Molecular and epigenetic basis of macrophage polarized activation

Seminars in Immunology 27 (2015) 237–248

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Molecular and epigenetic basis of macrophage polarized activation Chiara Porta a,∗ , Elena Riboldi a , Alessandro Ippolito a , Antonio Sica a,b a b

Department of Pharmaceutical Sciences, Università del Piemonte Orientale “Amedeo Avogadro”, via Bovio 6, Novara, Italy Humanitas Clinical and Research Center, Via Manzoni 56, Rozzano, Milan 20089, Italy

a r t i c l e

i n f o

Article history: Received 11 June 2015 Received in revised form 16 October 2015 Accepted 19 October 2015 Keywords: Macrophage Activation Plasticity Metabolism Epigenetic Gene expression

a b s t r a c t Macrophages are unique cells for origin, heterogeneity and plasticity. At steady state most of macrophages are derived from fetal sources and maintained in adulthood through self-renewing. Despite sharing common progenitors, a remarkable heterogeneity characterized tissue–resident macrophages indicating that local signals educate them to express organ-specific functions. Macrophages are extremely plastic: chromatin landscape and transcriptional programs can be dynamically re-shaped in response to microenvironmental changes. Owing to their ductility, macrophages are crucial orchestrators of both initiation and resolution of immune responses and key supporters of tissue development and functions in homeostatic and pathological conditions. Herein, we describe current understanding of heterogeneity and plasticity of macrophages using the M1–M2 dichotomy as operationally useful simplification of polarized activation. We focused on the complex network of signaling cascades, metabolic pathways, transcription factors, and epigenetic changes that control macrophage activation. In particular, this network was addressed in sepsis, as a paradigm of a pathological condition determining dynamic macrophage reprogramming. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction: Macrophages are unique immune cells for origin, heterogeneity and plasticity

Abbreviations: HSCs, hematopoietic stem cells; EMPs, erythro-myeloid progenitors; LCs, Langerhans cells; BM, bone marrow; TRAP, Tartrate-resistant acid phosphatase; RP, red pulp; MZ, marginal zone; AMs, alveolar macrophages; IMs, interstitial macrophages; PIMs, pulmonary intravascular macrophages; LPS, Lipopolysaccharide; SOCS, suppressor of cytokine signaling; PGE2, prostaglandin E2; STAT, Signal Transducer and Activator of Transcription; DMs, dermal macrophages; LDTFs, lineage-determining transcription factors; GATA6, GATA-binding protein 6; GM-CSF, Granulocyte-macrophage colony-stimulating factor; PPAR␥, peroxisome proliferator-activated receptor-␥; RANK, Receptor activator of nuclear factor kappa-B; LXR␣, transcription factors liver X receptor ␣; iNOS, inducible nitric oxide synthase; NO, nitric oxide; ROS, reactive oxygen species; IRF, Interferon Regulatory Factor; PFK2, 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase; CARKL, carbohydrate kinase-like protein; IDH, isocitrate dehydrogenase; FFAs, free fatty acids; FAO, fatty acid oxidation; PGC-1␤, PPAR␥-coactivator-1␤; COX, cyclooxygenase; HIFs, Hypoxia Inducible Factors; KLFs, Kruppel-like transcription factors; TLRs, Toll like receptors; TAMs, tumor associated macrophages; PGK, Phosphoglycerate kinase; LAL, lysosomal acid lipase; PDK1, pyruvate dehydrogenate kinase 1; AMPK, adenosine monophosphate kinase; PTEN, tensin homolog deleted on chromosome 10; mTOR, Mechanistic Target of Rapamycin; HDAC, Histone deacetylases; MLL, Myeloid Lymphoid Leukaemia; SHIP-1, phosphatidylinositol3,4,5-trisphosphate 5-phosphatase 1; SIRT1, sirtuin 1. ∗ Corresponding author. Tel.: +39 0321 375 883; fax: +39 0321 375 821. E-mail addresses: [email protected] (C. Porta), [email protected] (E. Riboldi), [email protected] (A. Ippolito), [email protected] (A. Sica). http://dx.doi.org/10.1016/j.smim.2015.10.003 1044-5323/© 2015 Elsevier Ltd. All rights reserved.

Macrophages are crucial orchestrators of both initiation and resolution of immune responses, and also key supporters of tissue development and functions under both homeostatic and pathological conditions [1–3]. Macrophages can coordinate this broad spectrum of diverse activities because they are endowed of an extraordinary plasticity and diversity [4]. Over the last five years, the development of genetic fate-mapping techniques has allowed to re-write macrophages ontogeny [5–10]. Hitherto, macrophages emerge as the only immune cell type that, in different proportion depending on the organ, are established during embryonic development and maintained in adulthood through longevity and local self-renewal [1,3,5–13]. These prenatal-derived macrophages represent the majority of steady state macrophages in tissues such as liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells), lungs (alveolar macrophages) and heart [5–10,12,13]. In few tissues, like dermis and gut, tissue macrophages are constantly replenished by adult hematopoietic stem cells (HSC)-derived monocytes [14–16]. Macrophages with dual origin (embryonic and adult HSCs-derived) co-exist in organs such as spleen, kidney and pancreas [11]. Some controversial results are reported in tissue like peritoneum, where both embryonic [7,8] and adult bone marrow (BM)-derived [12] origin are suggested. Further, with the exception of microglia, widely recognized as yolk sac derived, the nature of

