**3. Polarization of ATM and the link to IR**

Macrophages are extremely heterogenic in function and phenotype, and have historically been characterized into two phenotypes; M1 and M2. M1 macrophages are often defined as 'classically activated' and are generally pro-inflammatory in function, with a vital role in eliminating pathogens and virus-infected cells. Whereas, M2 macrophages and termed 'alternatively activated' are anti-inflammatory in function and promote tissue repair and wound healing. This is very simplified and dated model however, evidence now suggests that ATMs include highly plastic cell populations, with their phenotype largely dependent on the microenvironment of the AT. Whilst the exact number and function of ATM in the AT is evolving, it is clear there are distinct populations in the lean and obese AT, with unique tissue distribution, marker expression, transcriptional profiles and functions. Indeed, obese ATM display markers that are largely induced by their metabolic state rather than cytokine stimuli that classically polarise M1 and M2 cells [32].

#### **3.1 M1 and M2 macrophage phenotypes**

M1 macrophages are activated by signals associated with infection such as IFN-ɣ as well as bacterial-derived products such lipopolysaccharide (LPS) and free fatty acids (FFA). M1 macrophages are loosely identified by surface expression of F4/80+ CD11C+ with high levels of MHC-II, CD68, CD80 and CD86 costimulatory molecules in addition to release of TNF-α and inducible nitric oxide synthase (iNOS) [36]. M2 macrophages are activated via Th2 cytokines, IL-4 and IL-13 as well as by parasitic products. M2 macrophages are loosely identified as F4/80+ CD2 06<sup>+</sup> CD301+ CD11C<sup>−</sup> and express genes encoding anti-inflammatory proteins such as *Chil3*, *Arg1* and *Il10* in mice [34]. A crucial transcription factor in M2 macrophage polarisation is peroxisome proliferator-activated receptor (PPAR-γ/δ), which can be driven by adipocyte derived IL-4 and IL-13 [37–39] (**Figure 2**). Some markers vary between mice and human macrophages. For example, there are no human homologues of the M2-associated genes *Chil3*, *Arg1 and Fizz1,* with human M2 identified based on expression of tranglutaminase-2 (TGM2) and CD68 [40].

#### **3.2 MMe and Mox macrophage phenotypes**

Macrophages with a phenotype associated with obesity are induced by several metabolic stimuli such as FFA, high insulin and glucose, oxidised phospholipids and low-density lipoproteins. These macrophages display surface markers that are neither representative of typical M1 or M2 macrophages and give rise to a population of metabolic activated (MMe) and oxidised (Mox) macrophages [41, 42] (**Figure 2**). Both these macrophage phenotypes are associated with a state of IR. MMe macrophages cellsurface markers express ABCA1, CD36 and PLIN2 and are involved in the clearance of dead adipocytes through lysosomal exocytosis as well as potentiating inflammation. NADPH-oxidase-2 (NOX2) has been identified as a key driver of the functions of MMe macrophages, with *Nox2*-deficient mice displaying attenuated ATM inflammation

#### **Figure 2.**

*Adipose tissue macrophage (ATM) polarisation in lean and obese state. ATM originate from polarisation of 'M0' macrophages. Depending on the stimuli and local environment facilitates the macrophage polarisation. Th1 cytokines, LPS, IFN-ɣ, TNF-α and IL-1β promote a M1 macrophage that is characterised by F4/80+ CD11c+ . In contrast, Th2 cytokines, IL-4 and IL-13 promote an M2 macrophage that is characterised by F4/80<sup>+</sup> CD206+ CD301+ CD11c<sup>−</sup>. Interestingly, in an obese state, the hypoxic environment and metabolic cues such as excess free fatty acids, high insulin and glucose, oxidised phospholipids and low-density lipoprotein, which promotes a metabolic activated (MME) or oxidised (MOX) macrophage. In the obese state, macrophages promote inflammation, IR and tissue damage. In contrast, in the lean state, macrophages promote ant inflammation, insulin sensitivity and tissue repair.*

and improved glucose sensitivity in a model of diet-induced obesity, when compared to WT animals [41]. Mox macrophages are driven by oxidised phospholipids derived from oxidised low density lipoproteins (LDL) [43] and express surface markers Srnx-1 and Txnrd-1 [42]. Mox macrophages have been studied primarily in the context of atherosclerosis, where the oxidation of accumulated LDL leads to enrichment of the tissue with oxidised lipids, causing the polarisation of macrophages towards a phenotype dependent on the transcription factor Nrf2 [43]. However, a recent paper identified that ATM with a Mox phenotype (CX3CR1negF4/80lowTxnrd1+ HO1+ ) are the predominant phenotype present the AT of lean mice, driven by individual oxidised phospholipids in the AT [44]. It will be an interesting further area study to appraise the role of these novel macrophage phenotypes within the context of obesity and IR.

