Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage "Switch" Phenotype. — TEI-tagged view

Igor MalyshevpersName, Yuri MalyshevpersName (2015date). “Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage "Switch" Phenotype..” BioMed Research Internationaltitle·j.

Object of study — macrophage plasticityterm·dom and reprogrammingterm·dom, and a proposed M3 switch phenotype

Research question — how switching between M1term·dom and M2 phenotypesterm·key under the alternative reprogrammingterm·dom factor occurs

Contribution — the hypothesis of an M3 switch phenotype through which M1term·dom and M2 phenotypesterm·key interchange

Full text — tagged

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Current Concept and Update of the Macrophage Plasticityterm·dom Concept: Intracellular Mechanisms of Reprogrammingterm·dom and M3 Macrophageterm·key "Switch" Phenotype.

Abstract Macrophagesterm·key play a key role in immunityterm·key. In this reviewterm·mth, we consider the traditional notion of macrophage plasticityterm·dom, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophageterm·key phenotype. Depending on the microenvironment, macrophagesterm·key can reprogram their phenotype toward the proinflammatory M1 phenotypeterm·key or toward the anti-inflammatory M2 phenotypeterm·key. Macrophageterm·key reprogrammingterm·dom involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophageterm·key reprogrammingterm·dom is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF- β -, TLR/NF- κ B-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1term·dom and M2 phenotypesterm·key, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogrammingterm·dom towards the anti-inflammatory M2 phenotypeterm·key or, contrarily, it responds to anti-inflammatory stimuli with reprogrammingterm·dom towards the proinflammatory M1 phenotypeterm·key. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophageterm·key reprogrammingterm·dom will assist in the selection of new therapeutic targets for correction of impaired immunityterm·key.

1. Introduction The immune system recognizes and eliminates pathogenic microbial products and tumor cells, thus preventing the progression of many diseases. Macrophagesterm·key play a crucial role in this system. Among all the immune cells, macrophagesterm·key have the greatest plasticity [ 1 – 3 ]. In various tissues, macrophagesterm·key acquire their long-term, respective phenotype [ 4 ] such as Kupffer cells in the liver, alveolar macrophagesterm·key in the lungs, microglia in the brain, osteoclasts in bone tissue, Langerhans cells in the skin, adipose tissue macrophagesterm·key in adipose tissue, and peritoneal macrophagesterm·key in the peritoneal cavity.

Macrophagesterm·key are the sensor cells of the immune system. They identify pathogenic cells with pattern recognition receptors (PRR), such as Toll-like receptors (TLR) [ 5 ], and water-soluble pattern recognition molecules (PRM), such as ficolins and collectins [ 6 ]. PRR identify different microbe-specific molecules, for example, bacterial carbohydrates such as lipopolysaccharide (LPS) and zymosan from the cell wall of yeast. In response to the binding, PRR activate intracellular signalling pathwaysterm·key of immune reactions. PRM represent evolutionarily ancient antibodies. They play an important role in the neutralization of such microbes as Aspergillus fumigates and Pseudomonas aeruginosa .

Macrophagesterm·key, together with dendrite cells, function as antigen-presenting cells (APC), whereas, together with natural killer cells (NK) and T- and B-lymphocytes, they eliminate microbes and tumor cells. In addition, macrophagesterm·key participate in tissue repair and remodelling, angiogenesis, and restoration of pathogen-disturbed homeostasis [ 4 , 7 ]. The efficacy of immune defence depends on the extent of macrophage plasticityterm·dom, that is, on how quickly and adequately the cells can change their functional phenotype in response to the microenvironment and pathogenic microbes.

Disturbances in macrophage plasticityterm·dom might compromise immune responses and the development of a number of diseases, such as cancer, bronchial asthmaterm·key, and atherosclerosis. Therefore, a more complete understanding of the mechanisms responsible for disorders of macrophage plasticityterm·dom that affect macrophageterm·key functional phenotypes is critical for advances in immunology, pathophysiology, and medicine.

In this reviewterm·mth, key phenomena and mechanisms of macrophage plasticityterm·dom will be addressed. In addition, we will consider published experimental data that do not conform to the current concept of macrophage plasticityterm·dom. These data lead us to hypothesize a new macrophageterm·key phenotype, which we name the switch phenotype.

2. Current Concept of Macrophage Plasticityterm·dom 2.1. Macrophageterm·key M1term·dom And M2 Phenotypesterm·key: The Concept of a Continuumterm·dom and Criticism of Some Aspects In response to viral or bacterial infection, macrophagesterm·key produce proinflammatory cytokinesterm such as IL-12 and TNF- α and chemokines [ 8 – 10 ], which attract neutrophils, NK, and Th- and T-lymphocytes [ 11 ]. By affecting NK and macrophagesterm·key, IL-12 and TNF- α stimulate IFN- γ secretion, which, in turn, results in a further increase in IL-12 and TNF- α production by macrophagesterm·key, thus enhancing their bactericidal, antiviral, and antitumoral activities. When parasites such as fungi or helminthes are recognized, macrophagesterm·key produce anti-inflammatory cytokinesterm·key, including IL-4, IL-10term, IL-13, and TGF- β [ 8 , 10 ]. These chemokines attract Th-lymphocytes, eosinophils, and basophils that produce IL-4 and IL-13 [ 12 ], which, in turn, further stimulate IL-10term secretion by macrophagesterm·key [ 12 ]. IL-10term suppresses the production of proinflammatory cytokinesterm [ 13 ], reactive oxygen species (ROS), and nitric oxide (NO). This reduces the bactericidal properties of macrophagesterm·key and enhances their antiparasitic properties.

The proinflammatory phenotype formed under the influence of LPS and/or IFN- γ was termed M1term·dom, and the anti-inflammatory phenotype formed under the influence of IL-4, IL-13, and IL-10term was termed M2term·dom [ 14 , 15 ]. The M1 phenotypeterm·key is characterized by TLR-4, the MARCO receptor, CD25, and CD80. The markers of the M2 phenotypeterm·key include the mannose receptor, SR-A, CD163, CD209, and FIZZ1 [ 15 ].

The current concept of macrophage plasticityterm·dom postulates that proinflammatory factors, such as LPS and IFN- γ , program the M1 phenotypeterm·key to enhance the production of proinflammatory cytokinesterm. These cytokinesterm·key shift the macrophageterm·key phenotype even further towards M1term·dom. As a result, a proinflammatory positive feedback mechanism is formed, enabling fast programming of the antimicrobial and antitumoral M1 phenotypeterm·key. Similarly, anti-inflammatory cytokinesterm·key, such as IL-10term, IL-13, or TGF- β , program the M2 phenotypeterm·key, which then intensively produces more anti-inflammatory cytokinesterm·key. These cytokinesterm·key shift the macrophageterm·key phenotype even further towards the M2term·dom type, thus again forming an anti-inflammatory positive feedback mechanism, enabling the fast programming of the antiparasitic M2 phenotypeterm·key of macrophagesterm·key [ 8 , 10 ].

When the microenvironment or active pathogen changes, tissue macrophagesterm·key can change their functional phenotype [ 8 , 9 , 16 ]. The process of changing the macrophageterm·key phenotype is called reprogrammingterm·dom . This process is also frequently referred to as polarizationterm·dom or alternate phenotype . Polarizationterm·dom and alternate phenotype assume a choice from two states. It was clear from the very beginning that these terms do not reflect the real nature of reprogrammingterm·dom. Therefore, additional phenotypes, М2a, M2b, and M2c, were recognized, and the concept of a continuumterm·dom soon appeared [ 1 , 17 , 18 ]. This concept implies that macrophageterm·key activation varies along a continuous proinflammatory spectrum: early stage M1term·dom macrophagesterm·key and later stage anti-inflammatory M2term·dom macrophagesterm·key. It was postulated that each phenotype is formed in response to action of certain inductors: M1term·dom in response to LPS and/or IFN- γ ; М2а in response to IL4/IL13; M2b in response to immune complexes; M2c in response to IL-10term or TGF- β [ 18 – 21 ]. It is important to note that, under in vivo conditions, there are usually several different reprogrammingterm·dom factors that affect macrophagesterm·key simultaneously. For example, those can be LPS, IL-4, TGF- β , and immune complexes. Which phenotype is formed in such a case, and how should it be referred to? Should it be referred to as М1/2аbс? Obviously, identifying the macrophageterm·key phenotype according to its reprogrammingterm·dom factor is not practical because (1) the same inductor, for instance, LPS or hypoxia, is able to program both the M1term·dom and M2 phenotypeterm·key depending on its concentration or extent [ 22 – 24 ]; (2) there are many reprogrammingterm·dom factors, which can act in different compositions; (3) as more new reprogrammingterm·dom factors are discovered, no more letters in the English alphabet may be available for identifying the new phenotype.

There are at least three categories of reprogrammingterm·dom factors. Among them are the following: (1) immune factors, such as immune complexes and cytokinesterm·key [ 25 ]; (2) pathogen-associated molecular patterns (PAMP), such as LPS, microbial nucleic acids, proteins, or carbohydrates that can be recognized by PRR of macrophagesterm·key; (3) physical and chemical factors, such as hypoxia [ 26 ], fever, and pH [ 27 ]. It has recently been proposed that factors which shift macrophageterm·key phenotype towards M1term·dom be referred to as reprogrammingterm·dom factor- (RF-) M1term·dom and those which shift the phenotype towards M2term·dom as RF-M2 [ 28 ]. For instance, RF-M1 is used for IFN- γ and low concentrations of serum [ 29 ], and RF-M2 is used for IL-4 and high concentrations of serum [ 14 ].