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the prenatal precursors is still under debate. Recently, using three different inducible fate mapping mice (Csf1rMercreMer , Tie2MercreMer and Flt3-Cre) Gomez Perdiguero et al. identified erythro-myeloid progenitors (EMPs) in the yolk sac as the precursor of all embryonicderived macrophages in the adult tissues [10]. In contrast using Kit (CD117) gene to create an inducible fate mapping mice, Sheng and colleagues indicate that brain microglia and, to some extent, epidermal LCs are the only yolk-sac derived macrophages because they are the unique cells that can be detected when EMPs are labeled before generation of pre-HSCs in the intra-embryonic mesoderm [12]. Labeling HSCs-dependent hematopoiesis, all adult non-microglial tissue-resident macrophages and peripheral blood cells are similarly detected, indicating an HSCs origin [12]. In agreement, combining in vivo yolk sac macrophages depletion with several fate-mapping models of yolk sac macrophages and/or fetal liver monocytes, Hoeffel et al. claim that only microglia derived from yolk sac ckit+ Csfr1+ EMPs without passing through fetal monocytes [13]. However, Hoeffel and colleagues indicate that a second wave of progenitors give rise to late definitive EMPs which, following the establishment of blood circulation, migrate to the liver where they acquire c-Myb expression and give rise to fetal monocytes [13]. Hence, these late c-Myb+ EMPs, instead of HSCs, are indicated as the principal source of fetal monocytes that then seeded embryonic tissues and differentiated into macrophages [13]. Pioneering studies suggest that human macrophages ontogeny resemble the murine one [17,18]. Indeed, the analysis of radioresistant Langerhans cells (LCs) from patients that underwent HSCs transplantation, as well as of human hand allograft biopsy, have showed that epidermal macrophages are maintained up to 10 years suggesting a great longevity or a self renewal capacity [17,19,20]. Moreover, the presence of a normal number of LCs and tissue macrophages in patients carrying a syndrome characterized by a defective hematopoiesis associated with monocytopenia and absence of peripheral DCs, B and NK lymphoid cells, suggests that human macrophages origin and maintenance are largely bone marrow (BM)-independent [21]. Despite sharing common progenitors, tissue-resident macrophages show distinct transcriptional [22,23] and epigenetic signatures [23,24], suggesting that local microenvironmental signals educate them to express organ-specific functions. Indeed, when transplanted BM precursors are seeded to the different tissues, their chromatin landscape is reshaped according to local signals to whom they are exposed [23]. Even tissue macrophages can be reprogramed when transferred into a new microenvironment, indicating that epigenetic chromatin marks are dynamic and that, macrophages retain plasticity, even if they are terminally differentiated cells [23]. Further, in a mouse model where host (embryonic) coexist with donor (post-natal)-derived AMs, the transcriptional profiles of both population are largely similar, suggesting that microenvironment dictates gene expression with the exception of few genes that remain linked to cellular origin [25]. Beside physiological local signals, macrophages can finely shape their functional phenotype in response to the micro environmental cues that they sense [26–30]. The identification of “latent” or “denovo” enhancers, which are distal regulatory elements that gain and retain histone modifications only upon stimulation [31,32], further supports the epigenetic plasticity of macrophages in response to external stimuli. Owing to their ductility, macrophages play a key role in both host defense and tissue homeostasis, however the ability of macrophages to reprogram their functions acts as a double edged sword in several pathological conditions [33–37]. Hence, dissecting the molecular basis of macrophages biology will enable new macrophage-centered strategies with promising therapeutic value for a wide range of human diseases [1,2,34]. Here, we will

describe current understanding of heterogeneity and plasticity of macrophages, focusing on the complex network of signaling cascades, metabolic pathways, transcription factors, and epigenetic changes that controls macrophage activation.

2. Hallmarks and regulation of physiological macrophage heterogeneity A remarkable heterogeneity characterizes tissue-resident macrophages. Indeed, not only multiple functional macrophage subtypes have been identified in different organs, but even within the same tissue, macrophages located in specific anatomical sites exhibit distinct trophic activities [4]. In the bone, Tartrate-resistant acid phosphatase (TRAP)+ F4/80+ osteoclasts participate in bone remodeling by reabsorbing bone tissue [38,39], whereas TRAP− CD169+ F4/80+ BM macrophages are crucial component of the erythroid niche, supporting red blood cells development and uptake of the nuclei extruded by erythroid precursors [40,41]. In the spleen, three different subsets of macrophages can be distinguished: (1) F4/80+ CD68+ macrophages, localized in the red pulp (RP) and specialized in the clearance of senescent erythrocytes and iron recycling; (2) F4/80l◦ CD68Hi MFG-E8Hi macrophages, situated in the white pulp (WP) and committed to the phagocytosis of apoptotic lymphocytes; (3) CD169+ SIGN-R1+ MARCO+ TIM-4+ macrophages, located at the marginal zone (MZ) where they initiate immune responses against blood-borne particulate antigens [42,43]. Accumulating evidence indicate that three different macrophage populations coexist even in lungs: (1) alveolar macrophages (AMs) are located on the luminal side of alveolar epithelium behind surfactant; (2) interstitial macrophages (IMs) reside in the narrow space limited by alveolar epithelium and vascular endothelium; (3) pulmonary intravascular macrophages (PIMs) are blood monocytes-derived macrophages that adhere to endothelium of pulmonary capillaries [44,45]. AMs originate from pre-natal precursor and are characterized by a rounded morphology, intense autofluorescence and the expression of F4/80, CD11c, CD64, lectins (e.g. CD206 and SiglecF) and scavenger receptors (e.g. SR-AI/II and MARCO), but not CD11b and CD24 [3,46–50]. In contrast, during both acute and chronic lung inflammation two additional populations of CD11chigh CD11b+ phagocytesand CD11c− CD11bhigh monocytes appear in the alveolar interspace [46]. Resident AMs are mainly involved in the clearance of surfactant [51] and of inhaled antigens [52] minimizing lung injury [53]. AMs are committed to limit local inflammation through different mechanisms, including regulatory circuits based on macrophages-epithelial communication. Using real-time alveolar imaging in situ, Westphalen and collegues showed that a subset of AMs is connected with the epithelium by connexin 43-containing gap junction channels and due to these interconnections, AMs spread immunosuppressive Ca2+ waves to restrain lipopolysaccharide (LPS)-induced inflammation [54]. The transcellular delivery of vesicular suppressor of cytokine signaling (SOCS) proteins from AMs to alveolar epithelial cells represents another inhibitory strategy [55]. At steady state, alveolar epithelial cells contribute to the production of anti-inflammatory signals such as prostaglandin E2 (PGE2) and IL-10, which induce AMs to secrete exosomes and microparticles containing SOCS1 and SOCS3 proteins. In turn the uptake of vesicular SOCS proteins by alveolar epithelial cells inhibits cytokines signaling by blocking Signal Transducer and Activator of Transcription (STAT) activation. In contrast, in response to LPS or cigarette smoke, SOCS secretion into airway fluid is reduced; the elimination of this brake enhances inflammation and likely contributes to the pathogenesis of inflammatory lung diseases [55].

