Perspective Chapter: Macrophages Plasticity and Immune Metabolism

Abstract

Macrophages are phagocytic cells that reside within body tissues. They can either be derived from circulating monocytes or can arise during the embryonic stage of fetal development. Tissue macrophages are predominantly of embryonic origin. But can result from differentiation of circulating monocytes to become resident macrophages either in pathological or physiological state. Macrophages are classified based on their tissue location and method of activation. Classically activated macrophages are the M1 phenotype while alternatively activated macrophages are M2 phenotype. M1 macrophages are pro-inflammatory since they secrete cytokines that attract inflammatory mediators. They are majorly activated by either interferon-gamma or lipopolysaccharide molecules. M2 macrophages are anti-inflammatory and mediate tissue healing and repair. They are activated by cytokines such as interleukin four, ten, and thirteen. The metabolic profiles of these classes of macrophages are intrinsically different and complex yet intertwined. M1 macrophages depend on aerobic glycolysis for energy production while M2 macrophages rely on aerobic fatty acid oxidation pathways. These metabolic pathways optimize macrophage functioning. Regulation of both activation and metabolism depends on transcriptional factors such as STAT 1 and 6, and IRF. Defects in these pathways lead to development of disorders related to macrophage activation and metabolism.

1. Introduction

The immune system is responsible for protection against infections and diseases. It is made up of a complex network of cells and proteins, working in harmony to protect the body against pathogens. White blood cells make up the larger component of this system comprising granulocytes and agranulocytes. Human macrophages can be formed through the differentiation of monocytes. Monocytes are a population of mononuclear leukocytes that are generated in the bone marrow. They constitute approximately 10% of peripheral blood cells in the human body. The monocytes move into the blood from the bone marrow, where they migrate to various body tissues through blood circulation. Once in the tissues, they differentiate into different types of macrophages depending on resident tissue. Some tissue-resident macrophages are non- monocyte-derived. However, their origin, proliferation, self-renewal, and mechanisms of replacement are vaguely well known and will be elaborated on later on in this chapter.

The term “macrophage” is a combination of two Greek words: makros which means “large” and phagein which means “to eat”. They are thus cells that carry out phagocytosis through engulfment and digestion of any pathogen or foreign particle that is opsonized. These include cellular debris, foreign substances, cancer cells, and microbes. Ilya Metchnikoff, a Russian Zoologist was the first to discover macrophages in the nineteenth century (1884) [1]. Van Furth in the 60s proposed that tissue macrophages were effector cells derived from circulating monocytes [1]. Evolutionary, macrophages are conserved phagocytes that have existed for the last 500 million years and belong to the Metazoan phylogeny. Studies in recent years have shown that the majority of resident-tissue macrophages are established at the embryonic stage and persist into adulthood [1]. The function of blood monocytes that get differentiated into macrophages is to replenish the lost pool or aid in situations such as inflammation.

2. Formation and development of macrophages

Macrophages form the major part of the mononuclear phagocytic system. These cells are distributed widely within the body in various organs displaying heterogeneity in terms of their structure and function. Their sizes range between 15 to 50 microns with an ovoid nucleus of about 6 to 12 microns [2]. The cytoplasm is granulated with vacuoles located towards the periphery of the cell. The granules are finely divided especially towards the periphery of the cytoplasm and azurophilic in nature [1]. Macrophages can either arise from the differentiation of recruited macrophages or early in life through macrophage progenitor cells of embryonic origin.

2.1 Blood derived macrophages

Macrophages can be derived from blood monocytes that have migrated from the circulatory system into the tissues. Monocytes are white blood cells formed through hematopoiesis in the bone marrow. They form approximately 10% of the white blood cell composition circulating within the blood, the spleen, and the bone marrow. Structurally, monocytes are irregularly shaped with a kidney-shaped nucleus besides a high ratio of cytoplasm to the nucleus.

Precursor cells for monocytes arise from the bone marrow in a series of steps activated by cytokines and stimulating factors. Sequentially four stages are involved in the formation and development of macrophages i.e. hematopoietic stem cells develop into the common myeloid progenitor cells which then differentiate into granulocyte-macrophage progenitor cells [3]. The latter group of cells differentiates into the common macrophage and dendritic cell precursor before finally committing to the committed monocyte progenitor group. The newly formed monocytes stay within the bone marrow for the first 24 hours before being recruited into circulation [3]. The mature monocyte remains in circulation for about 1 to 2 days before translocating into the tissues. If after that period they have not translocated, the circulating monocytes die and are removed from the blood circulation.

