**1. Introduction**

Macrophages are innate immune cells derived either from blood-circulating monocytes or tissue-resident persistent embryonic stem cells [1]. Monocytes are hematopoietic stem cells formed in adult bone marrow and once mature are released into systemic circulation from where they go into different body tissues [2]. Once in the tissue, monocytes differentiate into and become macrophages [2]. Depending on the body tissue invaded, monocyte-derived macrophages function primarily to add onto the pool of total tissue macrophages during an immune response [3]. Contrary, during embryogenesis, macrophage stem cells and monocyte progenitor cells remain localized within body tissues permanently till adulthood. These cells are capable of self-renewal and in adults result in formation of resident macrophages i.e. the progenitor cells of embryonic origin directly differentiate into tissue-specific resident

macrophage with signature molecules explicit for a given tissue [4]. Primarily, they serve homeostatic functions within tissues such as iron metabolism, removal of dead apoptotic cellular debris, synthesis of surfactant etc. [5]. They, however, are also involved in immune response against foreign particles in conjunction with monocytederived macrophages. In fact, during an immune response in a particular tissue, tissue-specific macrophages are the first type of macrophages to respond. As their population diminishes within the tissue, monocyte-derived macrophages migrate into the tissue to add to the pool and also fight the invading foreign particle.

In general, macrophages are phagocytic cells that function as antigen processing cells during a foreign invasion [6]. They use pattern recognition receptors (PRRs) to detect pathogen associated molecular patterns (PAMPs) in pathogens and damage associated molecular patterns (DAMPs) in damaged cells [7]. These patterns constitute molecules express on cellular surface that denote cellular damage or presence of a pathogenic microbe etc. Once detected, macrophages engulf the supposed recognized particle through a cell eating process termed phagocytosis [8]. The foreign materials are then digested in a sac-like organelle called the phagolysosomes into smaller particles [9]. These breakdown particles are then expressed on surface of macrophage membrane together with major histocompatibility molecules (MHC) for lymphocytes to recognized and trigger cytotoxic and humoral effects [10].

Although the functioning of macrophages seems straightforward, it is rather a complex process of active immune response. Presence of foreign particles within tissue surroundings alters the tissue microenvironment [11]. This microenvironment acts as a signaling pathway for activating of naïve macrophages [11]. Macrophages can either be polarized to be pro-inflammatory or anti-inflammatory in nature. Classically activated macrophages are pro-inflammatory in nature and function to eradicate foreign particle [12]. They are also known as M1 macrophages. Alternatively activated macrophages or M2 macrophages are anti-inflammatory in nature and are recruited during resolution of inflammation when invader has been eradicated to promote tissue healing and removal of debris [12].

Altogether, the entire process from activation, polarization, functioning and return to homeostatic steady-state, the macrophage cell is regulated by the microenvironment surrounding it. This consist of signaling molecules and metabolites, either intermediate or finished, that influence the working of the cell. While immune signaling molecules such as cytokines is discussed in detailed in the field of immunology, the latter has been recently recognized and its crucial role in chronic inflammation associated with specific chronic diseases; especially those inflammatory in nature. Noteworthy, it has led to emergence of the field of immunometabolism which focuses on how metabolism affects functioning of immune components. This chapter reviews current knowledge of how metabolic processes and metabolites influences functioning of macrophages.

### **2. Immunometabolic processes of macrophages**

The term metabolism sums up chemical processes that occur within the body of a living organism to maintain life. The basic unit of life, which is a cell, utilizes cellular processes to break down large organic compounds into energy depending on their biogenetics. Metabolism can be viewed as either cellular or systemic. Cellular metabolism occurs within a cell while the latter depicts metabolism in which specific organs/ tissues are regarded as producers of metabolites which are then utilized by consumer organs/tissues. For instance, the liver metabolizes much of carbohydrate and iron

*Immunometabolic Processes of Macrophages in Disease States DOI: http://dx.doi.org/10.5772/intechopen.109936*

to form metabolites e.g. glucose that other organs such as the brain and bone tissue utilize for their own cellular processes. However, the general outlook of metabolism predominates around cellular metabolism and for this review; five metabolic pathways will be discussed in relation to influence on macrophage function. These are: glycolysis, pentose phosphate pathway (PPP), Tri-carboxylic acid cycle (TCA), amino acid (AA) and fatty acid (FA) metabolism.