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IMs originate from bone marrow-derived monocytes and have a CD11b+ F4/80+ CD11c+ , MHCIIHi CD64+ CD24− phenotype [50]. In vivo studies suggest that IMs, defined asF4/80+ CD11c− MHCIIHi cells, can be crucial regulators of tissue fibrosis, inflammation, and antigen presentation [56]. Indeed, when mice are exposed to antigens along with low doses of LPS, IMs are the major producers of IL-10 that, by blocking maturation and migration of antigen loaded DCs, prevent airway allergy [56]. Hence, IMs seem to be key gatekeepers of lung immune homeostasis [56]. In apparent contradiction, as compared to AMs, isolated IMs are higher inducers of T cells proliferation and up-regulate more TNF␣ in response to IFN␥ plus LPS stimulation [44,56]. Finally, PIMs which are constitutively found in some animal species (e.g. cattle, horse, pig, sheep, goat, cats, and whales) but not in healthy humans, express pro-inflammatory functions [57]. Recently, the identification of inducible accumulation of PIMs in both rodent models of hepato-pulmonary syndrome and in patients suffering from liver dysfunction has rekindled the interest in their biology and impact on inflammatory lung diseases [57]. In the skin, two subsets of macrophages can be distinguished: (1) dermal macrophages (DMs), identified asF4/80+ CD11b+ CD11cl◦ CD206+ MHCIIl◦ CD169+ Dectin-1+ CD301+ , Dectin2+ , located in the deep layers of dermis where they can be found interspersed in the intervascular space or associated with blood or lymphatic vessel; (2) LCs, identified as F4/80+ CD11b+ CD11c+ Langerin+ Runx3+ cells, situated in the epidermis, the superficial avascular layer of the skin [4,58,59]. DMs are crucial gatekeepers of tissue homeostasis, scavenging self antigens, sensing and killing invading pathogens as well as promoting the healing of the damaged skin and the resolution of both sterile and microbial inflammation [59]. In contrast, LCs are a unique population of non-lymphoid tissue macrophages endowed of a migratory capability to the draining lymph nodes similar to that of non-lymphoid tissue conventional DCs [59]. Similar to intestinal macrophages [60], LCs extend their dendrites between keratinocytes to sample external antigens and due to their plasticity, they may exert tolerogenic or immunogenic functions according to the type of threats to whom they are exposed [58,59]. In blood, circulating monocytes can be classified in two subsets: (1) “classical” or “inflammatory” monocytes, which are Ly6C+ cells in mouse and CD14+ CD16− cells in humans, are recruited to tissue in a CCR2-dependent manner; (2) “nonclassical” or “resident” monocytes, which are Ly6C− cells in mouse and CD14lo CD16+ cells in humans, are committed to remain in the blood vessels [61–63]. Ly6Clo monocytesderive from Ly6Chi monocytes through a Nr4a1-dependent transcriptional program, patrol endothelium to maintain vessel integrity [62,64], whereas Ly6Chi monocytes, under homeostatic conditions, constantly replenish tissue macrophages in selected organs like gut [15] or, remaining relatively undifferentiated, scout extra vascular tissues and transport antigens to draining lymph nodes [65]. In inflammation, classical monocytes are recruited in injured tissues where they differentiate in macrophages. An aspect that deserves to be addressed is whether monocyte-derived and tissue-resident macrophages differentially contribute to inflammation, resolution, and pathology. New knowledge could allow selective interventions in macrophage-targeted therapies. Very recently, some studies analyzed macrophage populations in organs like brain, liver, heart, lungs and adipose tissue [9,66–68]. During macrophage development, local signals dictate a characteristic epigenetic landscape that, regulating the impact of signaling pathways and transcription factors, defines the pattern of gene expression and functional outcome. In particular, gene expression is regulated by a close collaboration between gene promoters and regulatory enhancer elements, which are bound by lineagedetermining transcription factors (LDTFs), mediating cell fate and

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identity [32]. In macrophages, the cooperation of pioneer PU.1 with additional LDTF like IRF-8, Maf and MafB [23,69] defines a cluster of macrophages specific enhancers, whereas transcription factors activated by local microenvironmental signals determine an additional group of enhancers which mark tissue specific macrophage identities [23,24]. Over the last few years, some transcriptional programs activated by specific tissue signals, underlying the development of different functional macrophages subsets, have been identified [11]. Peritoneal macrophages development requires the transcription factor GATA-binding protein 6 (GATA6) [70,71], whose expression is induced by omentum-produced factors, including retinoic acid [71]. GATA6 is also crucial for the expression of genes associated with specific peritoneal macrophage functions [70,71], localization into the peritoneal cavity [71] and proliferative renewal both at steady state and during resolution of inflammation [70]. Similarly, Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced peroxisome proliferator-activated receptor-␥ (PPAR␥) is essential for the development of AMs and for the expression of a specific transcriptional signature [48]. The mechanism underlying CSF1-CSF1R dependent osteoclasts formation [72] includes the up-regulation of Receptor activator of nuclear factor kappa-B (RANK) in osteoclast progenitors followed by RANK ligand-dependent activation of NF-␬B signaling [73]. Mirroring their different functional phenotype, the development of splenic MZ and RP macrophages is controlled by two transcription factors: liver X receptor ␣ (LXR␣) [43] and SPI-C [74], respectively. Noteworthy, SPI-C, which is induced by heme, a metabolite of erythrocyte degradation, is needed also for the generation of BM macrophages [75]. This evidence strengthen the importance of signals in ontology and functional macrophages differentiation.

3. Macrophage polarized activation Starting from the “M1–M2 paradigm”, introduced to describe the different programs of macrophage activation triggered by IFN␥ and IL-4, respectively [76], the great complexity underlying functional macrophage polarization has been broadly recognized and addressed [77]. Within the dynamic spectrum of functional states of macrophages activation, inflammatory cytokines (IFN␥, TNF␣), pathogen-associated molecular patterns (e.g. LPS) and damage associated molecular patterns (e.g. High Mobility Group Box 1, Heat Shock Proteins, adenosine triphosphate, Fetuin A) induce states of classic or M1 polarized activation. On the contrary, Th2 cytokines (IL-4, IL-13), anti-inflammatory molecules (IL-10, glucocorticoids, adenosine monophosphate), immune complexes (IC) and apoptotic cells promote states of alternative or M2 polarized activation [26,77]. M1 macrophages express copious amount of inflammatory cytokines (TNF␣, IL-1␤, IL-6) and promote cytotoxic adaptive immunity by up-regulating MHCII in conjunction with co-stimulatory molecules (CD40, CD80, CD86), Th1- and Th17orienting cytokines (IL-12, IL-27, IL-23) and Th1-recruiting chemokines (CXCL9, CXCL10, CXCL11) [77]. In contrast M2 macrophages support resolution of inflammation by switching gene expression toward anti-inflammatory molecules (IL-10, TGF␤, IL-1R type II, IL-1Ra) and by expressing high levels of endocytic receptors including scavenger (CD163, Stabilin-1) and c-type lectin (CD206, CD301, dectin-1) receptors [77]. Further, M2 macrophages recruit Th2, Tregs, as well as eosinophils and basophils through the secretion of the chemokines CCL17, CCL18, CCL22, CCL24 [78]. A hugely different metabolic profile, involving iron, amino acids, glucose and lipids, distinguishes M1 and M2 polarized macrophages and greatly impacts on their different functional activities [36,79–81]. Indeed,M1 macrophages express molecules needed for intracellular iron import and storage (ferritin,