To date, no pure macrophageterm·key phenotype having only M1term·dom or M2term·dom markers has been described. Therefore, it would be correct to consider the M1 phenotypeterm·key as having more M1term·dom than M2term·dom markers and vice versa. Thus, reprogrammingterm·dom is more appropriate for describing the formation of any cell phenotype, whereas the terms polarizationterm·dom and alternate phenotype should be used to describe only the pure phenotypes, which, strictly speaking, do not exist. Therefore, we will use the term reprogrammingterm·dom rather than polarizationterm·dom or alternate phenotype .

The difference between macrophageterm·key phenotypes is most obvious during macrophageterm·key activation in response to the same stimulus, as demonstrated for the first time by Morrison [ 22 , 23 ]. In particular, it was shown that the preliminarily reprogrammed (preprogrammed) M1term·dom and M2term·dom macrophagesterm·key produced different amounts of cytokinesterm·key and NO in response to the same dose of LPS.

Reprogrammed macrophagesterm·key and APC activate the cellular or the humoral type of the immune response. When the cellular type of the immune response occurs, APC and M1term·dom macrophagesterm·key stimulate Th0 cells to differentiate into Th1 cells and T cells into CTL (cytotoxic T-lymphocytes). M1term·dom macrophagesterm·key, Th1, and CTL kill bacteria, viruses, and tumor cells [ 30 , 31 ]. When the humoral type of the immune response occurs, APC and M2term·dom macrophagesterm·key stimulate Th0 cells to differentiate into Th2 cells [ 32 ]. M2term·dom macrophagesterm·key, together with Th2 cells, remove parasites and toxins by releasing IL-4, which promotes the activation of B-cells and the production of antigen-specific antibodies [ 8 , 33 ]. In addition, M2term·dom macrophagesterm·key participate in tissue repair, angiogenesis, and phagocytosis of apoptotic cells [ 34 – 36 ].

Thus, the reprogrammingterm·dom of macrophagesterm·key causes changes not only in the functioning of the macrophagesterm·key themselves but also in the functioning of other immune cells, thus providing the required plasticity of the immune response required for eliminating pathogenic factors.

2.2. The Macrophageterm·key Reprogrammingterm·dom Phenomena The macrophageterm·key reprogrammingterm·dom process is characterized by four phenomena.

(1) The Phenomenon of an Amplified Macrophageterm·key Response to Both the Reprogrammingterm·dom Factor (Direct Amplification) and Another Factor (Cross Amplification). For instance, the reprogrammingterm·dom due to IFN- γ enhances the following macrophageterm·key response to IFN- γ itself (direct amplification) and to LPS (crisscross amplification).

(2) The Phenomenon of Reciprocal Suppression of the Alternate Phenotype. M1 phenotypeterm·key reprogrammingterm·dom enhances production of proinflammatory cytokinesterm while suppressing production of anti-inflammatory cytokinesterm·key and formation of the M2 phenotypeterm·key. M2 phenotypeterm·key reprogrammingterm·dom enhances production of anti-inflammatory cytokinesterm·key while suppressing production of proinflammatory cytokinesterm and formation of the M1 phenotypeterm·key.

(3) The Cascade Activation of the Reprogrammingterm·dom Mechanisms Phenomenon. This phenomenon provides the rapid formation of the desired macrophageterm·key phenotype.

(4) The Feedback Phenomenon. Positive feedback provides rapid formation of the desired macrophageterm·key phenotype. For instance, the M1 phenotypeterm·key is produced if there is a need to kill a virus, bacteria, or a tumor cell. Negative feedback prevents excessive M1 phenotypeterm·key formation, which might result in excessive inflammation followed by the development of inflammatory diseases.

The occurrence of these phenomena is triggered by various intracellular signalling pathwaysterm·key.

2.3. The Reprogrammingterm·dom Signaling Pathways The JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF- β /SMAD-/non-SMAD-, TLR/NF- κ B-, and hypoxia-dependent intracellular signalling pathwaysterm·key [ 37 ] are involved in macrophageterm·key reprogrammingterm·dom.

2.3.1. The JNK-Signalling Pathway in Macrophageterm·key Reprogrammingterm·dom The JNK- (C-Jun N-terminal kinase-) signalling pathwayterm·key can be activated via growth factor receptors, cytokinesterm·key receptors, and G-protein-associated receptors. The role of the JNK pathway in macrophageterm·key reprogrammingterm·dom has been demonstrated on adipose tissue macrophagesterm·key (ATMs). ATMs from control mice have the M2 phenotypeterm·key, whereas ATMs from obese mice have the M1 phenotypeterm·key [ 38 ]. The ATM M1 phenotypeterm·key contributes to the development of insulin resistance [ 38 ].

In obesity, saturated fatty acids trigger JNK via mixed-lineage kinase 3 (MLK3) activation. In turn, JNK activates proinflammatory gene expression and thereby reprograms macrophagesterm·key to the M1 phenotypeterm·key [ 37 ] ( Figure 1 ). However, JNK can also activate the M2 phenotypeterm·key transcription factor SMAD3 [ 39 ]. This suggests that under certain circumstances JNK can be involved in M2 phenotypeterm·key formation.

Maintaining the ATM M2 phenotypeterm·key in a lean organism is associated with the fact that normal adipocytes produce RF-М2, such as IL-13 and IL-4. These cytokinesterm·key activate the macrophageterm·key signal transducers and activators of transcription factor-6 (STAT6) which activates the M2 phenotypeterm·key and macrophageterm·key peroxisome proliferator-activated receptor gene expression (PPAR δ / β ). PPAR δ / β blocks the JNK-dependent reprogrammingterm·dom of the ATM M1 phenotypeterm·key [ 40 ] ( Figure 1 ).

Thus, the JNK-dependent signalling pathwayterm·key controls the macrophageterm·key response to growth factors, cytokinesterm·key, fatty acids, and ligands of the G-protein-associated receptors; is involved in the reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key; can activate the M2 phenotypeterm·key transcription factor SMAD3, thus restricting M1 phenotypeterm·key formation.

2.3.2. The PI3K/Akt-Signalling Pathway in Macrophageterm·key Reprogrammingterm·dom Phosphatidylinositol-3-kinase (PI3K) is activated via cytokineterm·key receptors and TLR. PI3K produces phosphatidylinositol-3,4,5-triphosphate (PIP3), which activates protein kinase Akt. Akt has three isoforms: Akt1, Akt2, and Akt3; Akt1 promotes M2 phenotypeterm·key formation, and Akt2 promotes M1 phenotypeterm·key formation [ 40 , 41 ] ( Figure 2 ). MicroRNA-155 (miR-155) and CAAT/enhancer-binding proteins β (C/EBP β ) play a key role in Akt-dependent macrophageterm·key reprogrammingterm·dom [ 41 , 42 ]. Akt2 enhances the expression of miR-155, which activates RelA/NF- κ B transcription factor and inhibits suppressor of cytokineterm·key signalling 1 (SOCS1). As a result, Akt2 increases expression of the M1 phenotypeterm·key genes iNOS and TNF-α . On the contrary, Akt1 inhibits miR-155, resulting in an increase in C/EBP β and the M2 phenotypeterm·key gene (Arg1 and IL-4) expression [ 43 ]. Switching the translation signal between Akt1 and Akt2 seems to be one of the mechanisms responsible for high macrophage plasticityterm·dom. Arranz et al. [ 41 ] and Androulidaki et al. [ 42 ] have shown that LPS activates Akt1, thereby enhancing production of anti-inflammatory mediators.

Thus, the PI3K/Akt-dependent signalling pathwayterm·key mediates the macrophageterm·key response to cytokinesterm·key and ligands of TLR; promotes the reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key via Akt2 and to the M2 phenotypeterm·key via Akt1; can induce formation of such a macrophageterm·key phenotype that will produce anti-inflammatory mediators in response to LPS.

2.3.3. The Notch-Signalling Pathway in Macrophageterm·key Reprogrammingterm·dom In mammals, there are four transmembrane Notch receptors (Notch-1, Notch-2, Notch-3, and Notch-4) and five transmembrane ligands referred to as Delta-like 1 (Dll1), Dll3, Dll4, Jagged-1, and Jagged-2 [ 44 ]. The ligand proteins induce ADAM-proteinase- and γ -secretase-mediated splitting of the Notch protein and release of the NICD domain. NICD enters the cell nucleus, binds to RBP-J, and modifies gene expression ( Figure 3 ) [ 45 ].

It has been shown that LPS increases the Dll4 content on the surface of macrophagesterm·key via the TLR4/NF- κ B-dependent pathway [ 46 ]. Incubation of intact macrophagesterm·key with macrophagesterm·key expressing Dll4 induced Notch proteolysis in the intact macrophagesterm·key ( Figure 3 ). This led to increased activity of genes such as IL-12 and i NOS, activation of Akt- and NF- κ B-signalling pathways, and the reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key. Activation of the Notch-signalling pathway provides rapid reprogrammingterm·dom to the M1term·dom macrophageterm·key phenotype and is an example of cascade activation of the macrophageterm·key reprogrammingterm·dom pathways, that is, from the TLR4/NF- κ B-dependent pathway to the Notch-signalling pathway.

Xu et al. [ 45 ] have shown that Notch1 activation enhances RBP-J formation. RBP-J increases the expression of interferon regulatory factor 8 (IRF8) transcription factor in macrophagesterm·key. In turn, IRF8 contributes to the activation of inflammatory genes, such as IL-12 [ 47 ]. IRF8 is involved not only in the IFN- γ and Notch pathways, but also in the TLR-4-signaling pathway of activation of proinflammatory M1term·dom cytokinesterm·key [ 48 ]. Therefore, the binding of ligands to TLR4 on reprogrammed macrophagesterm·key with an increased content of IRF8 will lead to a more prominent inflammatory response compared to that of macrophagesterm·key which were not reprogrammed. This could explain the mechanism of an enhanced proinflammatory M1term·dom response of macrophagesterm·key after the reprogrammingterm·dom, based on the convergence of the Notch1-RBP-J-, IFN- γ -, and TLR4-signaling pathways at the level of IRF8 ( Figure 3 ).