In terms of macrophage activation, hypoxia and danger signals induce HIF1-α which that stimulates the glycolytic pathway [13]. This leads to rapid generation of ATP and macrophage polarization to M1 phenotype. Contrary, helminths stimulate interleukin 4 release which in addition to normoxia activates the TCA and electron transport chain pathways typical of M2 macrophages [14].

#### **2.1 Glycolysis**

Glycolysis defines the breakdown of glucose into pyruvate. The process occurs within the cytosol. Glucose is a monosaccharide which represents the unit molecule for carbohydrate compounds. Glycolysis can occur under either aerobic or anaerobic conditions. In aerobic conditions, pyruvate is converted to acetyl CoA which enters the TCA cycle or FA synthesis [15]. In anaerobic conditions, pyruvate is converted to lactate which is removed from cells and taken to the liver to be converted back to pyruvate for gluconeogenesis. Glycolysis is a 10-step process that results in a net amount of 2-ATP molecules being formed per unit of glucose [15]. Additionally, NAD+ is reduced to NADH which is used as a cofactor in various anabolic conditions [16].

During an immune response, naïve macrophages are polarized to the M1 subset due to changes in the microenvironment. In particular, when macrophages are stimulated by lipopolysaccharide (LPS) and interferon γ (IFN-γ), expression of glucose transporter 1 (GLUT1) increases significantly unlike that of succinate dehydrogenase A (SDHA), which is a component of electron transport chain [17]. High levels of intracellular glucose activates the glycolytic process which has been seen to be significantly ongoing during infectious conditions and carcinogenesis [18]. Inhibition of hexokinase using 2-deoxy-D-glucose, the first glycolytic enzyme that phosphorylates glucose leads to a reduction in the amount of TNF-α and interleukin-12 (IL-12) produced [19]. Notably, the cytokines stated previous mediate pro-inflammatory reaction. In addition, hexokinase inhibition also reduces expression of CD80, CD86 and inducible nitric oxide syntheses (iNOS) which are co-stimulatory molecules in macrophages and marker of M1 differentiation respectively [20, 21]. Hexokinase 1 also functions as a regulator of NOD-LRR and pyrin domain-containing 3 (NLRP3) found in the outer membrane of mitochondria [22]. NLRP3 functions to regulate activity of caspase 1 which is involved in generation of active and mature IL-18 and -1β respectively. Increased glycolytic activity activates hexokinase 1 which in turn activates NLRP3 to induce cell death via pyroptosis in macrophages [22].

Noteworthy is the effect of isomeric forms of pyruvate kinase on macrophage polarization. Pyruvate kinase mediates last step of glycolysis by converting intermediate metabolite- phosphoenolpyruvate to pyruvate with concomitant generation of ATP. The enzyme exists as two isoforms either as pyruvate kinase M1 (PKM1) or M2 (PKM2) [23]. Macrophage stimulation by LPS induces expression of PKM2 which is associated with slowing the rate of pyruvate formation unlike PKM1 [24]. This allows glycolytic intermediate metabolites to accumulate and divert to other pathways. For instance, glucose-6-phosphate is diverted to the PPP pathway to generate ribose for nucleotide synthesis while 3-phosphoglycerate is shunted to serine synthesis process.

In addition, the PKM2 enzyme translocate into the nucleus where it interacts with HIF1-α and induce expression of HIF1-α genes [24]. These genes are responsible for expression of inflammatory proteins such as IL-1β and glycolytic enzymes [23]. At times however, the PKM2 enzyme is stabilized in the tetrameric form rather than dimeric. This inhibits its translocation into the nucleus [24]. The enzyme then becomes more concentrated in cytosol and predominantly participate in glycolysis reducing its immune-related function.

Ultimately, interference with glycolytic process in macrophages during an immune response reduces their pro-inflammatory activity. This is because, M1 macrophages depend largely on glycolysis for energy production and subsequent use of such energy for survival and synthesis of mediator molecules [25]. Furthermore, it would seem that in organisms with dysfunctional glycolytic process, a chronic inflammatory process is likely to ensue with fibrotic resolution and formation of granulomas; as the pathogen is not sufficiently eradicated.