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ceruloplasmin, natural resistance-associated macrophage protein 1, divalent metal transporter-1), thus limiting iron availability for extracellular bacteria growth [82]. At the opposite, M2 macrophages up-regulate molecules mediatingiron recycling and release (CD163, CD94, transferrin receptor, hemeoxygenase-1, ferroportin), therefore promoting cell proliferation and wound healing [82]. M1 macrophages express high levels of inducible nitric oxide synthase (iNOS) that converts l-arginine to l-citrulline and nitric oxide (NO). The latter, interacting with reactive oxygen species (ROS), generates additional anti-microbial species [83] or, performing nitration of tyrosine residues of Interferon Regulatory Factor5 (IRF5) protein, limits M1 gene expression [84]. Hence, upregulation of iNOS in M1 macrophages seems to be crucial for both the production of cytotoxic effector molecules and for the activation of regulatory circuits to prevent overwhelming inflammation. In contrast, M2 macrophages up-regulate enzymes (Arginase 1, Arginase 2, ornithine decarboxylase, spermidine oxidase)that skewarginine catabolism toward the production of ornithine and polyamines, which support the repair of damaged tissue [83]. M1 macrophagesincrease glucose uptake and switch from the liver-form of 6-phosphofructose-2-kinase/fructose-2,6bisphosphatase (L-PFK2) to the more active ubiquitous form (u-PFK2); this event promotes the aerobic glycolytic pathway and rapid ATP fuel [85]. Concomitantly, the down-regulation of the carbohydrate kinase-like protein (CARKL) induces the pentose phosphate pathway that, in conjunction with attenuation of respiratory chain, supports ROS production [86,87]. Recently, combining transcriptomic with metabolomics, two breaks in Krebs cycle have been identified in M1 macrophages, in close association with their functional phenotype [88]. First, down-regulation of isocitrate dehydrogenase [88] leads to accumulation of citrate, which in turn promotes the production of several anti-microbial and inflammatory molecules including NO, itaconate and free fatty acids (FFAs), which support the synthesis of prostaglandins [89,90]. The second break, which occurs after succinate, enhances inflammation and host defense through multiple mechanisms [88]. The accumulation of succinate enhances IL-1␤ production in HIF-1␣-dependent manner [91], whereas the induction of the arginosuccinate shunt results in the production of fumarate and malate, but also of NO and IL-6 [88]. Conversely, M2 macrophages supply sustained energy demand by high rates of oxidative glucose metabolism and fatty acid oxidation (FAO) [36,80,81]. Indeed, M2 macrophages induce PPAR␥-coactivator-1␤ (PGC-1␤), which enhances mitochondrial respiration and biogenesis [92]. Further, M2 macrophages are characterized by an increased glutamine metabolism, which fuels CCL22 synthesis and UDP-N-acetyl-alpha-d-glucosamine accumulation [88]. The latter, enhancing protein glycosylation, promotes the expression of typical M2 receptors such as CD206 and CD301 [88]. Moreover, M1- and M2-polarized human macrophages express different genes encoding arachidonate metabolism related enzymes [93]. M1 macrophages showed up-regulation of cyclooxygenase (COX) 2 and down-regulation of COX1, leukotriene A4 hydrolase, thromboxane A synthase 1, and arachidonate 5lipoxygenase (ALOX5), whereas M2 macrophages express high levels of arachidonate 15-lipoxygenase (ALOX15) and COX1 [93]. Overall, M1 macrophages have powerful cytotoxic activity, thus they are capable of killing pathogens (bacteria, virus and protozoa) and tumor cells, but also of destroying tissues and of impairing metabolic homeostasis by inducing insulin resistance [2,27,88]. On the other hand, M2 macrophages have immunoregulatory functions: they promote immunity against extracellular parasites (helminths), angiogenesis, repair of damaged tissues and metabolic homeostasis, but also support allergic inflammation, fibrosis, tumor growth and progression [1,2,27]. Hence, understanding the molecular network underlying macrophage polarization represents a

crucial challenge to identify new targets for therapeutic approaches aimed to macrophages reprogramming.