#### **3.3 Regulators of macrophage polarization**

Macrophage polarization has been well studied over the past decade leading to the discovery of several key regulators which orchestrate macrophage polarization, such as the Signal Transducer and Activator of Transcription (STAT) family, interferons, regulators of lipid metabolism, transcription factor families, microRNAs (miRNAs) and long non-coding RNAs [45] (**Figure 3**).

#### *3.3.1 STAT family members*

The Janus Kinase (JAK)/STAT signalling pathway transmits signals from extracellular cytokines into the nucleus. Indeed, JAK/STATs are arguably the most widely studied pathway within the context of macrophage polarisation, with IFNɣ binding

*The Pivotal Role of Macrophages in Metabolic Distress DOI: http://dx.doi.org/10.5772/intechopen.86474*

to its receptor triggering activation of JAK1/2-mediated tyrosine phosphorylation and subsequent dimerization of STAT1 one of the first pathways to M1 polarisation identified [46]. In addition, LPS binding to TLR4 induces autocrine production of IFN-ß that activates the type 1 IFN receptor triggering STAT1 and STAT2 phosphorylation and heterodimerisation [47]. STAT3 has a dichotomous role in macrophage polarisation, it is the key transcriptional regulator in the production of the anti-inflammatory cytokine IL-10, which can drive an anti-inflammatory macrophage phenotype, however, STAT3 can also be activated by IL-6 and IFN-ß, inducing a pro-inflammatory phenotype [48]. Conversely, IL-4 and IL-13 induces M2 macrophage polarisation largely through induction of STAT6 and KLF4 via the dual catalytic activities of MCP-1-induced protein and inducing PPAR-ɣ [49–51].

#### *3.3.2 Interferon regulatory factors*

Interferon regulatory factors (IRFs) are intracellular proteins that regulate immune cell maturation and play a pivotal role in macrophage polarization. Two key and opposing IRFs in macrophage polarisation are IRF4 and IRF5, which directly compete for binding to MyD88 and subsequent transcription factors such as NFκB. Interestingly, IRF5 was shown to promote M1 macrophages, while IRF5 expression was upregulated in obese individuals compared to lean individuals at both the mRNA and protein levels and is negatively associated with insulin sensitivity [52–54]. It was also shown that IRF5 promotes inflammatory macrophage polarization by activating the transcription of IL-12 and repressing IL-10 [55]. Conversely, IRF4 acts as an antagonist of M1 macrophage polarisation, promoting M2 macrophages [53, 54, 56]. In the context of obesity, macrophage-specific knockout of IRF4 resulted in significant IR and an increase in expression of pro-inflammatory genes [57]. Furthermore, IRF6 has also been implicated in

#### **Figure 3.**

*Intracellular signalling of M1 and M2 macrophage polarisation. Respective intracellular key regulators of M1 and M2 macrophage polarisation. Signal transducer and activator of transcription (STAT) family, interferons (IRFs) and microRNA (miRNAs).*

macrophage polarisation, promoting M1 macrophages due to suppression of PPARɣ expression, a critical regulator of M2 macrophages. Overexpression of IRF6 reduced M2 activation, whilst IRF6 knockdown enhanced M2 macrophage activation [58]. The impact of IRFs on macrophage polarisation and plasticity is clearly quite complex and further studies will hopefully provide information on how IRFs function in different microenvironments, for example, in lean versus obese AT.