Wang et al. [ 49 ] have shown that expression of SOCS3, which is an inhibitor of the M2term·dom transcription factor of STAT3 genes, increases in Notch/RBP-J-dependent reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key ( Figure 3 ). These findings could explain the mechanism linking an increase in proinflammatory cytokinesterm production with a decrease in anti-inflammatory cytokinesterm·key production.

Other investigators, using the macrophagesterm·key from mice with systemic lupus erythematosus, have shown that the Notch1-signaling pathway can be involved also in the formation of the pathogenic M2 phenotypeterm·key [ 50 ]. In this case, the Notch1 pathway translates the signal via the PI3K and MAPK pathways and accelerates the translocation of NF- κ B p50 to the nucleus. As the dimer p50/p50 activates genes of the M2 phenotypeterm·key [ 51 ], the Notch1/NF- κ B p50 pathway reprograms macrophagesterm·key to the M2 phenotypeterm·key.

Thus, the Notch-dependent signalling pathwayterm·key mediates the reaction of macrophagesterm·key to Delta-like and JAG ligands and, indirectly, to TLR4 ligands LPS; reprograms the M1term·dom macrophageterm·key phenotype via NICD, RBP-J, and IRF8 activation; in systemic lupus erythematosus seems to be able to participate in the M2 phenotypeterm·key formation via the activation of PI3K/MAPК/NF- κ B p50 pathways; can participate in key macrophageterm·key reprogrammingterm·dom phenomena: (a) by enhancing the response of the M1term·dom macrophagesterm·key to proinflammatory stimuli due to the convergence of Notch1-RBP-J, IFN- γ , and TLR4 pathways at the level of IRF8, (b) by linking an increase in production of proinflammatory cytokinesterm with a decrease in production of anti-inflammatory cytokinesterm·key since Notch/RBP-J-dependent reprogrammingterm·dom to the M1 phenotypeterm·key increases expression of inhibitor of M2term·dom transcription factor of STAT3 genes (SOCS3), and (c) by cascade activation of the reprogrammingterm·dom pathways, due to the fact that the TLR4/NF- κ B pathway increases the amount of Dll4, ligands of the Notch pathway.

2.3.4. The JAK/STAT-Signalling Pathway in Macrophageterm·key Reprogrammingterm·dom In immune cells, the JAK/STAT-signalling pathway transmits signals from IFN- γ , IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21 via cytokinesterm·key receptors [ 52 ]. The JAK/STAT pathway employs four JAK (just another kinase or Janus kinase), namely, JAK1, JAK2, JAK3, and Tyk2, plus 7 transcription factors, STAT, namely, STAT1–4, STAT5A, STAT5B, and STAT6 [ 53 ]. The receptors of the JAK/STAT pathway are activated by RF-M1, for example, IFN- γ , as well as by RF-M2, for example, IL-4 ( Figure 4 ) [ 53 ].

IFN- γ via the JAK/STAT pathway induces STAT1 activation, which results in an increase in the production of proinflammatory cytokinesterm and, therefore, programs macrophagesterm·key to the M1 phenotypeterm·key [ 54 ] ( Figure 4 ). The IFN- γ /JAK/STAT1 pathway is under control of IRF5 and IRF4 [ 55 ]. IRF5 is activated by proinflammatory factors [ 56 ], whereas IRF4 is activated by anti-inflammatory factors [ 57 ]. IRF5 enhances the IFN- γ /JAK/STAT1-dependent production of the M1term·dom cytokineterm·key IL-12 [ 58 ], whereas IRF4 suppresses the effect of IRF5 [ 57 ]. Thus, IRF4 and IRF5 exert opposite effect on the IFN- γ /JAK/STAT1 pathway and on the macrophageterm·key phenotype.

The IFN- γ /JAK/STAT1 pathway is involved in the increase of the macrophageterm·key response to TLR ligands after preconditioning with IFN- γ . The basis of this phenomenon is the convergence of the IFN- γ /JAK- and TLR4-signaling pathways at the level of STAT1 [ 59 ]. Therefore, the increased activity of STAT1 in the IFN- γ -reprogrammed macrophagesterm·key produces a more intensive response to TLR4 ligands.

IL-4, IL-13, and IL-10term reprogram macrophagesterm·key to the M2 phenotypeterm·key ( Figure 4 ) via the JAK/STAT-signalling pathways. The binding of IL-4 with its receptor activates JAK. Subsequently, however, in contrast to IFN- γ , the phosphorylation and activation of the transcription factors of the M2 phenotypeterm·key gene, STAT3 and STAT6, occur [ 60 ]. In addition, IL-4 induces expression of c-Myc, which increases the expression of the M2 phenotypeterm·key genes, such as Scarb1 and Mrc1, and the activity of STAT6 and PPAR- γ [ 61 ].

Binding of IL-13 with its receptor activates JAK1, JAK2, and Tyk2 kinases with subsequent activation of STAT1, STAT3, and STAT6 [ 60 ]. After that, STAT3 and STAT6 activate expression of M2 phenotypeterm·key genes, such as mannose receptor, Fizz1, Ym1, and anti-inflammatory cytokinesterm·key [ 19 ], whereas STAT1 activates proinflammatory cytokinesterm ( Figure 4 ). The macrophageterm·key anti-inflammatory response to IL-13 seems to be predetermined by the prevalence of the activation of STAT3 and STAT6 over STAT1. However, it could be suggested that the blocking of STAT3 and STAT6 might lead to the formation of such a macrophageterm·key phenotype which could produce proinflammatory cytokinesterm in response to the anti-inflammatory cytokineterm·key IL-13.

Binding of IL-10term with its receptor via activation of JAK1 and STAT3 induces expression of the M2 phenotypeterm·key genes, such as TGF- β and IL-10term [ 19 ], as well as the genes which inhibit production of M1term·dom cytokinesterm·key, such as TNF- α [ 62 ]. In addition, IL-10term stimulates the relocation of p50/p50 into the nucleus, where it blocks the proinflammatory gene expression [ 63 ].

There are two important regulators of cross M1term·dom/M2term·dom macrophageterm·key reprogrammingterm·dom inherent to the JAK/STAT-signalling pathway. These are the SOCS1 and SOCS3 proteins [ 64 ]. IL-4 activates the synthesis of SOCS1, which blocks STAT1, thereby preventing the M1 phenotypeterm·key formation. IFN- γ and the TLR4 ligands activate the synthesis of SOCS3, which blocks STAT3, thereby preventing formation of the M2 phenotypeterm·key [ 65 ] ( Figure 4 ). Concurrently, SOCS1 activates STAT6, the M2 phenotypeterm·key transcription factor, while SOCS3 activates STAT1, the M1 phenotypeterm·key transcription factor. Such interactions between SOCS and STAT provide a further explanation of the link between the rise in the production of proinflammatory cytokinesterm and the decrease in the production of anti-inflammatory cytokinesterm·key during the reprogrammingterm·dom to the M1 phenotypeterm·key, and vice versa.

Thus, the JAK/STAT-dependent signalling pathwayterm·key mediates the response of the macrophagesterm·key to IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21, and IFN- γ ; translates signals into the nucleus from the M1term·dom cytokineterm·key IFN- γ and, via STAT1, reprograms macrophagesterm·key to the M1 phenotypeterm·key; this pathway also translates signals from the M2term·dom cytokinesterm·key, for example, IL-4, IL-10term, and IL-13, and via STAT3 and STAT6 reprograms macrophagesterm·key to the M2 phenotypeterm·key; this allows the macrophageterm·key to integrate the reprogrammingterm·dom effect of the microenvironment containing different cytokinesterm·key; triggered from the IL-13 receptors transmits signals to the M1-reprogramming STAT1 and the M2-reprogramming STAT3 and STAT6; due to this, the JAK/STAT signalling pathwayterm·key seems to be able under the M2-reprogramming pathways suppression to form a phenotype, which in response to RF-M2 IL-13 will increase the M1term·dom cytokinesterm·key production and form the M1 phenotypeterm·key; participates in the phenomenon of the augmentation of the macrophageterm·key proinflammatory response to different inflammatory stimuli after the macrophageterm·key reprogrammingterm·dom due to the IFN- γ /JAK and TLR4 pathway convergence at the level of STAT1; participates in the phenomenon of the link between the rise in the proinflammatory cytokinesterm production and the decrease in the anti-inflammatory cytokinesterm·key production during the reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key, and vice versa, due to the SOCS and STAT interactions.

2.3.5. The TGF- β -Signalling Pathway in Macrophageterm·key Reprogrammingterm·dom The family of secretory TGF- β proteins includes TGF- β 1, TGF- β 2, TGF- β 3, activins, and growth factors. The immune cells mostly produce TGF- β 1. TGF- β receptor consists of two transmembrane subunits of type I (T β RI) and two subunits of type II (T β RII) with the cytoplasmic kinase domains. After TGF- β binding to its receptor, the T β RII subunit autophosphorylates and phosphorylates T β RI. As a result, the T β RI kinase domain binds to the SMAD2 and SMAD3 transcription factors ( Figure 5 ). The SMAD Anchor for Receptor Activation (SARA) protein helps to attract SMAD2/3 to T β RI [ 66 ]. After that, T β RI phosphorylates and activates SMAD2/3. The activated SMAD2/3 binds to SMAD4, and the SMAD2/3/4 complex is translocated into the nucleus.