4. Molecular pathways driving macrophage polarization M1 and M2 promoting signals engage signaling pathways that lead to the activation of specific transcriptional programs controlled by distinct members of the transcription factor families STAT, IRF, NF-␬B, Hypoxia Inducible Factor (HIF), Kruppel-like transcription factor (KLF) (Fig. 1) [27]. Downstream of IFNs and TLRs signaling, activation of STAT1 crucially drives the expression of an M1 transcriptional program (e.g. CXCL9, CXCL10, CXCL11, CCL5, NOS2), whereas IL-4/IL-13 and IL-10 skew macrophages toward the M2 phenotype (e.g. IL-1RA, ARG1, YM1, FIZZ1, MR, IL-10, IL4R␣, ARG2) by the activation of STAT6 and STAT3, respectively [27,94–96]. Both M1 and M2 signals also induce feed-back inhibitors, including members of the SOCS family (e.g. SOCS1, SOCS3) whose potential role in regulating macrophages polarized activation is still a matter of debate. Indeed, genetic-based studies indicate that induction of SOCS1 in response to either IFN␥/TLR or IL-4 stimulation acts as feed-back inhibitor of both M1 and M2 activation [97,98], In contrast, using a siRNA-mediated knock-down strategy, Whyte and collegues suggest that SOCS1 restrains M1 and promotes M2 inflammation [99]. SOCS3 expression is also induced either by M1 (IFN␥) or by M2 (IL-10) signals, and studies based on different approaches reported SOCS3 both as an essential determinant [100] and as a brake [101,102] for M1 polarization. Following TLR engagement, the TRIF dependent pathway leads to IRF3 activation and IFN␤ production; in turn, type I IFN enhances M1 polarization by activating STAT1 and IRF5, the latter being a crucial regulator of cytokines (IL-12, TNF␣, IL-23,) involved in Th1 and Th17 differentiation/expansion [103,104]. On the contrary, IL-4 polarized macrophages up-regulate IRF4, which induces the expression of selected M2 genes (e.g. ARG1, YM1, FIZZ1 and MR), with concomitant inhibition of IRF5 activation [105]. Several M1 signals, including inflammatory cytokines (TNF␣ and IL-1␤), TLRs and NOD1/NOD2 ligands, trigger the canonical pathway of NF-␬B activation, resulting in p65-p50 NF-␬B dependent transcription of a large number of inflammatory genes associated with M1 macrophages [106]. However, NF-␬B activation can also induce a genetic program essential for resolution of inflammation [107]. In particular, induction of p50 NF-␬B homodimers in tumor associated macrophages (TAMs) [108] and endotoxintolerant macrophages [109] inhibits M1 genes expression, whereas it is required for M2 macrophages polarized activation in vitro and in vivo [110]. Hypoxia profoundly impacts on macrophages gene expression through the activation of the inducible transcription factors HIF-1␣ and HIF-2␣ [111]. In addition, IFN␥ and IL-4/IL-13 promote selective accumulation of HIF-1␣ and HIF-2␣, which in turn enhance the expression of M1 and M2 genes, respectively [112]. Indeed, HIF-1␣ induces the transcription of several genes encoding for inflammatory cytokines (e.g. TNF␣, IL-1␤, IL-6, IL-12), anti-microbial effectors (iNOS), glycolytic enzymes (e.g. Phosphoglycerate kinase) and glucose transporters [112–115]. Accordingly, inflammatory stimuli activate HIF-1␣ gene transcription through NF-␬B [116] and hypoxia enhances glycolytic flux and pro-inflammatory genes expression through HIF-1␣ [117]. In contrast, higher expression of HIF-2␣ in TAMs [118], which are mainly M2-oriented [119], suggested a role of this isoform in alternative macrophages polarization. In agreement, M2-orienting cytokines induce HIF-2␣, which in turn activates the transcription of ARG1 gene (M2) [112]. However, controversial results were reported for both HIF isoforms. Indeed, it has been shown that tumor-derived factors including lactic acid [120] and cytokines (Oncostatin M and Eotaxin) [121]

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Fig. 1. Mechanisms of macrophage polarized activation. The major pathways downstream engagement of macrophages receptors by M1 (yellow) and M2 (blue) signals are summarized (black arrows). Both negative feedback loops and cross-talks between M1 and M2-polarizing pathways are also outlined (blue lines). The balance between M1 (red) and M2 (turquoise) transcription factors defines macrophages polarized activation. In particular, STAT1 and p65/p50 NF-␬B are crucial orchestrators of M1 transcriptional program whereas STAT3 and STAT6 are key inducers of M2 gene transcription. Further, different members of IRF family enhance selected M1 or M2 gene expression. Accumulation of p50 NF-␬B homodimers upon IL-10 or prolonged LPS stimulation (LPS-tolerance) is associated with both inhibition of M1 and induction of M2 genes. Similarly, PPAR␥ and PPAR␦ control distinct aspects of M2 macrophage activation and oxidative metabolism. KLF4 and KLF2 also participate in the promotion of M2 macrophage functions by cooperating with STAT6 and suppressing the NF-␬B/HIF-1␣-dependent transcription, respectively. In contrast, KLF6 enhances NF-␬B-driven M1 gene expression. Distinct miRNAs, also participate in skewing macrophage activation. LPS-induced miR-155 is associated with inhibition of M2 and promotion of M1 polarization, while IL-4/IL-13-induced miR-124 is linked with enhanced M2 gene expression by an unknown mechanism. Genetic ablation of different proteins (fuchsia/violet*) involved in anabolic growth indicates potential roles in M1 (fuchsia) and M2 (violet) polarized activation. IL-4-induced c-Myc also controls a subset of M2-related genes. HIF-1␣ and HIF2-␣ are mainly associated with M1 and M2 activation, respectively. Along with inflammatory cytokines, HIF-1␣ enhances the glycolytic pathway, which is a hallmark of M1 macrophages. At the opposite, IL-10-induced AMPK␣1 promotes both fatty acid oxidation (FAO) and M2 oriented functions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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skew TAM polarization toward the M2 phenotype in a HIF-1␣dependent manner. In pulmonary hypertension model, induction of HIF1␣ in response to adventitial fibroblast-derived IL-6, drives ARG1 gene expression and in conjunction with STAT3 and C/EBP␤ signaling, is crucial in skewing macrophages activation toward the pro-fibrotic (M2) phenotype [122]. Further, mice carrying a conditional deletion of HIF-2␣ in myeloid cells are resistant to LPS-induced endotoxemia, chemical-induced skin and intestinal inflammation [123]. Hence, the role of HIF isoforms in macrophages polarization required further studies to be fully clarified. Accumulating evidence point out that metabolic differences between M1 and M2 macrophages are not only a simple adaptation to different micro environmental conditions (e.g. hypoxia) or energy demand, but are an essential part of the signaling network controlling the expression of specific transcriptional programs. Indeed, pushing oxidative metabolism in M1 macrophages enhances the M2 phenotype [85] whereas blocking oxidative metabolism actually drives the macrophage into an M1 state [92]. Downstream STAT6, activation of PGC-1␤ is crucial for the M2 switch [92]; whereas PPAR␥ [124] and PPAR␦ [124,125] have a key role in maintaining the M2 phenotype by inducing distinct subsets of genes associated with anti-inflammatory functions and FAO. M2 macrophages get FFA, required to supply their energy demand, through CD36-dependent uptake of triacylglycerol substrates, followed by lysosomal lipolysis [126]. Genetic ablation of either CD36 or lysosomal acid lipase (LAL) resulted in impaired oxidative phosphorylation, survival and expression of M2-related genes, further supporting the causal relationship between metabolism and functional polarized phenotype [126]. Again, in line with this concept, but on the other front of polarized activation, pyruvate dehydrogenate kinase 1 (PDK1) comes up as a crucial regulator of aerobic glycolysis and M1 gene expression [127]. Concomitantly, PDK1 restrains M2 activation by inhibiting mitochondrial oxidative respiration, that, in the early phase of M2 activation, appears to be strictly dependent on glucose rather than on lipids [128]. Further, several evidence indicate that adenosine monophosphate kinase (AMPK), which is a central regulator of fatty acid, cholesterol, and glucose homeostasis, is also a crucial regulator of macrophages polarization. Indeed, in the presence of FFA, ablation of AMPK ␤1 in macrophages inhibits FAO and enhances insulin resistance and M1 polarized activation [129]. In agreement, activation of AMPK by the omega-7 monounsaturated fatty acid cis-palmitoleate is able to antagonize or reverse pro-inflammatory macrophage polarization induced by saturated fatty acid [130]. In line, it has been reported that AMPK is activated by M2 (IL-10 and TGF␤) and inhibited by M1 (LPS) signals and in turn promotes alternative macrophage polarization with concomitant suppression of pro-inflammatory responses [131]. Indeed, ablation of AMPK␣1 in macrophages impairs STAT3 phosphorylation in response to IL-10, leading to reduced SOCS3 expression and defective IL-10 capacity to suppress LPS-induced M1 cytokine production [132]. Accordingly, during skeletal muscle regeneration, AMPK␣1 is crucial for the M2 switch of macrophages polarized activation that leads to the resolution of inflammation [133]. KLF4 cooperates with Stat6 to induce M2 genes (ARG1, MRC, FIZZ1, PPAR␥) and inhibit M1 genes (TNF␣, COX2, CCL5, NOS2) by restraining NF-␬B activation [134,135]. KLF2 also limits M1 macrophage activation by inhibiting NF-␬B/HIF-1␣ activities [136]. Conversely, KLF6 cooperates with NF-␬B to potentiate the expression of LPS-induced pro-inflammatory genes and limits M2 genes expression by inhibiting PPAR␥ [137]. Interestingly, IL-4 and other M2 signals up-regulate the transcription factor c-Myc, which contributes to alternative macrophages activation by directly triggering the expression of some IL-4-induced genes (SCARB1, ALOX15, and MRC) as well as of STAT6 and PPAR␥ [138]. Hence, in addition to metabolic