Sequential formation of these cells is governed by cytokines and stimuli factors resulting in either expression of new or loss of specific surface proteins. Colony-stimulating factor 1 (CSF-1) (also known as monocyte colony-stimulating factor- M-CSF) largely influences the development of monocytes under normal healthy hemostatic conditions. The factor is secreted by bone marrow stromal cells and also in tissues. CSF-1 is removed from circulation by the mononuclear phagocytes reducing hematopoiesis of monocytes. Interleukin 34 (IL-34) found in the central nervous system and the epidermal tissues is responsible for the proliferation of macrophages from monocytes within that specific tissue type. Interleukin 1 (IL-1) and tumor necrosis factor (TNF) on the other hand induce endothelial cells and fibroblast to secrete CSF-1 and CSF-2 [4]. During inflammatory periods, the formation and development of monocytes till their differentiation into macrophages is governed by the granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF-2) and interleukin 3 [4].

Mature monocytes differ in terms of expression of CD16 and CD14 molecules [3, 4]. Classical monocytes express a high CD14 but lack expressed CD 16. They form the major portion of the total monocytes (approx. 90%). Intermediate monocytes lowly express CD16 molecules but high CD14 expression. Alternative monocytes highly express CD16 molecules but CD14 in low amounts. Classical and intermediate monocytes are inflammatory mediators highly expressing the chemokine receptor CCR2 while the alternative monocytes function to patrol the circulatory system for foreign bodies and express the chemokine receptor CX3CR1 [3].

Monocytes that migrate into tissues from the circulatory system differentiate to macrophages. Translocation of monocytes to tissue spaces involves endothelium adherence, diapedesis in between the endothelium, and subsequent migration through the sub-endothelial layer into interstitial space. Monocytes express ligands e.g.CD11a/CD18 (lymphocyte function-associated antigen 1) that bind receptors e.g.CD54 (intercellular adhesion molecule-1) located on the surface of endothelial cells [3]. Interleukin 1 and interferon-gamma (IFN-γ) enhance the expression of CD54 further increasing the margination of monocytes. Upon arrival at the target site, these monocytes differentiate into macrophages. Differentiation largely affects cellular respiration and phagocytic activity. Mitochondria increase in number and their enzymes also show an enhanced rate of activity which ultimately leads to an increased rate of respiration. In addition, lysosomes and their enzymatic activity also increase.

2.2 Macrophages derived from embryonic tissue

Macrophages can also arise from progenitor cells of embryonic origin. These cells are found in almost all tissue types forming approximately 12% of the tissue cells [4]. Depending on the tissues, the macrophages are named differently and epigenetics studies illustrate each of these different tissue macrophages have different transcriptional profiles but all perform similar functions [1, 4]. Their gene expression profile is different from that expressed by bone-marrow-derived macrophages. Primitive hematopoiesis that occurs in the ectoderm of the yolk sac during embryogenesis results in the formation of macrophages that do not follow the monocytic system of development. During subsequent fetal development, definitive hematopoiesis occurs in the fetal liver to give rise to hematopoietic progenitors and stem cells. These cells afterward colonize the spleen and bone marrow taking residency. In adults, the bone marrow becomes the major site of hematopoiesis.

Adult tissue macrophages that were formed from the progenitor cells in the yolk sac and fetal liver have the capacity for self-renewal. CSF-1 and IL-34 still mediate activation and proliferation of these progenitor cells into macrophages depending on the transcription factor PU.1 [1]. These cells upon stimulation directly convert into macrophages. As such, the pool of tissue macrophages is minimally dependent on the translocation of monocytes during steady-state conditions. Macrophages irrespective of origin have to be activated before they can function properly. Macrophages can be activated classically or alternatively.