The TGF- β -activated, SMAD-dependent pathways upregulate the activity of the M2 phenotypeterm·key genes, such as arg1 and mgl2 [ 67 ], and reprogram the macrophageterm·key to the M2 phenotypeterm·key. The TGF- β -dependent reprogrammingterm·dom to the M2 phenotypeterm·key is under the SMAD7 control. SMAD7 can bind to T β RI, thereby preventing SMAD2/3 phosphorylation, or it can direct SMAD2/3 to the proteasomes for degradation. IFN- γ and TNF- α can upregulate the SMAD7 expression leading to the inhibition of the TGF- β /SMAD pathway and the decrease in the anti-inflammatory cytokinesterm·key production in response to TGF- β . This mechanism leads to understanding of the link between the increase in proinflammatory cytokinesterm production and the decrease in anti-inflammatory cytokinesterm·key production.

In addition to the SMAD-dependent pathway, TGF- β can activate SMAD-independent pathways, including Ras/MAPK/Erk, PI3K/Akt, p38, and JNK as well as Rho-like GTPases [ 39 ] pathways. In SMAD-independent pathways, the TGF- β activated kinase 1 protein (TAK1) transduces the signal from TGF- β to several downstream signalling cascades, including MAP kinase pathways, JNK, p38, and NF- κ B [ 68 ] ( Figure 5 ). It can be suggested that SMAD-independent pathways, which activate proinflammatory proteins and transcription factors such as JNK, p38, and NF- κ B, are able to reprogram macrophagesterm·key to the M1 phenotypeterm·key, particularly when the SMAD-dependent pathway is blocked.

Thus, we observe the following in the TGF- β -dependent signalling pathwayterm·key: It mediates the reaction of macrophagesterm·key to TGF- β ligands. It translates the signal from TGF- β via the SMAD2/3/4-dependent pathway, thereby reprogrammingterm·dom macrophagesterm·key to the M2 phenotypeterm·key. TGF- β seems to be able to form the macrophageterm·key phenotype, which in case of the suppression of SMAD-dependent pathways will increase production of the M1term·dom cytokinesterm·key and form the M1 phenotypeterm·key in response to RF-М2 TGF- β via the SMAD-independent TAK1/JNK/p38/NF- κ B-dependent pathways. It contributes to the link between an increase in proinflammatory cytokinesterm production and a decrease in anti-inflammatory cytokinesterm·key production in the M1 phenotypeterm·key. This results from the fact that the IFN- γ - or TNF- α -mediated macrophageterm·key reprogrammingterm·dom to the M1 phenotypeterm·key increases the expression of SMAD7, which blocks SMAD2/3/4 formation and anti-inflammatory M2term·dom cytokinesterm·key production.

2.3.6. The TLR/NF- κ B-Signalling Pathway in Macrophageterm·key Reprogrammingterm·dom Toll-like receptors (TLR) belong to the family of transmembrane PRR, which recognize specific PAMP on the molecules of microbes. The binding of PAMP to TLR triggers the signalling cascades, which induce the expression of proinflammatory cytokinesterm. Six TLR (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) have been identified on the surface of macrophagesterm·key. Each TLR consists of ligand-sensitive, transmembrane, and TIR domains [ 69 ]. When a ligand binds to a TLR, it forms a dimer. As a result, the TIR domains become closer to each other and start to interact with myeloid differentiation primary-response gene 88 (MyD88) [ 70 ]. MyD88 binds to the members of the IL-1R associated kinase (IRAK) family leading to the formation of the oligomeric Myddosome complex [ 71 ] ( Figure 6 ). Phosphorylation of IRAK occurs in the Myddosome complex. Phosphorylated IRAK attract tumor necrosis factor receptor-associated factor-6 (TRAF6) to the membrane [ 72 ]. In turn, TRAF6 attracts the TAK1 complex.

TAK transduces the signal from TLR and the TGF- β , TNF- α , and IL-1 receptors [ 68 ]. There is the convergence of the signals from pro- (TNF- α ) and anti-inflammatory (TGF- β ) cytokinesterm·key at the level of TAK. Due to this, macrophagesterm·key are probably able to respond to proinflammatory factors (RF-M1), for instance, TNF- α , by producing anti-inflammatory cytokinesterm·key and, conversely, by producing proinflammatory cytokinesterm in response to anti-inflammatory factors (RF-M2) such as TGF- β . Obviously, in the first case, this mechanism prevents the development of excessive inflammation, whereas in the second case a critical decrease in bactericidal, antiviral, and antitumoral properties of macrophagesterm·key occurs during the Th2 response development. In both cases, it could be concluded that TAK can form a special macrophageterm·key phenotype.

The attraction of TAK complexes to TRAF6 is accompanied by the convergence of the TAK kinase domains. This causes the autophosphorylation and activation of TAK1-kinase, which, in turn, activates the complex containing I κ B-kinase (IKK) [ 73 ] ( Figure 6 ). IKK phosphorylates the inhibitory subunit I κ B, which is associated with the NF- κ B transcription factor in the cytoplasm of inactive macrophagesterm·key. Phosphorylation of I κ B leads to its degradation in proteasomes. As a result, free NF- κ B is transported into the nucleus, where it activates the genes involved in inflammation, immune responses, and cellular growth [ 74 ]. The proteins belonging to the NF- κ B RelA (p65), RelB, and c-Rel families can activate the expression of proinflammatory genes whereas those belonging to the p50, p52 Relish families cannot. Proinflammatory cytokinesterm produced by the NF- κ B-dependent pathway can repeatedly activate the NF- κ B-dependent pathway. This is how a positive feedback is formed, which provides rapid formation of the proinflammatory phenotype. In addition, NF- κ B activates the I κ B genes. This mechanism restricts the excessive transportation of NF- κ B into the nucleus and represents a negative feedback, which prevents the development of an excessive inflammation.

In most cases, the TLR/NF- κ B-signalling pathway reprograms macrophagesterm·key to the M1 phenotypeterm·key in response to microbial invasion. There are three interesting and important features of TLR/NF- κ B-dependent reprogrammingterm·dom.

Firstly, during LPS-dependent macrophageterm·key reprogrammingterm·dom to the M1 phenotypeterm·key, the NF- κ B activation occurs in the form of p65/p50, which enhances the production of proinflammatory cytokinesterm. Simultaneously, LPS enhances the expression of the nuclear I κ B ζ genes [ 75 ]. I κ B ζ blocks the p50/p50 form of NF- κ B, which stimulates the production of anti-inflammatory cytokinesterm·key [ 76 ]. This can serve as another mechanism of the link between an increase in the proinflammatory cytokinesterm production and a decrease in the anti-inflammatory cytokinesterm·key production in the M1 phenotypeterm·key.

Secondly, the TLR/NF- κ B pathway can switch the phenotype reprogrammingterm·dom due to a change in the NF- κ B subunit composition. If the activation of NF- κ B occurs in the form of p50/p65, then the production of proinflammatory cytokinesterm increases [ 77 ], and the M1 phenotypeterm·key is formed. If the activation of NF- κ B occurs in the form of p50/p50, then the M2 phenotypeterm·key is formed as it occurs in tumor-associated and LPS-tolerant macrophagesterm·key [ 76 ]. This is one of the mechanisms regulating the macrophage plasticityterm·dom, which allows for a rapid response to changes in infection and microenvironment. The possibility to switch between the M2term·dom genes transcription factor p50/p50 [ 76 ] and the M1term·dom genes transcription factor p50/p65 [ 77 ] in response to the action of the same ligand, LPS, suggests the possibility of forming a macrophageterm·key phenotype that will produce anti-inflammatory factors and, thereby, manifest itself as the M2 phenotypeterm·key in response to RF-M1 (LPS).

Thirdly, it has been shown that the binding of LPS to TLR4 via TRAF6 induces phosphorylation of the STAT1 transcription factor [ 78 ]. After its activation, STAT1 is translocated into the nucleus where it activates proinflammatory genes, such as TNF- α . These data show the possibility of switching between different pathways, namely, from the TLR- to the STAT1-dependent pathway. This mechanism reflects the phenomenon of cascade activation of macrophageterm·key reprogrammingterm·dom pathways and might well contribute to the phenomenon of the increased production of proinflammatory cytokinesterm after the reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key. An example would be the increased production of TNF- α in response to the ligands of the JAK/STAT pathway after the TLR-dependent reprogrammingterm·dom.

Thus, we observe the following in the TLR-dependent signalling pathwayterm·key: It mediates the reprogrammingterm·dom of macrophagesterm·key in response to ligands of TLR. It activates NF- κ B and STAT1 and, thereby, upregulates the production of proinflammatory genes and reprograms macrophagesterm·key to the M1 phenotypeterm·key. Due to the possibility of producing NF- κ B in two different forms such as p65/p50, which is the proinflammatory M1term·dom form, or p50/p50, which is the anti-inflammatory M2term·dom form, the TLR-dependent pathway seems to be able to program the macrophageterm·key phenotype, which will then increase the production of the M2term·dom cytokinesterm·key in response to RF-M1 such as LPS. It contributes to the phenomenon of reprogrammingterm·dom: (a) contributes to the link between an increase in the proinflammatory cytokinesterm production and a decrease in the anti-inflammatory cytokinesterm·key production in the M1 phenotypeterm·key (the reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key due to LPS upregulates the expression of the nuclear I κ B ζ , which blocks the p50/p50 anti-inflammatory form of NF- κ B); (b) contributes to the increase in the production of proinflammatory cytokinesterm in response to the ligands of the JAK/STAT pathway after TLR-dependent macrophageterm·key reprogrammingterm·dom to the M1 phenotypeterm·key (the phenomenon is based on the TLR-dependent activation of STAT1, which is a transcription factor of the JAK/STAT pathway); (c) contributes to the cascade activation of the macrophageterm·key reprogrammingterm·dom pathways as a result of signal switching from the TLR-dependent pathway to the STAT1-dependent pathway via TRAF6; (d) contributes to positive and negative feedback due to proinflammatory cytokinesterm, which can repeatedly activate NF- κ B, and due to NF- κ B-dependent synthesis of the inhibitor I κ B, respectively.