pathways, proliferation and survival pathways may regulate macrophages polarized activation. In agreement, both PI3K/Akt and mTOR signaling cascades emerge as new regulators of functional macrophage differentiation [139–141]. Genetic ablation of Akt1 and Akt2, are associated respectively with increased and decreased capacity to mount inflammatory responses in vivo, suggesting that the two Akt isoforms control different programs of polarized activation [139]. Accordingly, Akt2 deficient macrophages showed an increased expression of selected M2 genes (ARG1, YM1, and FIZZ1), which are associated with reduced levels of miR-155 and consequent elevated amount of its targets, including C/EBP␤ [139], a transcription factor that selectively drives the expression of M2 genes [142]. The importance of the PI3K/Akt pathway in the regulation of macrophages activation is strengthened by the observation that ablation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) enhances M2 macrophages activation followingTLR4 stimulation [140]. However, whether PTEN can differentially regulates Akt 1 and Akt 2 isoforms is unknown. Further, ablation of Mechanistic Target of Rapamycin (mTOR) in myeloid cells impairs M2 polarized activation in vitro and vivo, while enhances the expression of LPS-induced inflammatory genes [141]. Interestingly, mTORC1-mediated attenuation of Akt activity contributes to the observed phenotype [141].

5. Epigenetic changes controlling macrophage polarization Epigenetic regulation of chromatin activity through distinct histone-modifying enzymes emerges as a crucial event controlling multiple aspects of macrophages biology, including polarized activation [143,144]. Multiple evidence indicate that Histone deacetylases (HDAC) are crucial regulators of inflammation and immunity. In particular HDCA3 is a key promoter of M1 and brake of M2 polarized activation [145–147]. Indeed, in LPS-stimulated macrophages, ablation of HDAC3 impairs the activation of hundreds of inflammatory genes, mainly STAT1-dependent [145]. Concomitantly, the absence of HDAC3 is sufficient for skewing macrophages toward the M2 phenotype and further enhanced Th2 cytokines driven alternative activation, in vitro and in vivo [146]. Brd4 and Brd2, which are members of bromodomain and extra terminal domain (BET) family of proteins, also promote inflammatory gene expression by interacting with acetylated histones and inducing transcriptional elongation by RNA polymerase II [148,149]. Accordingly, a small molecule, I-BET, by blocking the binding of Brd4 to chromatine, dampens LPS-induced inflammation in vitro and in vivo [148]. Further, either genetic ablation of Brd2 or inhibition of Brd2-Brd4 interaction by the small molecule JQ1 impairs inflammatory cytokine genes expression and confers resistance to endotoxin shock [149]. The Myeloid Lymphoid Leukaemia (MLL) methyltransferase is induced in LPS/IFN␥-stimulated macrophages and is essential for CXCL10 gene expression suggesting that this histone-modifying enzyme could be both a marker and a potential promoter of M1 activation [150]. In contrast the H3K27 demethylase Jumonji domain containing 3 (Jmjd3) has been mainly associated with M2 polarized activation [105,151]. Indeed, in response to different signals including IL-4 and chitinin, or during helminth infections, induction of Jmjd3 was associated with decrease H3K27 dimethylation and trimethylation (H3K27me2/3) marks and consequent induction of M2 genes expression [105,151]. However, Jmjd3 is also involved in the induction of selected LPS[152] and serum amyloid A-induced inflammatory gene expression [153] and, in conjunction with related UTX, is required for effective transcription of multiple M1 genes in human macrophages [154]. Moreover, Jmjd3 regulates RANKL-driven osteoclast differentiation by promoting Nfatc1 expression [155]. Overall, these evidence suggest that Jmjd3, rather than being selective for M2 polarization,