3. Activation of macrophages

Activation of macrophages comprises an enhanced cellular metabolism, lysosomal activity, mobility, and cytotoxic activity. Activation of macrophages requires a prior recognition of pathogens. Macrophages express an array of receptors that do not normally occur in a healthy cell. These receptors are generally termed pattern recognition receptors (PRRs). They include the toll-like receptors (TLRs), clathrin receptors (CLRs), NLRs, scavenger receptors, and, retinoic acid-inducible gene 1-like helicase receptors. These receptors receive a signal from the damage-associated molecular patterns (DAMPS) and pathogen-associated molecular patterns (PAMPS) present on damaged cells and invading pathogens respectively [5]. PRRs such as mannose receptors are crucial for the process of pathogen binding and phagocytosis. NLRs, TLRs, and RLRs on the other hand are key for the recognition of microbial by-products expressed on other cell surfaces. Though there exists a variety of the PRRs, upon stimulation, the downstream mechanisms result in activation of transcriptional pathways involving mitogen-activated protein kinases, interferon regulatory factors (IRF), and nuclear Factor-kappa B (NF-Κb) [5]. These pathways ultimately lead to the expression and secretion of specific cytokines, phagocytosis, chemokine secretion, cellular activation, and secretion of inflammatory mediators.

Macrophages are activated through two pathways depending on their polarization. Microphage polarization refers to the ability of macrophages to differentiate into functionally different phenotypes [5]. Activation through polarization enables macrophages to optimize the cell to perform a particular function. Macrophages can be polarized to result in the formation of two distinct groups of cells: M1 macrophages and M2 macrophages [6]. The latter is further divided into M2a, b, and c types. M1 macrophages are classically activated while M2 cells are alternatively activated. This classification is based on the metabolic functions of macrophages that can broadly be classified as either pro-inflammatory in nature or anti-inflammatory in nature.

Metabolism of arginine in M1 cells results in the formation of citrulline and nitric oxide. Nitric oxide has a microbicidal activity besides inhibiting cellular proliferation. On the other hand, in M2 macrophages, the metabolism of arginine shifts to result in the formation of polyamines and ornithine. Ornithine enhances cellular proliferation and healing by mediating collagen synthesis, cell repair through polyamines, and fibrosis. By default, resident macrophages are usually polarized to form predominantly M2 macrophages. M1 macrophages are formed in situations where inflammation is warranted such as in cases of infections [7].

3.1 Classical activation

Classical activation of macrophages results in the formation of M1 macrophages which are pro-inflammatory in nature. Ligands such as lipopolysaccharide (LPS) and IFN-γ are key stimulators of macrophages towards the M1 pathway [8]. These ligands stimulate TLRs present on the surface of macrophage membranes resulting in a downstream pathway that leads to the production of chemokines (CXCL2, CCL5, CXCL4, and CCL8), and cytokines (IL-6, TNF-alpha, IL-23, IL-1β, IL-12) [8, 9]. In addition, the pathways activate the expression of inducible nitric oxide synthase (iNOS) leading to the production of nitric oxide. M1 macrophages also express high levels of major histocompatibility molecules II (MHC II) molecules and CD80/86 making them functionally potent antigen-presenting cells (APCs). Six transcriptional factors are activated upon stimulation of TLRs by PAMPS. These are the Signal transducer and activator of transcription 1 (STAT-1), NF-κB, PU.1, IRF5, Activator protein 1 (AP-1), and CCAAT/enhancer-binding protein alpha (C/EBP-α) [5, 8, 9]. NF-kB is activated upon stimulation of macrophages by infectious agents, stress, or inflammatory cytokines. Activated NF-kB activates genes that ultimately lead to the relocation and activation of macrophages in the affected region [10] through modulation of the expression of mediators of inflammation and also macrophage differentiation.

When macrophages are exposed to growth factors, infectious agents, cytokines, apoptotic products, and oncogenic stimuli, the transcription factor AP-1 is activated [9]. This factor reduces angiogenesis in a tumor by reducing the production of reactive oxygen species (ROS). The factor also induces the expression of TNF-α during inflammatory responses and also induces mitogen-activated protein kinases (MAPK) thereby stabilizing mRNA that encodes inflammatory cytokines.

PU.1 is a key transcriptional factor during hematopoiesis. It upregulates the expression of the M-CSF receptor thereby enhancing the proliferation of macrophages [3, 5]. In addition, the factor also modulates the expression of genes responsible for the differentiation of macrophages by regulating the expression of GM-CSF receptors thereby affecting the maturation of such cells. In cases where the factor is inappropriately expressed or deficient, carcinogenesis may occur leading to the development of lymphomas.