2.3.7. The Hypoxia-Dependent Pathway of Macrophageterm·key Reprogrammingterm·dom The intracellular protein complex of the oxygen sensor [ 79 ] consisting of the prolyl-4-hydroxylase domain (PHD) enzyme and the hypoxia-inducible transcription factor (HIF) plays a key role in the hypoxic reprogrammingterm·dom of the macrophageterm·key phenotype. HIF consists of two subunits: a constitutive HIF- β subunit and an oxygen-sensitive subunit, HIF-1 α , HIF-2 α , or HIF-3 α . Under normoxic conditions, HIF- β is constantly present in the cell, whereas HIF- α is constantly synthesized but not accumulated because of its PHD-mediated binding to the hydroxyl group [ 80 ] with subsequent relocation to proteasomes and degradation therein.

Under hypoxia, the PHD activity decreases and, as a result, HIF- α accumulates. Then HIF- α binds to HIF- β and forms the HIF- α /HIF- β dimer. This dimer penetrates the nucleus and activates the gene [ 79 ], which increase the endurance of macrophagesterm·key to hypoxia. Either HIF-1 α or HIF-2 α is activated in macrophagesterm·key depending on the extent of the hypoxia. HIF-1 α induces i NOS synthesis and promotes the M1 phenotypeterm·key formation, whereas HIF-2 α activates arginase 1 and promotes the M2 phenotypeterm·key formation [ 24 ].

2.3.8. The Conclusions regarding the Signalling Mechanisms of Macrophageterm·key Reprogrammingterm·dom There is a relative specialization of the signalling pathwaysterm·key of macrophageterm·key reprogrammingterm·dom in response to different components of the microenvironment. In particular, growth factors, fatty acids, and the ligands of G-protein-associated receptors reprogram macrophagesterm·key via the JNK-dependent pathway; microbial invasion, namely, PAMP via the TLR-, PI3K/Akt-, and Notch-dependent pathways; changes in the Delta-like and JAG ligand content in the microenvironment via the Notch-dependent pathways; changes in the cytokineterm·key microenvironment via the JNK-, PI3K/Akt-, and JAK/STAT-dependent signalling pathwaysterm·key; changes in the TGF- β content via the SMAD-dependent and SMAD-independent signalling pathwaysterm·key; and changes in the oxygen content via the hypoxia/HIF-dependent pathways. There are two types of macrophageterm·key reprogrammingterm·dom signalling pathwaysterm·key: pathways which mainly program the M1 phenotypeterm·key such as JNK, Notch, TLR/NF- κ B (р65/р50), PI3K/Akt2, JAK/STAT1, and HIF1 α pathways and those pathways which mainly program the M2 phenotypeterm·key such as PI3K/Akt1, JAK/STAT3/6, TGF- β /SMAD, TLR/NF- κ B (р50/р50), and HIF2 α pathways. An understanding of the reprogrammingterm·dom mechanisms allows us to explain the four main phenomena observed during the course of macrophageterm·key reprogrammingterm·dom: (1) the phenomenon of an amplified macrophageterm·key response to both the reprogrammingterm·dom factor (direct amplification) and another factor (cross amplification), (2) the phenomenon of reciprocal suppression of the alternate phenotype, (3) the cascade activation of the reprogrammingterm·dom mechanisms phenomenon, and (4) the feedback phenomenon. The enhanced macrophageterm·key response to the factor with which the macrophagesterm·key have been reprogrammed (direct amplification) or their response to another factor (cross amplification) is based on the convergence of signalling pathwaysterm·key ( Figure 7(a) ) at the level of a certain protein. Therefore, macrophageterm·key reprogrammingterm·dom via a particular signalling pathwayterm·key that leads to an increased activity or content of this protein results in an increase in the subsequent response of the macrophagesterm·key to the ligands of another pathway. IRF8 is the convergence protein for the M1-reprogramming Notch1-RBP-J, IFN- γ , and TLR4 pathways. STAT1 is the convergence protein for the IFN- γ /JAK and TLR4 pathways. The phenomenon of reciprocal suppression of an alternative phenotype is caused by the fact that the formation of a phenotype is accompanied by an increase in synthesis of the molecules suppressing the formation of the alternative phenotype ( Figure 7(b) ). In particular, the M1 phenotypeterm·key formation via (1) the Notch/RBP-J, IFN- γ , or TLR4 pathway is accompanied by an increase in the expression of SOCS3, which suppresses the activity of the M2term·dom genes transcription factor STAT3; (2) the IFN- γ and TNF- α pathways by an increase in the expression of SMAD7 blocks the activation of the M2term·dom genes transcription factors SMAD2/3/4; (3) the TLR/NF- κ B (p65/p50) pathways increases the expression of inhibitors of the M2term·dom genes. The M2 phenotypeterm·key formation via the IL-4/JAK/STAT pathway is accompanied by an increase in SOCS1 synthesis, which blocks the M1term·dom genes transcription factor STAT1. The phenomenon of cascade activation of the reprogrammingterm·dom pathways is based on the ability of one reprogrammingterm·dom pathway to transmit the signal to another pathway. In particular, the LPS/TLR4/NF- κ B pathway of the M1term·dom reprogrammingterm·dom increases the amount of Dll4, which is a ligand of the Notch pathway of the M1term·dom reprogrammingterm·dom, whereas the TLR-dependent pathway via TRAF6 transmits the signal to the STAT1-dependent pathway ( Figure 7(c) ). The phenomenon of positive and negative feedback is based on the ability of a signalling pathwayterm·key to upregulate both its own activators and inhibitors. In particular, the NF- κ B-dependent pathway of the M1term·dom reprogrammingterm·dom upregulates the amount of both proinflammatory cytokinesterm, which activate this pathway, thereby forming positive feedback, and I κ B, which inhibits NF- κ B, thereby forming negative feedback ( Figure 7(d) ). These phenomena, on the one hand, provide rapid formation of M1term·dom macrophagesterm·key if it is necessary to eliminate bacteria or viruses or formation of M2term·dom macrophagesterm·key if there is a need to kill parasites, enhance angiogenesis, or repair tissue. At the same time, these phenomena prevent excessive activation of the phenotype. The signalling pathwaysterm·key that program the proinflammatory M1term·dom macrophageterm·key phenotype often have an offshoot, which, if activated, can upregulate the production of the anti-inflammatory M2term·dom cytokinesterm·key, and vice versa. In particular, the JNK-dependent pathways activate the M1 phenotypeterm·key transcription factors, but they can also activate the M2term·dom genes transcription factor SMAD3. The PI3K/Akt-dependent pathway programs the M1 phenotypeterm·key via Akt2 and the M2 phenotypeterm·key via Akt1. The Notch pathway increases the production of the M1term·dom cytokinesterm·key via NICD/RBP-J/IRF8 and the production of M2term·dom cytokinesterm·key via PI3K/MAPК/NF- κ B p50. IL-13 programs the M2 phenotypeterm·key via JAK2/STAT3 and Tyk2/STAT6 pathways and the M1 phenotypeterm·key via Tyk2/STAT1 pathway. TGF- β programs the M2 phenotypeterm·key via SMAD2/3/4 and the M1 phenotypeterm·key via TAK1/JNK/p38/NF- κ B. Lastly, the TLR-dependent pathway programs the M1 phenotypeterm·key via p65/p50 and the M2 phenotypeterm·key via p50/p50. These signalling intracellular pathways play a crucial but not the sole role in macrophageterm·key reprogrammingterm·dom. They can activate or inhibit the mechanisms of other levels of cell activity and cell phenotype regulation, namely, posttranscriptional ones.

2.4. The Posttranscriptional Mechanisms of Macrophageterm·key Reprogrammingterm·dom The key element of posttranscriptional regulation of many cellular processes, including macrophageterm·key reprogrammingterm·dom, is microRNA (miR) [ 81 ]. miRs are small, noncoding molecules of RNA comprised of 18–25 nucleotides. miRs bind with mRNA and inhibit translation of a protein [ 82 ]. One miR can control several mRNA. In addition, miR can interact with DNA during the course of methylation of DNA that suppresses activity of genes [ 83 ].

2.4.1. RF-M1s, Specific Sets of miRs, and Their Effects on the Phenotype of Macrophagesterm·key In macrophagesterm·key, factors of macrophageterm·key reprogrammingterm·dom activate the synthesis of specific sets of miRs that control balance of pro- and anti-inflammatory processes. In particular, RF-M1s induce the synthesis of miR-155, miR-21, miR-125, miR-9, and miR-146 ( Figure 8 ) via TLR, whereas RF-M2s induce the synthesis of miR-511 via the glucocorticoid receptor, the synthesis of miR-187 via the IL-10term receptor, and the synthesis of miR-378 via the IL-4 receptor [ 17 , 84 ] ( Figure 9 ).