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enables responses to transcription factors that actually, skew macrophage activation in response to specific signals. Macrophage polarized activation can be also epigenetically controlled by miRNAs [156]. M1 and M2 signals modulate the expression of several miRNA [157,158]. However the majority of them are involved in negative feedback circuits aimed to control inflammatory genes expression [159] rather than in specific modulation of downstream program of polarized activation. For example, LPS-stimulated human monocytic cells up-regulate miR146a, which in turn acts as a negative feedback regulator of TLR signaling, by targeting TNF receptor–associated factor 6 and IL-1 receptor–associated kinase 1 [160]. Similarly, LPS-induced miR9 restrains inflammatory responses in human neutrophils and monocytes by targeting NFKB1 transcript [161] and miR-21 is upregulated in response to LPS and in turn limits NF-␬B activation and IL-6 expression with concomitant promotion of IL-10 expression [162]. In contrast, few miRNA promote a polarized transcriptional program according to the type of its own inducing signals and actually represent crucial determinants of macrophage plasticity. In particular, miR-155 is up-regulated in response to different TLR ligands and in turn supports M1 inflammation by targeting negative regulators, such as SOCS1 [163] and phosphatidylinositol-3,4,5trisphosphate 5-phosphatase 1 (SHIP-1) [164], and by increasing the half-life of TNF␣ [165]. In agreement, IL-10 selectively restrains the LPS-induced expression of miR-155, but not of the antiinflammatory miR-21 or miR-146a [166]. The importance of miR-155 in skewing macrophages toward the M1 phenotype is further supported by the observation of miR-155 expression in atherosclerotic plaques and pro-inflammatory macrophages [167,168]. According, ablation of miR-155 decreased atherogenesis in ApoE-deficient mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an anti-atherogenic leukocyte profile [167,168]. Noteworthy, miR-155 plays an opposite role on M2 polarized activation. Indeed, it inhibits M2 gene expression by targeting the transcription factor C/EBP␤ [139,142] and by limiting IL-13 and TGF␤ signaling through the down-regulation of IL-13R␣1 [169] and of SMAD2/3 [170], respectively. Over expression of miR-155 is capable to reshape TAMs from the M2, tumor-promoting, to the M1, tumor-killing, phenotype, further strengthening the importance of miR-155 in M1-oriented activation [171]. In contrast, miR-124 emerges as a crucial driver of M2 polarized activation [172]. Indeed, miR124 enhances M2 (e.g. CD206 and YM1) and restrains M1 (e.g. CD86, NOS2, TNF␣) gene expression [172]. In line, in vitro and in vivo, miR-124 expression increases during M2 and decreases during M1 polarized activation [172,173]. Indeed, in both mice and humans, allergic lung inflammation is associated with increased amount of miR124 in monocytes/macrophages [172]. In contrast, down-regulation of miR-124 was observed in activated microglia cells from the central nerve system of mice with experimental autoimmune encephalomyelitis, while systemic administration of miR-124 was capable to dampen inflammation and improve the disease [173]. Although CEBP/␣ was identified as a target of miR124, this transcription factor is actually involved in the expression of both M1 and M2 polarized programs [174,175]. Additional potential regulators of macrophage activation are miRNA, which are differentially expressed in M1 as compared to M2 macrophages. For example, miR-223 is induced by LPS and repressed by IL-4 and actually regulates both M1 and M2 responses [176]. Indeed, miR-223-deficient macrophages are hypersensitive to LPS stimulation, whereas such macrophages exhibit a delayed responses to IL-4 compared with controls [176]. The inhibitory role of miR-223 in M1 polarized activation was further strengthened by the observation that adipose tissue inflammatory response and insulin resistance increase in chimeric mice with miR-223-deficient

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hematopoietic cells [176]. Hence, up-regulation of miR-233 in response to LPS likely represents a brake to limit inflammation. In contrast, miR-125b, which is down-regulated by TLR4 signaling, enhances M1 macrophages activation by increasing the expression of costimulatory factors and the responsiveness to IFN-␥ [177]. Since miR-125b targets the M2 transcription factor IRF4 [177], this miRNA can also inhibit macrophage alternative activation. At the opposite, both miR-125a-5p [178] and Let-c [179] are selective promoters of M2-polarized activation because they enhance IL-4induced gene expression and phagocytosis of apoptotic cells, while they inhibit LPS-induced genes expression and bactericidal activity. These M2-skewing activities, along with their late induction upon LPS treatment, prompt to speculate a potential involvement of both miR-125a-5p and Let-c in the circuits underlying macrophages reprogramming during endotoxin tolerance.

6. Sepsis as a paradigm of a pathological condition associated with dynamic macrophage reprogramming Functional macrophage polarization has been reported in vivo, under both physiological (embryogenesis and pregnancy and normal maintenance of selected tissues, such as testis and adipose) and pathological conditions (chronic inflammation and tissue repair, metabolic and vascular disorders, infection, and cancer). However, mirroring the plethora of different signals to whom they are exposed, macrophages with mixed phenotypes or populations with different phenotypes can coexist in different diseases such as neurodegenerative disorders [180], atherosclerotic plaques [181], and Helicobacter Pilory infection [182]. Further, the dynamic changes that occur during evolution of pathology reshape macrophage polarized activation, with classically activated M1 cells implicated in the genesis of acute inflammation and M2 cells associated with resolution or smoldering chronic inflammation [27]. Sepsis is a paradigm of such macrophages reprogramming. Indeed, the inflammation occurring during the initial phase of the “Systemic Inflammatory Response Syndrome” (SIRS), associated with severe sepsis, is followed by a state of tolerance, that supports the “Compensatory Anti-inflammatory Response Syndrome” (CARS) [183,184]. Although this functional re-education represents a protective mechanism to counteract overwhelming inflammation, it also associates with increased risk of relapse and susceptibility to secondary infections and mortality [185,186]. Hence, understanding the molecular network underlying monocyte/macrophages re-programming is a milestone to develop new therapeutic approaches tailored on the specific phase of disease. Whereas several negative regulators of TLR signaling (e.g. short version of MyD88, IRAK-M, ST2, SIGIRR/TIR8, SOCS1, A20, miR-146a) have been found up-regulated in LPS-tolerant monocytes/macrophages and causally linked with inhibition of M1 genes expression [187], few molecules involved in the M1–M2 switch of polarized activation that paralleled the transition from SIRS to CARS have been identified [110,188]. Using an in vitro model of LPS-induced tolerance, we demonstrated that accumulation of p50 NF-␬B in monocyte/macrophage is a crucial event not only to inhibit inflammatory genes expression but also to drive the expression of a cluster of genes (IL-10, TGF␤, ARG1, CCL2, CCL17, CCL22) associated with M2 polarized activation [110]. Recently, upregulation of HIF1␣ expression and activity in monocytes isolated from septic patients was found essential to dampen inflammatory phenotype by inducing IRAK-M, as well as to activate the transcription of genes associated with tissue remodeling (MMP9, MMP19, VEGFA) and anti-microbial activity (HAMP) [188]. Noteworthy, these findings strengthen the concept that LPS-tolerant monocytes/macrophages rather than being inert cells, express an