#### **2.2 Pentose phosphate pathway**

The pentose phosphate pathway is a shunt from the glycolytic process occurring in the cytosol and leads to formation of pentoses and a major contributor of total NADPH produced in the human body. The pathway is branched into oxidative branch leading to generation of NADPH and non-oxidative branch leading to formation of pentoses [26]. Pentose sugars are ultimately utilized in the synthesis of nucleotides and amino acids. Pentose phosphate pathway starts with glucose-6-phosphate branching off from glycolytic process to form 6-phosphogluconolactone that is oxidized via intermediates to form ribulose-5-phosphate and NADPH molecules. Ribulose-5-phosphate in turn is converted to ribose- and xylulose-5-phosphate. These metabolites can be further metabolizes into intermediate molecules that enter the glycolytic pathway.

Macrophages and neutrophils are phagocytic in nature and during phagocytosis induce a respiratory burst that synthesizes reactive oxygen species (ROS) which oxidizes and interfere with integrity of biological structures of a pathogen. Macrophages utilize NADPH oxidase to synthesize ROS from NADPH [27]. Additionally, NADPH is used to generate glutathione which is an antioxidant that minimizes oxidative stress subjected to tissues [28]. Macrophage activation via LPS increases activity of the PPP shunt [29]. Noteworthy, the enzyme carbohydrate kinase-like protein (CARKL) which is a sedoheptulose kinase is critical in macrophage polarization. The enzyme phosphorylates sedoheptulose to sedoheptulose-7-phosphate which is an intermediate metabolite in the PPP pathway. This reaction is coupled to conversion of glyceraldehyde-3-phosphate into the non-oxidative branch of PPP. LPS activation of macrophages represses expression of CARKL genes leading to the intermediate product being used to synthesized pentose phosphates [30]. It remains unclear why such a regulatory activity occurs in M1 macrophages which for the larger part are not proliferative in nature and would not require much of the nucleotides. On the other hand, IL-4 activated macrophages (M2 polarization), show enhanced expression of the CARKL genes thus the enzyme's catalyzed reactions increase in M2 macrophages [29].

#### **2.3 Tri-carboxylic cycle**

The TCA cycle or commonly known as Krebs cycle is an energy efficient mode of energy generation in form of ATP. Comparatively, from a single glucose molecule, glycolysis yields two ATP molecules while Krebs cycle yields a total of 36 ATP molecules. The process occurs within the mitochondrion and is commonly utilized by nonproliferative cells for energy generation. Krebs cycle has multiple input points notably via acetyl CoA and α-ketoglutarate (α-KG). Acetyl CoA together with oxaloacetate undergoes aldol condensation to form citrate while glutamate is converted to α-KG and altogether join the TCA. Ultimately, the cycle is meant to generate NADH and FADH2 by sequentially reducing the carbon atoms from acetyl CoA. Formed NADH and FADH2 enters ETC to generate ATP molecules.

M2 macrophages utilizes more or less the intact TCA cycle for energy production and further allows intermediates of UDP-GlcNAc to be generated which are utilized for glycosylation of M2-associated proteins e.g. mannose receptors [31]. Contrary, polarization of macrophages to the M1-phenotype is associated with break points in the intracellular TCA cycle [32]. First, conversion of citrate to isocitrate and secondly succinate to fumarate. This results in accumulation of citrate and succinate [32]. Citrate molecules are then directed to synthesis of fatty acid and itaconate and nitric oxide formation while the latter activates HIF1-α and production of IL-1β [33].

The first breakpoint occurs during the third reaction of Krebs cycle where isocitrate is converted to α-KG by the enzyme isocitrate dehydrogenase (IDH). M1 polarized macrophages transcriptional represses levels of IDH mRNA responsible for synthesis of IDH1 [34]. This leads to accumulation of isocitrate which isomerizes back to citrate via a two-step reaction process: isocitrate is first dehydrated to cisaconitate which is then rehydrated to citrate. Ultimately, it is citrate and cis-aconitate that accumulates within mitochondrial matrix. As a result, cis-aconitate is diverted to synthesis of itaconate through decarboxylation reaction catalyzed by the immuneresponsive gene 1 (IRG1). Itaconate is bactericidal in nature especially against *Salmonella typhimurium* and *Mycobacterium tuberculosis* [35]. Additionally, itaconate inhibits succinyl dehydrogenase (SDH) [36] which converts succinate to fumarate, the second break point in TCA cycle. Itaconate also alkylates the Kelch-like ECHassociated protein 1 which subsequently activates nuclear factor erythroid 2-related factor 2 (NRF2) that has anti-inflammatory activity [37].