### *3.3.3 MicroRNAs*

miRNA are short inhibitory non-coding RNAs (~22 nucleotides) that degrade specific mRNA targets or block RNA translation, and have also been implicated in driving macrophage polarisation. Indeed, miR-125b expression was shown to be upregulated in murine macrophages following IFN-ɣ stimulation and was identified to promote M1 macrophage polarisation, while suppressing IRF4, an important M2 transcription factor [59]. Additionally, miR-155 was also shown to promote M1 polarisation with expression in murine macrophages increased upon TLR activation or stimulation with pro-inflammatory cytokines (TNF-α, IFN-β or IFN-ɣ) [45]. Whilst in human macrophages, miR-155 was shown to target IL-13Rα1 and inhibit STAT6 activation, thus inhibiting M2 macrophage polarisation [60]. Additionally, miR-9 enhances M1 macrophage polarisation by suppressing PPARδ [61]. Whilst miR-127 suppresses B-Cell lymphoma protein (Bc6), which promotes M1 polarisation [62]. Conversely, miR-124 promotes M2 polarisation via LPS-induced cytokine production by targeting STAT3 to decrease IL-6 production and reduce TNF-α [63]. Furthermore, miR-132, miR-146a and miR-223 induce M2 macrophages by inhibiting NF-κB [64–66].

### **3.4 Hypoxia and macrophage polarisation**

Of interest in the context of obesity is the potential for hypoxia to influence macrophage polarisation of macrophages (**Figure 2**). Indeed, hypoxic areas in adipose tissue occur in obese individuals when rapid tissue expansion occurs without sufficient accompanying blood flow to these areas. M1 macrophages display high expression levels of Hypoxia-related genes including *Hif1α*, which has been shown to induce a pro-inflammatory phenotype in macrophages via TLR4 activation, involving the PI3K/Akt signalling pathway [67, 68]. TLR4 expression in macrophages was shown to increase in a hypoxic environment [69]. *Hif1α* enhances the transcriptional activity of NF-κB, driving production of pro-inflammatory cytokines and decreasing the induction of immune regulatory mediators [70]. In contrast, M2-like macrophages in obese individuals express *Hif2α* [71]. Indeed, HIF-2α is also upregulated under low oxygen levels [5]. It was shown that HIF-2α overexpressing macrophages suppressed pro-inflammatory responses and improved IR. Whilst knockdown of HIF-2α in macrophages induced pro-inflammatory gene expression in adipocytes [71]. Thus, it was suggested that HIF-2α counteracts the pro-inflammatory responses to relieve obesity induced IR in AT [71].

#### **3.5 IR and macrophage polarisation**

Insulin acts in the adipose tissue to promote uptake and storage of fatty acids, stored as triglycerides, and inhibits the lipolysis of stored triglycerides. IR is a reduced response to insulin in the liver, muscle and AT, due to an impairment in the insulin-signalling pathway, leading to hyperglycaemia [72]. In obesity, the increased recruitment of macrophages to AT positively correlates with IR [4]. Indeed, obese mice show increased macrophage-mediated inflammation that resulted in

#### *The Pivotal Role of Macrophages in Metabolic Distress DOI: http://dx.doi.org/10.5772/intechopen.86474*

long-term IR [73], with elevated levels of FFA which activated CD36 expressing macrophages and adipocytes, inducing inflammation and impairing insulin signalling [74]. In AT from lean states, the presence of M2 macrophages maintains insulin sensitivity via anti-inflammatory actions of IL-10 and STAT3 [25]. In obese individuals, there is a change in adipocyte metabolism and gene expression. Consequently, there is increased lipolysis and release of FFA which can lead to TLR4 activation of M1 macrophages [75]. Indeed, TLR4 expression is increased on macrophages in obese individuals. In co-culture studies, it was shown that macrophage activation through TLR4 signalling increased secretion of pro-inflammatory cytokines, blocking the insulin signalling cascade [76, 77]. In obese individuals, the IκB kinase (IKK) complex is activated in macrophages resulting in phosphorylation of IκBα on Ser32 and 36, degrading IκBα and allowing NF-κB to translocate to the nucleus and upregulated target genes such as pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 [78].

Increased expression of pro-inflammatory cytokines negatively affects insulin signalling pathways [79]. The effect of these cytokines on IR are seen locally in AT, but also systemically as they are released into circulation [76]. TNF-α phosphorylates insulin receptor substrates (IRS), consequently preventing downstream signalling via inhibiting IKK, c-Jun n-terminal kinase (JNK) and atypical protein kinase C (aPKC) [80]. Interestingly, males have been shown to have increased TNF-α plasma concentration compared to females, leading to the suggestion that obese males are more susceptible to develop IR [81]. IL-6 is increased in the serum of obese individuals and mice, with weight loss reducing circulating IL-6, which improves insulin sensitivity [82]. Systemic IR has also observed during pregnancy, puberty and during infection such as sepsis driven by TNF-α and IL-6 [83]. It is clear from such studies that the presence of pro-inflammatory cells and the release of pro-inflammatory cytokines lead to a loss of sensitivity to insulin and a state of IR. This is possibly the biggest incentive to therapeutically target the macrophages in cases of metabolic disease.