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M2-oriented program including molecules enable to support protective responses [188,189]. In line with the importance of metabolic pathways in defining functional macrophages activation, up-regulation of the protein TNF␣-induced protein 8-like 2 in response to long term stimulation with LPS emerge as an additional molecular event that, by switching arginine catabolism from iNOS to ARG1, participates in the M1/M2 shift of macrophages polarization [190]. The transcriptional reprogramming associated with LPStolerance mirrors the epigenetic remodeling of histone marks that lead to decreased and increased chromatin accessibility of tolerized and non-tolerized genes, respectively [189]. The epigenetic mechanisms underlying the inhibition of inflammatory genes include members of the transcription factor NF-␬B in conjunction with distinct histone modifying enzymes [191–194]. Indeed, in LPS tolerance, the interaction of the NF-␬B protein RelB with the histone H3 lysine 9 methyltransferase G9a induces facultative heterochromatin formation leading to repression of M1 genes [191]. In contrast IFN␥ abrogates LPS-tolerance by preserving the chromatin of inflammatory genes in an “open” status and by inhibiting the degradation of receptor-interacting protein 140 (RIP140), a p65 NF-␬B coactivator [194,195]. The selective inhibition of M1 genes in LPS-tolerant macrophages is also achieved through NF-␬B DNA binding motifs-dependent recruitment of NcoR–Hdac3–deacetylated-p50 repressor complexes to the promoters of inflammatory cytokines genes [192]. Strikingly, the accumulation of the energy sensor and deacetylase sirtuin 1 (SIRT1) to M1 genes also emerge as crucial event that, by coupling metabolic pathways with epigenetic regulation, drives the inhibition of inflammatory genes expression [193]. Indeed, the binding of SIRT1 to M1 promoters requires NAD+ whose amount increases in endotoxin tolerant blood leukocytes of septic patients probably due to impaired mitochondria respiration and reduced ATP levels [193]. In turn, SIRT1 inactivates M1 gene expression by deacetylating p65 NF-␬B and histone H4, as well as by promoting the recruitment of the above mentioned RelB-dependent transcription repressor complex [193]. On the other side, the molecular determinants driving the chromatin remodeling associated with expression of the LPS-tolerant transcriptional program are still largely unknown. Noteworthy, epigenetic chromatin remodeling represents also the molecular base of “trained” immunity, a term referred to the capacity of pathogens to increase monocytes nonspecific responsiveness against secondary infection [196,197]. Compared to “tolerant”, “trained” macrophages showed a hugely different epigenetic and transcriptional profile, including genes involved in immune signaling pathways and cellular metabolism [196]. Most importantly, interfering with cellular metabolism prevented “trained” immunity [196,197], thus strengthening the importance of epigenetic and metabolic changes in translation environmental signals in functional macrophages activation.

7. Conclusions Macrophages play a central role in the progression of chronic inflammation in several pathologies, indicating that macrophagecentered therapeutic strategies hold promises for a wide range of diseases. Considering macrophage heterogeneity, owed that microenvironment instead of origin largely dictates their functional differentiation, approaches of organotopic macrophages transplantation could be successfully adopted, opening new hopes for the treatment of human diseases like hereditary pulmonary alveolar proteinosis. In fact, in a preclinical model of this disease, pulmonary transplantation of BM-differentiated macrophages [198] or macrophages progenitors [199] carrying functional GM-CSF

receptors resulted in long-term pulmonary engraftment, differentiation into functional alveolar macrophages and correction of the lung disease. The “re-education” of already differentiated macrophages represents an alternative therapeutic strategy for several diseases (e.g. cancer, allergic and chronic inflammation, fibrosis, metabolic and vascular disorders, infection), in which macrophages express a wrong program of polarized activation. Along with a great body of experimental evidence, the therapeutic value of macrophage reprogramming has already been proven to be successful clinically. Indeed, triggering M1 activation through administration of IFN␥ showed beneficial effects in patients with ovarian carcinoma [200,201] and combination of gemcitabine chemotherapy with CD40 agonist antibody to induced M1 activation elicited partial clinical effects in patients with advanced pancreatic cancer [202]. Exploring the molecular determinants underlying macrophage polarized activation represents a crucial challenge to identify new targets for more powerful therapeutic approaches. Over the last years, several efforts in the study of macrophage biology have highlighted a complex network of signaling cascades, metabolic pathways, transcription factors, and epigenetic regulators that by shaping gene expression define the functional output. In different tumor models, type I TNFR signaling is able to restrain M2 polarized activation by inhibiting both IL13 production by eosinophils and macrophages responsiveness to IL-13 [203]. An inverse correlation between the amount of TNF␣ and M2 polarized activation can be also found in diseases such as obesity [204], Mycobacterium tuberculosis [205] and Schistosoma mansoni infections [206], further indicating that the balance between TNF and IL-13 is a crucial regulator of macrophage polarized activation in pathology [203]. Although macrophages plasticity encompasses chromatin landscape, determining the accessibility for binding of transcription factors that are activated by acute signals, epigenetic marks emerged as a sort of transcriptional memory that modulates macrophage reprogramming in response to subsequent environmental challenges [144,207]. Since epigenetic marks are typically more stable and induce longer-lived effects than transcription factors or upstream signaling molecules, they represent very attracting targets for long–lasting therapeutic approaches. Beside sensing environmental cues, chromatin modifying enzymes activity requires metabolites generated by cellular metabolism, indicating that these epigenetic regulators are also potentially able to integrate external and metabolic changes in a polarized activation program. As an example, in LPS-tolerant macrophages the energy sensor and deacetylase SIRT1 represents a link between bioenergy and epigenetic shifts [193]. Further, beneficial results have been obtained by HDAC inhibitors in a Phase I trial in juvenile inflammatory arthritis [73], proposing chromatin-regulating enzymes as novel potential targets for therapeutic approaches aimed to macrophage reprograming. Acknowledgments This work was supported by Ministero della Salute, Italy; Fondazione Cariplo, Italy; Associazione Italiana Ricerca sul Cancro (AIRC), Italy; Ministero Università Ricerca (MIUR), Italy. References [1] T.A. Wynn, A. Chawla, J.W. Pollard, Macrophage biology in development, homeostasis and disease, Nature 496 (7446) (2013) 445–455. [2] P.J. Murray, T.A. Wynn, Protective and pathogenic functions of macrophage subsets, Nat. Rev. Immunol. 11 (11) (2011) 723–737. [3] S. Gordon, A. Pluddemann, F. Martinez Estrada, Macrophage heterogeneity in tissues: phenotypic diversity and functions, Immunol. Rev. 262 (1) (2014) 36–55. [4] L.C. Davies, et al., Tissue-resident macrophages, Nat. Immunol. 14 (10) (2013) 986–995.

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