3.2 Alternative activation

Activation of macrophages in the direction of M2 polarization is predominantly done by cytokines including IL-4, IL-33, IL-13, and IL-10 [3, 5, 8]. M2 macrophages have anti-inflammatory and tissue repair roles within the body. During tissue injury or in response to fungal and parasitic chitin or as a result of adaptive immune responses, IL-4 is released by cells such as basophil and mast cells as one of the earlier mediators of wound healing. This cytokine binds to its receptors on the surface of macrophages inducing a downstream signaling pathway that results in stimulation of arginase activity. Arginase enzyme converts arginine in macrophages to ornithine which is a precursor for the formation of polyamines and collagen. As a result, the increased levels of ornithine are used to form collagen fibers necessary for the wound healing process [7].

In response to parasitic and fungal chitin, the downstream pathway leads to the activation of chitinase enzymes that aid in the interruption of parasitic or fungal cellular integrity. However, Dysregulated activation of macrophages via this pathway can lead to excessive deposition of extracellular matrix leading to fibrosis [7]. Activated M2 macrophages thus secrete IL-1 antagonist, TGF-β, and IL-10 besides blocking iNOS. IL-4 and -13 induce the development of M2a macrophages responsible for wound healing. Immune complexes formed from microbial ligands binding to TLR activate macrophages to develop into the M2b phenotype. However, such cells in presence of IL-10, glucocorticoids, and TGF-βdifferentiate into M2cphenotypes [5].

Five transcriptional factors enhance the activation of macrophages towards the M2 phenotype. These factors include STAT6, IRF4, Krüppel-like factor 4 (KLF4), C/EBP-β, and peroxisome proliferator-activated receptors (PPAR-γ) [11]. KLF4 is part of the zinc family transcription factors and regulates the development, differentiation, and activation of cells. IRF4 regulates the expression of interferon genes. IRF4 has a pivotal role during the polarization of macrophages towards the M2 phenotype. The binding of IL-4/IL-13 to their receptors induces phosphorylation and subsequent dimerization of STAT6 that in turn recruits IRF4 stimulating expression of M2-associated genes such as mannose receptor and chitinase receptor. In addition, the IL4-STAT6 pathway induces expression of nuclear receptor peroxisome proliferator-activated gamma which inhibits the transcriptional factors AP-1, STAT, and NF-kB. The IL4-STAT6 phosphorylation also promotes KLF4 expression which leads to the expression of the M2 gene.

4. Types of macrophages

Microphages can be classified based on the method of activation or based on the resident tissues where they are located. Though macrophages, in general, have similar functions, they are specialized to perform specific tissue functions [12].

4.1 Classification based on location

Macrophages are given different names depending on the tissue they reside in. Table 1 below shows the different macrophages, their location, and their functions.

Type of macrophage	Residing body tissue	functions
Microglia	Brains (central nervous system)	Development of brain,; Immune surveillance,; Remodeling of brain synapses
Osteoclasts	Bone marrow tissue	Remodeling and resorption of bone tissue.; Hematopoiesis [12]
Heart macrophages	Cardiovascular system	Immune surveillance
Kupffer cells	Liver	Remove toxins; Lipid metabolism; Recycling of iron; Clearance of microbes, debris, and red blood cells.
Alveolar macrophages	Lungs	Clearance of surfactant,; Immune surveillance
Adipose tissue-associated macrophages	Adipose tissue	Lipid metabolism,; Adipogenesis,; Thermogenesis [13]
Bone marrow macrophages	Bone marrow tissue	Reservoir of monocytes waste disposal [12]
Intestinal macrophages	Gut tissue	Development of microbiota tolerance,; Intestinal homeostasis,; Defense against pathogens [12]
Langerhans cells	Skin tissue	Immune surveillance
Marginal zone macrophages	Spleen	Trapping of microbes from blood,; Red blood cell clearance,; Iron processing
Tumor-associated macrophages(TAM)	Tumors	Create an immunosuppressive tumor microenvironment through the production of cytokines, chemokines, and growth factors
CD169^+ macrophages	Lymphoid organs and tissue	involved in immune tolerance; antigen presentation; immune system regulation; erythropoiesis regulation [13]
TCR^+ macrophages		Strong phagocytic activity

Table 1.

Classification of macrophages based on their location.

4.2 Classification based on activation

Microphages can be either activated classically or alternatively. The activation depends on the inducing stimuli. In addition, specific cytokines also activate macrophages as shown in the Table 2.

Type of microphage	Method of activation	Activators
M1 macrophages	Classical activation	IFN-γ from Th1 cells, cytotoxic T lymphocytes, Nk cells.; Lipopolysaccharide [8]
M2 macrophages	Alternative activation	exposure to IL-4, IL-10 and IL-13, CSF-1, TFG-beta; Fungal and helminthic infections [11]

Table 2.