Among all miRs, the synthesis of which increases under the influence of ligands of TLR, miR-155 has been the most investigated ( Figure 8 ). miR-155 increases stability of the TNF- α transcript [ 85 ], decreases synthesis of SOCS1, and, thereby, promotes the development of antiviral immunityterm·key [ 86 ]. It blocks IL-13 and IL-3 receptors [ 87 , 88 ], blocks SMAD2, and, thereby, decreases the M2-reprogramming effect of TGF- β [ 89 ]. miR-155 also blocks the translation of transcription factor BCL6 and, as a result, promotes the development of atherosclerosis [ 90 ]. In addition, miR-155 blocks transcription factor C/EBP- β and, thereby, decreases production of cytokineterm·key IL-10term [ 91 ], which is RF-M2. miR-155 can also shift the M2 phenotypeterm·key of TAM towards the M1 phenotypeterm·key and, thereby, enhance antitumoral properties of macrophagesterm·key [ 92 ].

miR-29b and miR-125, induced via TLR, increase proinflammatory NF- κ B signalling [ 93 , 94 ] and block the translation of signal from the IL-4 receptor at the level of IRF4 [ 95 ] ( Figure 8 ). These miRs also promote the formation of the M1 phenotypeterm·key. At the same time, miR-125a and miR-125b can decrease stability of the TNF- α transcript [ 96 ].

miR-146a [ 97 ], miR-9 [ 98 ], miR-21 [ 99 ], and miR-147 [ 100 ], induced in macrophagesterm·key via TLR, prevent M1 phenotypeterm·key reprogrammingterm·dom ( Figure 8 ). miR-146a blocks IRAK-1 and TRAF6 in the TLR-signalling pathway [ 97 ]. Due to this action miR-146a plays an important role in the formation of LPS-tolerant macrophagesterm·key [ 101 ]. miR-9 suppresses the proinflammatory activity of NF- κ B directly [ 98 ], whereas miR-21 indirectly suppresses this activity via PDCD4 [ 99 ].

Thus, the miRs induced via TLR form a regulatory network, in which two functional contours can be distinctly differentiated ( Figure 8 ): (1) the positive feedback mechanism, which is formed by miR-155 and miR-125, which provides rapid formation of the M1 phenotypeterm·key to eliminate an infection, and (2) the negative feedback mechanism, formed by miR-146a, miR-9, miR-21, and miR-147, which seems to prevent excessive inflammation in the immune response to an infection.

2.4.2. RF-M2, Specific Sets of miRs, and Their Effects on the Phenotype of Macrophagesterm·key Similar to RF-M1, RF-M2 induces another specific set of miRs in macrophagesterm·key ( Figure 9 ). IL-10term increases the synthesis of miR-187, which promotes the termination of inflammation by blocking the transcripts of TNF- α and IL-12 [ 102 ]. IL-10term also increases the synthesis of miR-146b, which suppresses the production of proinflammatory cytokinesterm by affecting the key points of the TLR pathway [ 103 ]. IL-4 increases the expression of miR-378 [ 102 ]. miR-378 is coexpressed together with the M2term·dom gene PPAR- γ but acts as an inhibitor of the M2term·dom responses, because it blocks the IL-4/PI3K/Akt-signaling pathway [ 102 ]. Glucocorticoids increase synthesis of miR-511 in M2term·dom macrophagesterm·key. miR-511-3p disturbs the translation of Rock2, which is the factor that promotes the reprogrammingterm·dom of macrophagesterm·key to the M2 phenotypeterm·key [ 104 ]. Thus, miR-511 and miR-378 are negative regulators of the macrophageterm·key M2term·dom response.

Thus, the miRs induced by RF-M2 form a regulatory network, in which, similar to RF-M1, two functional contours can be distinctly differentiated ( Figure 9 ): (1) the positive feedback mechanism, formed by miR-187 and miR-146b, which provides rapid formation of the M2 phenotypeterm·key to eliminate a parasitic infection or to prevent excessive inflammation induced by viruses and bacteria, and (2) the negative feedback mechanism, formed by miR-378 and miR-511, which limits a significant decrease in the bactericidal and antiviral activity of the immunityterm·key during the development of the Th2 response.

2.4.3. Conclusions on the Posttranscriptional Mechanisms of Macrophageterm·key Reprogrammingterm·dom In general, the intracellular network of miRs possesses all the features of a regulatory mechanism of macrophageterm·key reprogrammingterm·dom, including both positive and negative feedbacks, and a specificity of action on the receptors and proteins participating in macrophageterm·key reprogrammingterm·dom. A few generalities can be made concerning the miR-dependent regulation of the phenotype of macrophagesterm·key: There is a certain specificity in the expression of miRs; miR-155, miR-21, miR-29b, miR-125, miR-9, miR-146a, and miR-147 are expressed in response to RF-M1, whereas miR-146b, miR-511, miR-187, miR-378, miR-222, miR-27a, miR-125a-3p, and miR-125a-5p are expressed in response to RF-M2. miRs can form mechanisms of positive feedback to provide rapid reprogrammingterm·dom of macrophagesterm·key to a desired phenotype and of the negative feedback to limit excessive inflammation in case of the M1 phenotypeterm·key and a significant decrease in the bactericidal, antitumoral, and antiviral activity in case of the M2 phenotypeterm·key. Understanding of the functions of miRs allows us to explain the mechanisms of macrophageterm·key reprogrammingterm·dom.

The role of miRs in the enhanced response of the reprogrammed macrophagesterm·key is to form a positive feedback, which enhances the response of reprogrammed macrophagesterm·key. For instance, miR-155, the expression of which is initiated from TLR, blocks the translation of BCL6, which inhibits NF- κ B-dependent signalling [ 93 ]. With BCL6 blocked, the response of reprogrammed macrophagesterm·key to the ligands of TLR will be enhanced.

The contribution of miRs to the reciprocal suppression of an alternative macrophageterm·key phenotype is determined by the fact that the miR participating in the formation of one or another macrophageterm·key phenotype can suppress the signalling pathwaysterm·key producing an alternative phenotype. In particular, miR-155, which participates in the formation of the M1 phenotypeterm·key, blocks the signalling pathwaysterm·key leading to the M2 phenotypeterm·key, namely, the receptors of IL-13 [ 87 ] and IL-3 [ 79 ], SMAD2 [ 89 ], and C/EBP- β [ 105 ].

miRs are also involved in the phenomenon of the cascade activation of the reprogrammingterm·dom pathways. This is due to the fact that the synthesis of many of miRs is increased during the activation of the cytokine-dependent pathways of macrophageterm·key reprogrammingterm·dom. For instance, this occurs when IL-10term mediated synthesis of miR-146 increases.

3. Paradoxical Plasticityterm·dom of Macrophagesterm·key: The M3 Phenotypeterm·dom (Update) 3.1. The Current Concept of Macrophageterm·key Reprogrammingterm·dom Cannot Explain the Mechanism of the Transition between the M1term·dom and M2 Phenotypesterm·key A disturbance in the reprogrammingterm·dom of macrophagesterm·key during disease can be associated with an inadequate alteration of a specific phenotype. For instance, inadequate acquiring of the M2 phenotypeterm·key and excessive production of anti-inflammatory cytokinesterm·key can decrease bactericidal and antiviral functions of the immune response and provoke allergic and asthmatic Th2 reactions [ 106 , 107 ] and tumor growth [ 108 ]. On the other hand, if there is no change in phenotype when it is necessary, for instance, if phenotype does not change from the M1term·dom to the M2term·dom type at the end of an inflammatory reaction after the elimination of pathogenic microbes, excessive production of the proinflammatory M1term·dom mediators followed by tissue damage and development of proinflammatory diseases can occur [ 109 ].

Switching from the M1term·dom to the M2 phenotypeterm·key protects an organism from excessive inflammation, whereas switching from the M2term·dom to the M1 phenotypeterm·key protects from allergic and asthmatic Th2 reactions and a decrease in bactericidal properties of macrophagesterm·key [ 110 , 111 ]. The current concept says that the M1 phenotypeterm·key can be reprogrammed to the M2 phenotypeterm·key under the influence of RF-M2 and vice versa. However, it has been demonstrated on numerous occasions that the M1 phenotypeterm·key can be transformed to the M2 phenotypeterm·key under conditions of inflammation and the action of proinflammatory RF-M1, whereas the M2 phenotypeterm·key can be transformed to the M1 phenotypeterm·key under the influence of anti-inflammatory RF-M2.

Among the facts that contradict the current concept are the following: The reprogrammingterm·dom of macrophagesterm·key to the M2 phenotypeterm·key in tumor-induced inflammation [ 17 ]. Such paradoxical reprogrammingterm·dom of macrophagesterm·key promotes tumor survival and metastasis [ 112 ]. The reprogrammingterm·dom of macrophagesterm·key from the M1 phenotypeterm·key to M2term·dom at the end of inflammation or in chronic inflammation [ 9 ], when the proinflammatory RF-M1s continue to affect macrophagesterm·key. The reprogrammingterm·dom of macrophagesterm·key to the M2 phenotypeterm·key in vitro under the influence of classic proinflammatory (according to the current concept) RF-M1 LPS [ 23 ]. Switching of macrophagesterm·key from the M1 phenotypeterm·key to the M2 phenotypeterm·key in vivo under the influence of some LPS-containing microbes [ 9 , 113 ]. Such reprogrammingterm·dom of macrophagesterm·key helps bacteria to avoid the bactericidal action of the M1term·dom macrophagesterm·key. The reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key and an increased production of proinflammatory cytokinesterm under the influence of anti-inflammatory cytokinesterm·key IL-10term and IL-13, which are under certain conditions, in fact RF-M2 [ 60 , 114 ]. The reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key under the influence of a parasitic infection (RF-M2). Macrophagesterm·key react to parasites by the formation of the M2 phenotypeterm·key [ 8 ]. However, at the early stages of infection with Taenia crassiceps , Schistosoma mansoni , and Trypanosoma congolense , macrophagesterm·key have the M1 phenotypeterm·key and only at the later stages is the antiparasitic M2 phenotypeterm·key formed [ 33 , 115 ]. The existence of both M1term·dom and M2term·dom macrophagesterm·key in the same microenvironment. Allergy and an allergy-associated increase in the production of the anti-inflammatory RF-M2 cytokinesterm·key IL-4 and IL-13 program macrophagesterm·key to the M2 phenotypeterm·key [ 106 ]. However, in the area of the allergic inflammation, not only the M2term·dom but also the M1term·dom macrophagesterm·key can be found [ 116 ]. In obesity, despite the prevalence of M1term·dom macrophagesterm·key among ATMs, M2term·dom macrophagesterm·key can be also found [ 117 ]. There is a similar situation in alcoholic hepatitis; namely, there are both M1term·dom and M2term·dom macrophagesterm·key in the same microenvironment [ 118 ]. The current concept cannot explain how the macrophagesterm·key belonging to alternative phenotypes could be found in the same microenvironment.