Accumulation of citrate in M1 macrophages inhibits pyruvate dehydrogenase (PDH) and SDH ultimately decreasing formation of Acetyl CoA and FADH2 respectively. This additionally promotes consumption of ATP [38]. Accumulated citrate is transported from mitochondria matrix to cytosol via citrate transporter. Expression of citrate transporter is increased in proinflammatory macrophages in a NF-KB-dependent fashion [29]. Exported mitochondrial citrate is catabolized into products utilized in synthesis of inflammatory mediators e.g. nitric oxide and prostaglandins (PGE2) [29]. In M2 macrophages, since the TCA cycle is intact, much of α-KG is accumulated. α-KG has been shown to be immunosuppressive in nature by: preventing expression of proinflammatory IL-1β, inhibiting stabilization of HIF-1α and inactivating NF-Kβ signaling pathways [39]. Glutamate and glutamine provide precursors for formation of α-KG once deaminated via anaplerosis and will be discussed further under AA metabolism.

Conversion of α-KG to succinyl-CoA is a rate limiting step in the TCA cycle as it is one of the oxidoreductase reactions that result in formation of NADH. The reaction is catalyzed by the enzyme α-KG dehydrogenase (α-KGDH) which is highly sensitive to levels of ROS [40]. LPS activation and cytosolic accumulation of calcium within macrophages enhances activity of α-KGDH and limit production of anti-inflammatory IL-10 [29]. The intermediate product, succinyl-CoA is used to succinylate lysine residues within proteins such as SDH, PDH, Acyl-CoA and carbamoyl phosphate synthase 1 [32]. Notably, in LPS-activated macrophages, a lot of succinyl-CoA is produced which leads to succinylation of lysine 311 present on PKM2 [41–43]. As a result, the enzyme acquires a dimeric form which as earlier discussed facilitates entry of the enzyme into nucleus and consequent association with HIF-1α [41]. However, not all proteins succinylated activates pro-inflammatory activity as some have been shown to be immunosuppressive when succinylated. Formed succinyl-CoA is hydrolyzed by succinyl-CoA synthetase to succinate. Alternatively, succinate can also be produced via the gamma-aminobutyric acid shunt through which glutamine is deaminated to glutamate which is further catabolized to succinic semialdehyde and eventually succinate [17]. The latter product is of special importance in situations of inflammation and metabolic stress where it has been shown to regulate tumorigenesis, cellular inflammatory activity, signal transduction and epigenetics.

The second break occurs during conversion of succinate to fumarate by the enzyme SDH. Notably, the increased accumulation of succinate is not largely depended on SDH inhibition rather on glutamine anaplerosis as discussed above. Increased succinate is exported outside the mitochondria where it stabilizes HIF-1α and activate inflammatory genes leading to sustained production of IL-1β [32]. Within the mitochondrion, oxidation of succinate by SDH drives formation of ROS required during an immune response [44]. Although conversion of succinate to fumarate may be disrupted in M1 macrophages, LPS-activation has been shown to induce the aspartate-arginosuccinate shunt which feeds fumarate precursor molecules and leads to formation of fumarate [45]. Moreover, activation of the above stated shunt has been associated with upregulation of synthesis of NO and IL-6 [46] which are pro-inflammatory. At the same time, excess fumarate levels being formed inhibit pyroptosis increasing formation of gasdermin D [47]. Most likely, intermediates of aspartatearginosuccinate shunt drives pro-inflammatory activities while the final product fumarate is anti-inflammatory in nature. Notably, might be another reason behind the second break that prevents conversion of succinate to fumarate in M1 macrophages.

Overall, metabolites of the TCA cycle on their own and in homeostatic concentrations seems to be immunosuppressive but activity of such metabolites on other pathways within and out of mitochondria most likely activate inflammatory pathways.