Classification of macrophages based on method of activation.

5. Functions of macrophages

Macrophage’s main role involves maintenance of the integrity of an organism either by direct pathogen elimination or tissue repair under sterile inflammatory conditions.

Macrophages are immune cells tasked with eliminating pathogens within the body and damaged body cells. This they do in various specific ways but in general, will lead to pathogen elimination.

5.1 Immune surveillance

Macrophages recognize PAMPs and DAMPs through their wide array of receptors. As professional phagocytes, they phagocytose pathogenic microorganisms and cellular debris. The process involves phagosome formation through endocytosis. The phagosome is then fused with a lysosome forming phagolysosomes, where the pathogen is enzymatically broken down.

5.2 Induction of inflammation

Macrophages take part in the initiation of inflammatory responses. Once the macrophages have detected possible infectious agents and subsequently bind the PAMPs, they are activated to start producing proinflammatory cytokines. The resident macrophages together with other resident immune cells, such as mast cells, stromal cells, and dendritic cells, initiate an influx of inflammatory leukocytes into the site mainly neutrophils and cytotoxic T lymphocytes.

5.3 Adaptive immunity

Macrophages and dendritic cells are antigen-presenting cells (APCs). Damaged cells and pathogens, once ingested are broken down into smaller molecules. These molecules are complexed with MHC molecules and expressed on the surface of macrophages. The expressed molecules act as antigens that stimulate lymphocytes. Lymphocytes recognize the antigens, become activated, and proliferate to effector cells that eliminate the pathogen.

5.4 Wound healing

Functions of M2 macrophages predominantly involve wound repair and anti-inflammatory action. Monocytes from the bloodstream are recruited to a wounded tissue by growth factors secreted by platelets and other cells at the site and then mature into macrophages. They degrade pathogen or cellular debris available to clear them from the tissue. They also secrete growth factors and cytokines that attract fibroblast involved in healing. The low oxygen concentration of the wounded tissue surroundings stimulates macrophages to secrete cytokines that induce and speed up angiogenesis. They also stimulate granulation of affected tissue by laying down new extra-cellular matrices.

5.5 Tissue homeostasis

Tissue-resident macrophages are specialized both physically and functionally to the role they play in the specific tissues they reside. The resident macrophages are non-migratory residing permanently at the tissue they are adapted to function. They support the physiological function of the tissue by providing essential growth factors. They also actively protect the tissue from inflammatory damage.

5.6 Iron homeostasis

Red blood cells live up to 120 days after which they are destroyed and removed from the circulation in the spleen and liver. The macrophages carrying out this role can engulf macromolecules. They play a role in the control of the distribution of parenteral irons. Iron released from destroyed erythrocytes is stored internally in ferritin or released into circulation via ferroportin. During inflammation, the elevated level of hepcidin leads to the retention of iron in the macrophages. This is through the regulation of macrophage ferroportin channels.

5.7 Muscle regeneration

Phagocytic macrophages are recruited during periods of increased muscle use, causing muscle membrane lysis and membrane inflammation. Their peak concentration at the site is usually reached about 4 hours following the onset of some form of muscle cell injury or reloading. They degrade the contents of injured muscle fibers. M2 macrophages on the other hand are usually distributed near regenerative fibers. They release soluble substances that influence proliferation, differentiation, growth, repair, and regeneration of muscle [1]. The number of M2 macrophages remains elevated for several days until muscle tissue rebuilding is done.

5.8 Pigment retention

Melanophages which are a subset of macrophages can absorb and retain pigments. The pigment could either be native to the organism or exogenous from extracellular spaces such as tattoo ink. The pigment from dead dermal macrophages is phagocytosed by their successors thereby accumulating phagocytosed melanin in lysosome-like phagosomes. The ultimate effect is the retention of the pigment at the same place [14].

6. Macrophage immune metabolism

Macrophages can be found in nearly all the tissues in the body. The microenvironment around such tissues dictates the metabolic profile of macrophages which in turn affects their function. Moreover, the microenvironment also influences polarization that a microphage cell will undertake within a given tissue. The influence of tissue microenvironment on macrophage functions is indirect through modulation of their metabolic profile. The genetic makeup of macrophages regulating their metabolic pathway is highly plastic due to the influence of the micro-environment [7]. This makes microphages sensitive to metabolic changes both intracellularly and extracellularly. Moreover, incoming monocytes from systemic circulation that are translocating into the tissue are influenced by the niches within the various organs to differentiate into tissue-specific macrophages ensuring the macrophage pool of a given tissue is not exhausted.