The current concept can explain how the M1term·dom macrophagesterm·key phenotype could be formed under the influence of RF-M1 and the M2 phenotypeterm·key under the influence of RF-M2 [ 119 ]. However, the current concept can neither explain the facts listed above nor answer the question of how switching between different phenotypes under the influence of alternative RF occurs. In other words, how can the proinflammatory M1 phenotypeterm·key be formed under the influence of the anti-inflammatory RF-M2 and, contrarily, how can the anti-inflammatory M2 phenotypeterm·key be formed under the influence of the proinflammatory RF-M1?

3.2. The Interchange of the M1term·dom and the M2 Phenotypesterm·key Might Well Occur as a Result of the Formation of the M3 Switching Phenotypeterm·dom The aforementioned examples of paradoxical reprogrammingterm·dom of macrophagesterm·key suggest a very interesting hypothesis: In addition to the M1 phenotypeterm·key of macrophagesterm·key, which in response to RF-M1 induces an increase in the production of the proinflammatory cytokinesterm followed by a further shift towards the M1 phenotypeterm·key, and the M2 phenotypeterm·key, which in response to RF-M2 induces an increase in the production of the anti-inflammatory cytokinesterm·key with a further shift to the M2 phenotypeterm·key, there is the third phenotype of macrophagesterm·key , which in response to RF-M1 induces an increase in the production of the anti-inflammatory cytokinesterm·key and reprograms itself to the M2 phenotypeterm·key and, contrarily, in response to RF-M2 induces an increase in the production of the proinflammatory cytokinesterm and reprograms itself to the M1 phenotypeterm·key ( Figure 10 ). Such a phenotype could be referred to as the M3 phenotypeterm·dom or the switching phenotypeterm·dom. The M3 switching phenotypeterm·dom could be divided into two subtypes such as the M1term·dom/2 phenotype, which in response to RF-M1 induces the production of the M2term·dom mediators, and the M2term·dom/1 phenotype, which in response to RF-M2 induces the production of the M1term·dom mediators.

Experimental data indicate the existence of the M3 phenotypeterm·dom. Recently, we have obtained experimental data that support the hypothesis of the existence of the switching phenotypeterm·dom. We have found indicators of the formation of such a phenotype in lung diseases and during use of some medicines [ 120 , 121 ].

We have found the phenotype of switching in lung diseases. Experiments have been carried out on alveolar macrophagesterm·key isolated from patients suffering from chronic obstructive pulmonary diseaseterm·dom (COPDterm·dom) or bronchial asthmaterm·key (BA) and on healthy control subjects. The macrophagesterm·key were cultivated in RPMI-1640 medium with 10% fetal bovine serum (FBS). LPS was to activate the macrophagesterm·key. A 0% FBS medium was used as RF-M1, whereas a 40% FBS medium was used as RF-M2 [ 14 , 122 ]. The changes in phenotype were identified by the changes in the production of cytokinesterm·key by the macrophagesterm·key. In particular, an increased production of proinflammatory cytokinesterm compared to 10% FBS indicated a shift of phenotype towards the M1 phenotypeterm·key, whereas an increased production of anti-inflammatory cytokinesterm·key indicated a shift towards the M2 phenotypeterm·key.

We found that the macrophagesterm·key from healthy subjects increased the production of the proinflammatory M1term·dom cytokinesterm·key and decreased the production of the anti-inflammatory M2term·dom cytokinesterm·key in response to RF-M1, whereas in response to RF-M2 the macrophagesterm·key from healthy subjects increased the production of the anti-inflammatory M2term·dom cytokinesterm·key and decreased the production of the proinflammatory M1term·dom cytokinesterm·key. These changes in the production of specific cytokinesterm·key reflect the normal adequate plasticity of macrophagesterm·key from healthy subjects.

In contrast to the macrophagesterm·key from healthy subjects, the macrophagesterm·key from patients with COPDterm·dom demonstrated a decrease in the production of the proinflammatory M1term·dom cytokinesterm·key INF- γ , IL-1 β , and TNF- α in response to RF-M1, whereas in response to RF-M2 they demonstrated an increase in the production of the proinflammatory M1term·dom cytokinesterm·key TNF- α and IL-8. This situation could be termed as paradoxical/inverted reprogrammingterm·dom or paradoxical plasticityterm·dom. Thus, alveolar macrophagesterm·key from patients with COPDterm·dom possessed the characteristics of the switching phenotypeterm·dom. There was no qualitative change in the reprogrammingterm·dom of macrophagesterm·key in patients with BA in remission.

We have also found the phenotype of switching when prescribing inhaled glucocorticosteroids (IGCS). The effect of IGCS therapy on the macrophageterm·key phenotype and plasticity was evaluated in patients with BA. The results have shown that the macrophagesterm·key from the IGCS-treated patients with BA decreased production of most proinflammatory M1term·dom cytokinesterm·key, such as IL-12, IFN- γ , IL-8, IL-6, and IL-1 β , under normal conditions of cultivation with 10% FBS. However, macrophagesterm·key from the IGCS-treated patients with BA increased production of the anti-inflammatory cytokineterm·key IL-10term compared to the macrophagesterm·key from the healthy subjects and non-IGCS-treated patients with BA. These changes reflect the shift of the macrophageterm·key phenotype in patients with BA towards the anti-inflammatory M2 phenotypeterm·key under the influence of IGCS. The same conclusion was made by others earlier [ 123 ].

Prescribing IGCS to patients with BA qualitatively affected their macrophage plasticityterm·dom. In contrast to the healthy subjects and non-IGCS-treated patients with BA, the production of the proinflammatory cytokinesterm IL-12, IFN- γ , IL-8, IL-6, and TNF- α by the macrophagesterm·key from the IGCS-treated patients with BA decreased in response to RF-M1 and increased in response to RF-M2, whereas the production of the anti-inflammatory cytokinesterm·key IL-4, IL-5, and IL-10term decreased in response to RF-M2 and increased in response to RF-M1. These changes in cytokineterm·key production suggest the formation of inverted plasticity of the macrophageterm·key secretory activity in the IGCS-treated patients with BA. Thus, the switching phenotypeterm·dom forms in response to IGCS.

3.3. The Issues Raised by the M3 Switching Phenotypeterm·dom An analysis of the literature and the results of our experiments suggest the existence of a new immunologic phenomenon, which could be referred to as inverted reprogrammingterm·dom or, in other words, paradoxical plasticityterm·dom of macrophagesterm·key. The data described above support the hypothesis of the existence of a particular macrophageterm·key phenotype, which increases the production of the M2term·dom and decreases the production of the M1term·dom cytokinesterm·key in the proinflammatory microenvironment. On the contrary, this macrophageterm·key phenotype increases production of the M1term·dom and decreases the production of the M2term·dom cytokinesterm·key in anti-inflammatory microenvironment. This hypothesis significantly extends the current concept of macrophage plasticityterm·dom but immediately raises several important issues: What role can the switching phenotypeterm·dom play in disease development and drug effects? Our data have shown that the switching phenotypeterm·dom is formed in lung diseases, for instance, in COPDterm·dom [ 120 ]. Macrophagesterm·key with the switching phenotypeterm·dom found in COPDterm·dom did not increase but rather greatly decreased production of the proinflammatory cytokinesterm in response to RF-M1. It could be suggested that such a phenotype promotes the decrease in bactericidal and antiviral activity of the lung immune system and the development of an increased susceptibility to lung infection. Therefore, this M3 phenotypeterm·dom may possibly play a pathogenic role in COPDterm·dom. Furthermore, the M3 phenotypeterm·dom has been found in the alveolar macrophagesterm·key from IGCS-treated patients with BA [ 121 ]. As IGCS improves the condition of patients with BA, the formation of the M3 phenotypeterm·dom might be suggested to be a component of the therapeutic action of IGCS. Despite the fact that this suggestion is still to be verified, it is already clear that the M3 phenotypeterm·dom could be considered as an effective therapeutic target worthy of attention. Under what conditions can the M3 phenotypeterm·dom be formed? At present, there are no clear experimental or clinical data to answer this question. Nevertheless, based on the expediency of M3 phenotypeterm·dom formation, we suggest this phenotype would be formed when there is need for a negative feedback mechanism. When there is a danger of excessive inflammation as a result of the influence of RF-M1 on a macrophageterm·key, the M1term·dom/M2term·dom is formed, whereas when there is a risk of a significant decrease in bactericidal or antiviral properties of macrophagesterm·key as a result of the RF-M2 action, the M2term·dom/M1term·dom is formed. What intracellular mechanisms could be involved in the formation of the M3 phenotypeterm·dom? The findings presented above in The Reprogrammingterm·dom Signaling Pathways can help to answer this question, particularly the studies by Locati et al. [ 84 ] and by Zhou et al. [ 37 ]. Analysis of these studies has shown that there are seven intracellular signalling pathwaysterm·key involved in the reprogrammingterm·dom of macrophagesterm·key, specifically the JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF- β /SMAD/non-SMAD-, TLR/NF- κ B-, and hypoxia-induced signalling pathwaysterm·key. Almost every one of these pathways possesses an important constructive feature, namely, pathways, which transmit the signal from RF-M1 and, thereby, mediate the reprogrammingterm·dom of macrophagesterm·key to the M1 phenotypeterm·key; there is often an intracellular offshoot, which could result in an increase in the anti-inflammatory M2term·dom cytokinesterm·key. On the contrary, the M2term·dom signalling pathwaysterm·key often have an offshoot which could result in an increase in the proinflammatory M1term·dom cytokinesterm·key ( Figure 11 ). At the point of bifurcation, there is a protein switch that determines which way the reprogrammingterm·dom of macrophagesterm·key will go. It is clear that such a protein (enzyme or transcription factor) is of the greatest importance for manipulating the macrophageterm·key phenotype in order to correct the immune response.

