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Page 8 of 20 Orekhov et al. Vessel Plus 2019;3:10 I http://dx.doi.org/10.20517/2574-1209.2019.04

to prevent M2 differentiation. SOCS3 can induce STAT-3 degradation through ubiquitination-dependent
[74]
mechanisms or inhibit Jak-dependent activation of STAT-3 . STAT-3 and p38 mitogen-activated protein
kinases (MAPK) participate in the reciprocal control of macrophage response to activation with LPS. This
includes control of SOCS3 expression and p38 MAPK-dependent stimulation of protein kinases MK2 and MK3,
[73]
which mediate up-regulation of pro-inflammatory NF-κB or anti-inflammatory IRF-3 in response to LPS .
The crosstalk between STAT-3 and p38 MAPK is important for initiation of the pro-inflammatory macrophage
response and regulation of the inflammation resolution, which is largely mediated by IL-10 and STAT-3.

Role of IRFs in M2 differentiation
It was suggested that IRF-3 may play an important role mediating M2 differentiation of macrophages.
[75]
Dephosphorylated IRF-3 maintains self-inhibitory conformation and is inactive . TBK1 and/or IKK-ε
phosphorylate IRF-3 lead to conformational changes, which abolish self-inhibitory structure and allow
[76]
binding of coactivators CBP/p300 followed by nuclear translocation and activation of the factor . IKK-
β-dependent phosphorylation of IRF-3 abrogates its transcriptional activity and stimulates further
polyubiquitination and degradation mediated by E3 ubiquitin ligase cullin or RBCK1 [77,78] . M1 macrophages
induced by GM-CSF are characterized by inactive IRF-3 and up-regulated MyD88and active NF-κB and
[16]
AP-1 transcription factors . By contrast, macrophages stimulated by M-CSF are characterized by active
TLR-induced IRF-3 and decreased NF-κB activity [16,79] . These observations were confirmed in the glial
cells, which can be regarded as resident macrophages, where IRF-3 suppressed pro-inflammatory genes IL-
1, IL-6, IL-8, TNF-α and CXCL1 and activated anti-inflammatory genes, such as IL-1 receptor agonist, IL-
[80]
10 and IFN-β . In summary, IRF-3 mediates M-CSF-dependent polarization of alternatively-activated
macrophages. At the same time, it can also promote the expression of a number of pro-inflammatory genes,
[81]
such as IFN-β and chemokine CCL5 .
[82]
Together with IRF-8, IRF-4 belongs to ‘hematopoietic’ transcription factors . The activation of IRF-4 leads
to homodimerization and assembling with Pu.1, a Ets transcription factor, which can also bind IRF-8 [83,84] .
[85]
Complexes of IRF-4 and IRF-8 with Pu.1 can cooperate to enhance the expression of target genes .

[86]
The prominent role of IRF-4 in the alternative polarization of macrophages has been demonstrated .
M2-specific macrophage genes are controlled by epigenetic regulation, and JMJD3 is able to remove the
[66]
[87]
methylation marks and induce their expression . In macrophages, IL-4 activates both JMJD3 and IRF4 ,
which can, in their turn, activate each other. As a result of this activation, a set of M2-specific genes is up-
regulated, including arginase 1, Fizz1, Ym1, and mannose receptor (MR). Moreover, IRF-4 induces the
[88]
expression of IL-4 and IL-10 cytokines . In accordance with these data, IRF-4-deficient mice are susceptible
[89]
to LPS-induced sepsis and have increased expression of IL-6 and TNF-α in response to TLR ligands . IRF-
4 also prevents M1 polarization of macrophages by competing with IRF-5 for interaction with MyD88, a
[43]
potent activator of pro-inflammatory factors NF-κB .

Role of NF-κB and AP-1 in M1 differentiation
NF-κB and AP-1 are two key transcription factors that drive expression of a bulk of inflammatory genes in
macrophages.

NF- κB
NF-κB activates transcription of various inflammatory genes. In the absence of inflammatory stimuli,
NF-κB forms inactive complex with IkB. Upon inflammatory activation with LPS and other ligands, IkB
[90]
phosphorylation induces its dissociation and NF-κB transition to the nucleus . On the cell surface, LPS
[91]
binding protein (LBP) serves for LPS capture and delivery to the pattern recognition receptor CD14 .
CD14-LPS complex then binds to TLR-4 assembled with lymphocyte antigen 96 (also known as MD2) and
activates TLR-4-dependent intracellular signaling, which is mediated by MyD88 or in Myd88-independent
(TRIF-dependent) manner.

MyD88-dependent mechanism activates the expression of pro-inflammatory cytokines, while TRIF-