of STAT-1/2 and IRF-9 results in the induction of