The existence of the offshoots of the reprogrammingterm·dom signalling pathwaysterm·key allows for understanding of how the M3 phenotypeterm·dom increases the production of the anti-inflammatory M2term·dom cytokinesterm·key and shifts the phenotype towards M2term·dom in response to RF-M1 or, conversely, the M3 phenotypeterm·dom increases the production of the proinflammatory M1term·dom cytokinesterm·key and shifts the phenotype towards M1term·dom in response to RF-M2.

3.4. A Possible Role of the M3 Phenotypeterm·dom in Immune Phenomena and Disease Development The notion of M3 phenotypeterm·dom can be built perfectly into the concept of macrophageterm·key reprogrammingterm·dom and, thus, extends our understanding of the mechanisms of macrophage plasticityterm·dom. A possible role of the M3 phenotypeterm·dom in immune phenomena and disease development can be demonstrated by the next examples.

(1) Normal Termination of Inflammation and Immune Reactions Could Be Associated with the M3 Phenotypeterm·dom of Macrophagesterm·key. Excessive production of the proinflammatory or anti-inflammatory cytokinesterm·key could lead to the development of proinflammatory diseases or to a great decrease in bactericidal, antiviral, and antitumoral activity of the immune system, respectively. The M3 phenotypeterm·dom could be suggested to help the immune system in resolving these issues. In inflammation, when the infection is already eliminated, but there are still many proinflammatory mediators in the microenvironment, in this case, the M1term·dom/M2 phenotypeterm·key will promote termination of inflammation as a result of the negative feedback formation and increased production of the anti-inflammatory cytokinesterm·key. Similarly, as a result of negative feedback, the M2term·dom/M1 phenotypeterm·key can prevent a critical decrease in bactericidal, antiviral, and antitumoral functions of macrophagesterm·key during the M2 phenotypeterm·key reprogrammingterm·dom and development of an antiparasitic Th2 response. Indeed, the M2 phenotypeterm·key can always be found in an area of decreased inflammation at the termination stage of inflammation [ 9 ], whereas, in an area of allergic reactions, not only the M2term·dom but also the M1 phenotypeterm·key can be found [ 116 ].

In fact, the results reported by Bystrom et al. [ 124 ] support similar conclusions. They have found that macrophagesterm·key at normal termination of inflammation possess weak bactericidal activity compared to those at the initial stage of the inflammation. In other words, macrophagesterm·key at the termination of inflammation manifest the features of the M1term·dom and M2 phenotypesterm·key. The authors referred to this phenotype as the rM (resolution-phase macrophagesterm·key) phenotype. Within the context of our hypothesis, the rM phenotype is one of the switching phenotypesterm·dom, namely, the M1term·dom/M2 phenotypeterm·key.

(2) The Pathogenesis of Many Diseases Is in Agreement with the Activity of the Switching Phenotypeterm·dom. For instance, the switching phenotypeterm·dom could be involved in the reprogrammingterm·dom of the pathologic M2 phenotypeterm·key under conditions when many proinflammatory mediators are in the tumor microenvironment. In particular, proinflammatory TNF- α has been found to demonstrate the greatest concentration increase in mice peritoneal ascites with Ehrlich carcinoma [ 125 ], and this increase is accompanied by macrophagesterm·key having the M2 phenotypeterm·key (our unpublished data). It could be suggested that the formation of TAM-M2 in tumoral inflammation is associated with formation of the switching phenotypeterm·dom M1term·dom/M2term·dom.

In addition, the pathogenesis of inflammatory diseases could be suggested to be associated with a disturbance in the formation of the M3 switching phenotypeterm·dom. In particular, in patients with a chronic venous ulcer, a disturbed process of switching from the macrophageterm·key M1 phenotypeterm·key to the M2term·dom was observed [ 126 ]. Also, our data suggest that the M3 phenotypeterm·dom is involved in the development of COPDterm·dom.

(3) The Switching Phenotypeterm·dom May Be Involved in the Formation of the Phenomenon of LPS Tolerance. The existence of several isoforms of Akt allows macrophagesterm·key to be reprogrammed to both the M1term·dom and M2 phenotypeterm·key via the PI3K/Akt-signalling pathway in response to the same ligand of LPS [ 22 , 23 , 41 , 127 ]. This makes it clear that the “LPS-tolerant” phenotype is not a strict definition of the phenotype that decreases production of the proinflammatory cytokinesterm and increases production of anti-inflammatory cytokinesterm·key in response to the M1-inductor LPS. This phenotype does not become less sensitive to LPS; that is, it does not become LPS tolerant. This phenotype just changes its reaction to LPS. It has been established that the p50/р50 NF- κ B homodimers, which prevent activation of the M1 phenotypeterm·key genes and activate the M2 phenotypeterm·key genes, are accumulated in the nuclei of the “LPS-tolerant” macrophagesterm·key [ 76 ]. Within the context of our hypothesis, the “LPS-tolerant” phenotype is the switching phenotypeterm·dom M1term·dom/M2term·dom.

(4) The Switching Phenotypeterm·dom Could Be Involved in the Defence Strategy of Some Bacteria and Viruses. Most viruses and bacteria reprogram macrophagesterm·key to the M1 phenotypeterm·key [ 10 ], and then the M1term·dom macrophagesterm·key eliminate the infection. However, some microbes can suppress the bactericidal and antiviral properties of the innate immunityterm·key due to reprogrammingterm·dom of macrophagesterm·key to the M2 phenotypeterm·key [ 9 , 113 , 127 ]. This mechanism of microbial defence strategy could be suggested to involve the formation of the macrophageterm·key switching phenotypeterm·dom M1term·dom/M2term·dom, which, instead of producing bactericidal free radicals and proinflammatory cytokinesterm, produces anti-inflammatory cytokinesterm·key in response to microbes.

Among immune cells, there are some cells which behave similarly to the macrophagesterm·key with the switching phenotypeterm·dom. These are Treg lymphocytes, which are known to produce anti-inflammatory cytokinesterm·key in response to a proinflammatory microenvironment [ 128 , 129 ]. In fact, the Treg lymphocytes have a switching phenotypeterm·dom as proposed for macrophagesterm·key. The existence of Treg lymphocytes proves the possibility that intracellular mechanisms could form a switching phenotypeterm·dom in cells.

4. Conclusions The current concept of macrophageterm·key reprogrammingterm·dom describes the dynamics of changes from the M1term·dom to M2 phenotypeterm·key as a “continuumterm·dom” [ 1 , 17 , 80 ], that is, as a gradual and continuous process. The concept of a continuumterm·dom is obviously valid in a situation when the phenotype changes from M1term·dom to M2term·dom in response to RF-M2 or when it changes from M2term·dom to M1term·dom in response to RF-M1. The situation when the phenotype changes from M1term·dom to M2term·dom or, contrarily, from M2term·dom to M1term·dom in response to the alternative reprogrammingterm·dom microenvironment (RF-M1 or RF-M2, resp.) obviously does not comply with the concept of a continuumterm·dom. Our hypothesis suggests that the changes in phenotype from M1term·dom to M2term·dom and vice versa could occur via the formation of the M3 switching phenotypeterm·dom. If this is so, then the changes of phenotype should be described using the term of “discretuumterm·dom.”

Our hypothesis implies a new understanding of macrophage plasticityterm·dom and allows us to answer the question of how the paradoxical reprogrammingterm·dom of these cells occurs, that is, how the anti-inflammatory M2 phenotypeterm·key is formed in response to RF-M1 in inflammation and, contrarily, how the proinflammatory M1 phenotypeterm·key is formed in response to RF-M2. The understanding of the mechanism of the paradoxical reprogrammingterm·dom of macrophagesterm·key will extend our vision of the mechanisms of plasticity of the immune response. This understanding will also help to answer several important questions of modern immunology and medicine: How does the TAM reprogrammingterm·dom to the M2 phenotypeterm·key occur in tumor-induced inflammation [ 17 , 119 ]? How does the reprogrammingterm·dom of macrophagesterm·key from the M1term·dom to M2 phenotypeterm·key occur in normal termination of an inflammation [ 8 ] at the stage when macrophagesterm·key are still affected by the proinflammatory RF-M1? How does the formation of the M2 phenotypeterm·key in response to an RF-M1, such as LPS [ 23 ], occur? How do some bacteria switch the macrophageterm·key phenotype from M1term·dom to M2term·dom [ 9 , 113 ], so as to avoid the bactericidal action of the M1term·dom macrophagesterm·key? How can the anti-inflammatory M2term·dom cytokinesterm·key IL-10term and IL-13 program the M1 phenotypeterm·key and increase the production of the proinflammatory M1term·dom cytokinesterm·key [ 60 , 114 ]? Why can the alternate macrophageterm·key phenotypes be found in the same microenvironment during the development of allergic reactions and in obesity and alcoholic hepatitis [ 116 – 118 ]? How is the phenomenon of LPS tolerance formed? Why do some inflammatory diseases develop? What is the new mechanism of the action of glucocorticoids?

It seems evident that the switching phenotypeterm·dom may potentially play a significant role during the development of inflammatory, autoimmune, oncologic, and other diseases. Thus, the switching phenotypeterm·dom should be a therapeutic target in the development of new medicines and medical cell technologies for treating diseases associated with a disturbed immune response.