#### **2.4 Lipid metabolism**

Lipid metabolism functions to deliver lipid compounds to peripheral tissues and at the same time recycle lipids from peripheral tissues within the liver. It entails three pathways: exogenous, reverse cholesterol transport and endogenous pathways [48]. Dietary lipids are metabolized via exogenous pathway while endogenous pathways metabolize lipids synthesized in the liver. Reverse cholesterol transport pathway describes how cholesterol is removed from body tissues and transported to the liver for recycling since most peripheral tissue are not able to metabolize cholesterol.

Broadly, lipids entails triglycerides (TG), cholesterol and transporting molecules. Dietary lipids absorbed from intestinal lumen are package into chylomicrons and transported via lymphatic system where they become associated with apolipoproteins (ApoB, ApoC-II, -III, ApoE etc.) and enter systemic circulation to be delivered to various body tissues [49]. Apolipoproteins enable different body tissues to identify and uptake lipids; lipids bound to ApoC-II are recognized by adipose tissue which upon cellular entry are hydrolyzed to free fatty acids (FFA) and chylomicron remnants by lipoprotein lipase (LPL). Similarly, endogenously synthesized TGs and cholesterol get packaged into very low-density lipoprotein (VLDL) and ApoB respectively and then transported to body tissues. Adipocyte LDL hydrolyzes VLDL-bound TGs to FFAs and IDLs (VLDL remnants) [48]. The lipid transporting remnants are

#### *Immunometabolic Processes of Macrophages in Disease States DOI: http://dx.doi.org/10.5772/intechopen.109936*

transported to the liver where hepatic lipases convert IDLs to low density lipoproteins (LDLs) which transport cholesterol across body tissues. In conditions such as chronic high fat intake where LDL is secreted in high amounts, excess LDL binds to free ApoA and resulting compound binds extracellular matrix within walls of vessels [50]. This has been implicated in the pathogenesis of atherosclerosis.

Macrophages are well fashioned to metabolize lipids especially liver, lung and adipose macrophages. They readily absorb and release lipoproteins and cholesterol respectively from dying cells. Lipoproteins, LDL and VLDL are absorbed either via phagocytosis, micropinocytosis or scavenger receptors such as CD36 and digested in the lysosomes by action of lysosomal acid lipase to form free FAs or cholesterol molecules [48]. Cytosolic esterification of free cholesterol to esters results in formation of lipid droplets that makes macrophages look like foam cells [51]. Additionally, free cytosolic cholesterol activates transcription factors such as peroxisome proliferator-activated receptor (PPAR), liver X receptor (LXR) and retinoid X receptor (RXR) [52]. The latter two receptors regulate lipogenesis in a more complex manner and in terms of macrophage function, their deficiency increases susceptibility to infections by *Listeria monocytogenes* [53] and *mycobacterium tuberculosis* [54] but at the same time confers protection from leishmania infections [55]. Nevertheless, binding of LXR by agonists inhibits expression of inflammatory genes that lead to synthesis of NO, PG and IL-6 [56].

Formed FFA on the other hand, enters mitochondria where it is oxidized to generate acetyl CoA and the reducing agents FADH2 and NADH. This latter process is largely seen in M2 polarized macrophages where lipolysis is registered but also occurs in M1 macrophages [57]. Blocking of the fatty acid oxidation pathway, as seen by use of etomoxir which inhibits carnitine palmitoyl-transferase 1, leads to inhibition of M2 macrophages polarization [58]. Additionally, formation of reducing agents FADH2 and NADH enables metabolism within macrophages to take an oxidative shift and by so doing activates PPAR-γ which activates expression of M2 signature genes. Activated PPAR-γ also enhances oxidation of glutamine within via anaplerosis which activates the TCA cycle [57]. In M1 macrophages on the other hand, fatty acid synthesis (FAS) is a more predominantly seen feature when macrophages are activated by TLR, LPS or IFN-γ [59]. Fatty acid synthesis ultimately leads to lipid formation which are necessary for macrophage cell membrane expansion during remodeling. Additionally, activation of FAS pathway induces the NLRP3 inflammasome with subsequent secretion of IL-1β [60].