Cellular metabolism comprises a network of pathways that utilize fuels from nutrients for energy production and structural formation. Immuno-metabolism illustrates the sub-domain of the immune system that looks at how bioenergetics, usage of nutrients, and metabolite generation all influence and impact the function of immune mediators-in this case, the macrophages. As such, the functional phenotype of a macrophage expressed in a given tissue can be said to be the resultant effect of local environmental signals and metabolic settings. This versatility is attributable to a large number of metabolic and transcriptional profiles. Macrophages, just like any other cell, utilize fuels (glucose, amino acids, and lipids) to generate metabolites (pyruvate, TCA metabolites) and energy in form of ATP through biochemical processes such as glycolysis, and glutaminolysis.

6.1 Intracellular mechanism of macrophage metabolism

Three characteristics are manifested by M1 macrophages activation: secretion of proinflammatory cytokines, expression of iNOS, and cellular metabolism through glycolysis [12, 15]. The major stimulating signals are LPS and IFN-γ. These ligands bind to their receptors and upon ligation, such macrophages undergo increased uptake of glucose [16]. This activates the glycolytic pathway leading to increased formation of glycolytic intermediates. The intermediates are thus shifted into the pentose phosphate pathway (PPP) which leads to the regeneration of NADPH used for nucleotide synthesis and production of ROS. In addition, excess amounts of pyruvate molecules are produced. Some of these molecules are converted into lactate while others enter the Tri-carboxylic acid cycle. However, the pyruvate that enters the TCA cycle is not completely metabolized through the pathway rather two breaks occur within the cycle that shifts into other pathways [12, 17]. One of the breaks occurs during the conversion of isocitrate to alpha-ketoglutarate by the enzyme isocitrate dehydrogenase [12, 15, 18]. Macrophages downregulate this reaction step leading to the accumulation of citrate. The accumulated citrate is in turn shunted to the pathway used to form itaconate which is a potent microbicidal metabolite. The excess citrate is also converted to Acetyl-CoA which is used to acetylate histones within inflammatory genes. This acetylation activates the expression of inflammatory genes. The formed Acetyl-CoA is also used for de novo synthesis of fatty acids that are used to expand cell membranes, synthesize prostaglandins and arachidonic acids.

The second break occurs in the reaction step where succinate is converted into fumarate by succinate dehydrogenase [12]. The earlier formed itaconate mediates this inhibition leading to the accumulation of succinate metabolite. Increased amounts of succinate molecules function as stabilizers for the hypoxia-inducible factor-1α (HIF-1-α) [18]. This in turn decreases oxygen tension within tissues leading to a decrease in mitochondrial respiration. Thereby, macrophages have to be dependent on glycolysis for energy production. The accumulated HIF-1-αpromotes the transcription of IL-1β (a pro-inflammatory cytokine) and enhances the expression of hexokinase1 and glucose transporter1(GLUT1) [18]. This increases the uptake and metabolism of glucose via the glycolytic pathway. Moreover, the little oxidation of succinate by succinate dehydrogenase in macrophages stimulated by LPS induces the generation of mitochondrial reactive oxygen species (mtROS) [18, 19]. These byproducts are recruited into phagosomes for bacterial killing. In addition, the ROS cause oxidative damage to the DNA, and as a result, the poly (ADP-ribose) polymerase (PARP) enzymes are activated [19]. These enzymes consume a lot of NAD leaving the M1 macrophages to rely on salvage pathways for NAD⁺. Apart from succinate, the induced nitric oxide also reduces mitochondrial respiration. This leads to a reduction in the ATP: ADP ratio dampening the inflammatory process.