## **4. The metabolic signature of macrophages**

Metabolism is a series of highly interconnected pathways that generate metabolic products such as energy and macromolecules from nutrients in the microenvironment. Whilst the metabolic pathways are plastic, cells, in particular macrophages, tend to utilise a pathway that suits their immediate energy requirements. M1 macrophages have huge metabolic demands, and rely largely on glycolysis, conversely, M2 macrophages meet their energy requirements using oxidative phosphorylation (OXPHOS) pathways. During glycolysis, extracellular glucose is taken up by the cell and converted to two molecules of pyruvate and ATP; NAD+ is converted to NADH<sup>+</sup> H+ regenerated through the breakdown of pyruvate to lactate. Glycolysis also provides the first molecule in the pentose-phosphate pathway, glucose-6-phosphate, which provides NADPH to maintain the cellular redox balance and the production of fatty acids. In M1 macrophages, the increased glucose consumption is associated with the capacity for rapid cytokine production and antimicrobial activity through ROS generation [84, 85]. Conversely, in the presence of oxygen cells produce ATP via the electron transport chain (ETC), which is linked to the tricarboxylic acid (TCA) cycle. The TCA cycle uses carbon sources, such as Acetyl CoA, glutamine or fatty acids to fuel a cycle which generates the reducing agents NADH and FADH2 that serve as electron carriers for the ETC for OXPHOS. Glycolysis is a poor producer of energy, with only two molecules of ATP per glucose molecule, compared to 36 molecules produced by OXPHOS. However,

the use of each pathway will depend on the environment and functional requirements of the cells, as glycolysis provides energy rapidly.

The TCA cycle is truncated in M1 macrophages, resulting in a reduced production of alpha-ketoglutarate (α-KG) and accumulation of citrate and succinate metabolites [86]. The accumulation of citrate leads to the production of the macrophage specific metabolite itaconic acid, which is a major feature of LPS stimulated macrophages [86]. The build-up of itaconic acid has been identified as a driver for succinate accumulation, through its ability to inhibit succinate dehydrogenase [87, 88]. The excess succinate leads to the induction of IL-1β through the stabilization of HIF-1a further enhancing inflammation in the macrophages [89]. Indeed, blocking glycolysis reduces release of CCL2 from TNF-a or LPS stimulated adipocytes, providing further evidence for a link between metabolism and inflammation [90] (**Figure 4**).

Macrophage polarization is also influenced by the metabolism of arginine. M1 macrophages upregulated nitric oxide synthase (iNOS), which catabolize arginine to citrulline and nitric oxide (NO). This NO is important for intracellular killing of pathogens. In addition, M1 macrophages use the pentose phosphate pathway, which generates NADPH for the NADPH oxidase, which produces ROS and NO. Consequently, these metabolic pathways provide M1 macrophages with rapid energy. Conversely, in M2 macrophages, arginase-1 (Arg1) is induced which produces urea, ornithine and polyamines which are key in tissue repair [36, 91].

The metabolic profile of ATM alters dependent on the microenvironment of the AT. In mice, transcriptome and extracellular flux analysis have shown that in lean AT fatty acid oxidation, glycolysis and glutaminolysis all participate in cytokine release by ATM [92]. In obese AT, both glycolysis and OXPHOS are utilised, however glycolysis takes precedence, potentially due to the hypoxic environment in the AT [92].

#### **Figure 4.**

*Immunometabolic differences in M1 and M2 macrophages. M2 macrophages use OXPHOS and the TCA cycle to produce energy. They have increased ability to uptake free fatty acid (FFA) and fatty acid oxidation (FAO) to facilitate the TCA cycle. In contrast, M1 macrophages increase energy production oxidative glycolysis. M1 macrophages also have a 'broken' TCA cycle, resulting in accumulate of citrate and succinate. Increased succinate results in secretion of pro-inflammatory cytokine IL-1β via HIF-1α. TCA, tricarboxylic acid; HIF-1a, hypoxia-inducible factor-1a; a-KG, alpha-ketoglutarate.*