#### **2.5 Amino acid metabolism**

Amino acids are essential building blocks for proteins that constitutively have an amino and carboxyl group attached to a central carbon. In mammals, 20 amino acids are utilized in protein synthesis out of which 9 are essential while the rest are non-essential. Noteworthy, there exist more than 20 amino acids but consensually, the 20 essential and non-essential amino acids are mostly described in biochemistry for cell function. The human body cannot synthesize essential amino acids and have to be supplied by dietary intake unlike non-essential amino acids [61]. They include valine, phenylalanine, lysine, methionine, tryptophan, isoleucine, leucine and histidine. Contrary, non-essential amino acids include arginine, tyrosine, alanine, serine, aspartate, glycine, asparagine, proline, glutamine and glutamate [61]. The role of amino acids and their metabolites during immune response is quite complex, varied and still an enigma. Of importance in macrophage functioning is tryptophan, arginine, serine, methionine and glutamine metabolism.

Tryptophan can either be metabolized via kynurenine or serotonin pathway [62]. Kynurenine pathway is key for de novo synthesis of NAD+ which is essential during

redox reactions in glycolytic, TCA, fatty acid and electron transport chain pathways [63]. Contrary, serotonin pathway leads to formation of serotonin, a neurotransmitter, via action of tryptophan hydroxylase and aromatic amino acid decarboxylase [62]. Tryptophan can also be metabolized to indole-pyruvate that is skewed into TCA via anaplerosis. Kynurenine pathway starts with conversion of tryptophan to N-formylkynurenine mediated by indoleamine-2,3-dioxigenases (IDO) especially IDO1. Presences of infectious agents, TNF-α and IFN-γ stimulates expression of IDO genes which upregulates enzyme expression and tipping of tryptophan metabolism to the kynurenine pathway [64]. This additionally denies pathogens growth substrates within body tissues. In macrophages, serotonin formed from tryptophan is used to synthesize melatonin by action of N-acetyltransferase and O-methyltransferase. The product, melatonin has regulatory functions in terms of cytokine production by macrophages [65].

Serine and methionine are important during one-carbon metabolism in which methylation reactions occurs [62]. Serine, obtained exogenously or endogenously from 3-phosphoglycerate, donates one carbon atom to the folate and methionine cycles whereby in the latter, methionine acts as an intermediate donor in form of S-adenosylmethionine (SAM) thus not consumed. Macrophages utilize SAM to methylate histone molecules and synthesized IL-1beta in M1 phenotypes [66]. Additionally, LPS-activated macrophages utilize serine as a precursor molecule for synthesis of glycine which is subsequently used for glutathione synthesis. This enables rapid provision of the antioxidant as the NRF2-driven pathways generates glutathione molecules slowly [67].

Arginine can be catabolized either via arginase pathway or the nitric oxide synthesis pathway [62]. TNF-α and IFN-γ upregulate expression of inducible NO synthase (iNOS) in M1 macrophages to catabolize arginine into NO [68]. The enzyme iNOS mediates conversion of arginine to citrulline with concomitant production of NO which is an inflammatory mediator [68]. Upregulation of arginase enzyme, especially arginase 1, has been noted in macrophages stimulated by IL-4, -5 and -13 which are signature activators for naïve macrophages to M2 phenotype [69]. The enzyme converts arginine to ornithine limiting available substrate for iNOS within the cell. Ornithine is an important source of polyamines such as spermine which inhibits mitochondrial respiration and synthesis of pro-inflammatory cytokines [10].

In macrophages, glutamine is an important amino acid especially in M2 macrophages where it serves as a fuel source via anaplerotic processes [62]. In response to stimulation by IL-4, macrophages upregulate glutaminolysis to produce α-KG which mediates epigenetic reprogramming. Additionally, glutamine together with glycine and cysteine are used to synthesize glutathione which is a potent intracellular antioxidant. Glutamine is also a substrate for arginine biosynthesis [67] which is important for generation of NO in M1 macrophages.

Branched chain amino acids (leucine, isoleucine and valine) are major sources of carbon, and generation of glutamine, acetyl- and succinyl-CoA [67]. Increased uptake of leucine by macrophages activates the mTORC1 pathway leading to increased production of TNF-α and IL-1β in M1 macrophages [67].