Activation of macrophages towards the M2 phase is often induced by IL-4 and IL-13 [5, 17, 18]. Such cells have enhanced mitochondrial respiration, production of anti-inflammatory cytokines, and increased expression of arginase-1. The increased arginase-1 activity shifts the metabolism of arginine to result in the formation of ornithine metabolites. The ornithine is used in the biosynthesis of various polyamines such as spermidine [18]. Spermidine activates eukaryotic initiation factor 5A (EIF-5A) which is a translation factor. This factor facilitates the expression of mitochondrial proteins needed for the oxidative phosphorylation-dependent differentiation of M2 cells [20]. The overall effect is increased energetic profile of the macrophage and this energy is shifted towards uptake, transport, and oxidation of fatty acids besides increased glucose uptake. M2 macrophages unlike M1 macrophages rely on glutamine and fatty acid oxidation for metabolic processes, especially if the activating stimuli is IL-4.

Transcriptional factors such as STAT6, IRF-4, and PPARy are highly activated and expressed in M2 macrophages as earlier discussed [5, 18]. These factors in addition increase the expression of CD36 on surfaces of M2 macrophages. CD36 enhances the endocytosis of lipoproteins containing triglycerides. This increased uptake of lipids leads to an even higher rate of fatty acid oxidation and mitochondrial biogenesis enhancing the consumption rate of oxygen within cells.M2 cells also have a higher rate of glutamine catabolism resulting in the accumulation of alpha-ketoglutarate which is one of the metabolites of the TCA cycle. Alpha-ketoglutarate in turn activates the jumonji domain-containing protein D3 demethylases which promote histone demethylation on M2-specific gene promoters which as a result leads to macrophage polarization towards the M2 direction.

7. Role of IFN-γ in macrophage activation, metabolism, and functioning

Interferons are a group of potent cytokines that are pleiotropic in nature and carry out paracrine and autocrine actions [21]. Their secretion is majorly induced during viral infection but also to a lesser extent during bacterial or autoimmune disorders. Three types of interferons exist IFN α, -β, and -γ. The former two are grouped and known as type I interferons while the latter is commonly grouped under type II interferons [21]. The three molecules exhibit different primary molecular structures. IFN-α, and -β have more or less similar properties, unlike IFN-γ [21]. These different characteristics and properties are illustrated in the Table 3.

IFN α/β	IFN-γ
Their genes are not split by introns	The gene is split by introns
The molecule is stable at pH 2.0	It is acid-labile
Both have a common receptor different from that of IFN-γ (IFNAR1 and IFNAR2)	Its receptor is different from that of type I interferons (IFNGR1 and IFNGR2)
Stimulate mainly natural killer cells	Stimulates manly macrophages
Rapid induction of an antiviral state	Slow induction of an antiviral state
Mainly secreted by IFN-α (B-cells and macrophages), IFN-α(epithelial cells and fibroblasts)	Mainly Secreted by T cells and NK cells

Table 3.

Differences between type 1 interferons and type II interferons.

Type I interferons do not have much of important roles in macrophage activation and metabolism as seen it has on plasmacytoid dendritic cells [21]. IFN-γ on the other hand regulates the activation and metabolic profile of macrophages. IFN-γ does this indirectly by either activating macrophages towards the M1 direction and upregulating or downregulating the expression of genes that enhance energy production via the glycolytic pathway. IFN-γ binds to its receptor IFN-γR activating Janus Kinase 1 and 2 associated protein tyrosine kinase [21, 22]. This causes phosphorylation of the tyrosine residues and activation of signal transducer and activator of transcription-1 (STAT1). STAT1 translocates into the nucleus and activates IFN-y related interferon-stimulated genes. As a result, the activated genes encode for various proteins that manifest the downstream effect of IFN-y on macrophage metabolism.

IFN-γ acts on three major enzymes when it comes to modulating macrophage metabolism [22]. These are the mammalian target of rapamycin complex 1 (mTORC1), glycogen synthase kinase 3 (GSK3), and 5’-AMP-activated protein kinase (AMPK) [23]. IFN-γ inhibits the activity of mTORC1 in macrophages resulting in decreased mitochondrial function, decreased secretion of anti-inflammatory mediators, and synthesis of purine nucleotides. In addition, this inhibition to leads to more autophagy by macrophages enhancing the process of microbial killing. GSK3 upon stimulation by IFN-γ modulates the activity of NF-kB such that an increase in the production of inflammatory cytokines is seen [21]. AMPK acts as a control point. It senses energy deprivation during metabolic processes in M1 macrophages and shifts the metabolism towards the M2 direction [23]. As a result, aerobic respiration and energy production are revamped within the cells.

8. Disorders associated with dysfunctional macrophages

Macrophages just like any other cell can be dysfunctional. These disorders can directly affect the activation, metabolism, and functions of macrophages or indirectly aid in the development of systemic diseases such as metabolic syndromes.

Activation of macrophages leads to their expansion to different functional cell lines. Disordered expansion of macrophages leads to a collective group of disorders known as hemophagocytolymphohistiocytosis (HLH) [24]. This group of illnesses can either be familial/primary or secondary/reactive. Familial HLH is due to inherited autosomal recessive immune disorders caused by genetic defects of genes controlling the cytolytic pathway of macrophages. Secondary HLH is caused by infections particularly Epstein-Barr virus (EBV), Cytomegalovirus (CMV), and cancer [25].

One of the disordered related to macrophage activation is the macrophage activation syndrome [25]. This syndrome is caused by too much activation and subsequent differentiation of macrophages. Clinically it manifests as cytopenias, dysfunctional liver state, hyperferritinemia, and coagulopathy [26]. It is a life-threatening condition with mortality rates of between 20 and 30%. This syndrome occurs mostly and is associated with systemic lupus erythematosus (SLE), systemic juvenile idiopathic arthritis (SJIA), Kawasaki disease, and other rheumatic conditions [27]. Cytotoxic cells induce apoptosis of activated macrophages. Failure of this to occur leads to prolonged expansion of macrophages leading to excessive production of proinflammatory cytokines. This induces a state of cytokine storm and hemophagocytosis is related to changes in metabolic pathways of macrophages [13]. The alveolar macrophages first utilize glycolysis for energy production but then switch to fatty acid metabolism. This process is dependent on the mitochondrial calcium channel and levels of mitochondrial reactive oxygen species. This activation sustains fibrosis within the ling tissues. However, administration of itaconate or when systemized by the macrophages reverses the glycolytic process and protects from fibrosis [13].

Tuberculosis is one of the communicable diseases with a high mortality rate. It is caused by the bacteria mycobacterium tuberculosis. Usually, during infections, macrophages are activated to ingest and present these pathogens. Infection chronicity leads to the formation of granulomas that prevent the spread of the bacteria. However, a section of the population is highly susceptible to severe tuberculosis. This is because they have a defect in genes encoding receptors for IFN-γ on the surface of macrophage cells. As such, during infection, the macrophages are not activated to induce inflammation and present the pathogen for destruction but remain insensitive to IFN-y stimulation. The macrophages are thus unable to kill the bacteria. Moreover, the granulomas do not effectively form and systemic infection may occur.

In addition, macrophages contribute to diseases whose etiology does not primarily lie on defects associated with the activation, metabolism, and functioning of macrophages. These diseases include inflammatory diseases (inflammatory bowel disease, arthritis), infections (HIV), and metabolic diseases (such as atherosclerosis, obesity, and diabetes). The pathogenesis of such diseases usually utilizes the macrophage inflammatory process to develop its pathophysiology.

9. Conclusion

Macrophages are phagocytic immune cells. They function in both homeostasis and pathology. Macrophages are activated through two pathways depending on their polarization. Polarization refers to the ability of such cells to differentiate into functionally different phenotypes. Classically activated macrophages are also known as M1 macrophage, are pro-inflammatory in nature and thus mediates inflammatory reactions. M2 macrophages on the other hand results from alternative activation of macrophages. The M2 macrophages are anti-inflammatory in nature functioning in wound healing and tissue repair. Macrophages can be classified based on their mode of activation and based on the tissue they are located in. based on activation, macrophages can fall into either M1 or M2 macrophages as stated above.

Different tissues have different types of macrophages depending on their roles and phenotypic expression. Kupffer cells in the liver, alveolar macrophages in the lungs, osteoclasts in bone tissue, Langerhans cells in the skin, intraocular macrophages in the eye, microglia in the brain, splenic macrophage, intestinal macrophage, and subscapular sinusoidal macrophages in lymph nodes. These macrophages have specific tissue functions in addition to the generalized functions of a macrophage cell. The generalized functions of a macrophage cell include induction of inflammation, immune surveillance, antigen processing, presentation wound healing, iron metabolism, and muscle regeneration. All these functions either fall into anti-inflammatory or proinflammatory. The mechanism of these functions is integrated with the metabolic profile of a macrophage and the type of polarization it acquires. M1 macrophages are activated mainly by IFN-γ and lipopolysaccharide while M2 macrophages are activated by interleukin 4. M1 macrophages primarily utilize aerobic glycolysis for energy production through inhibition of the Krebs cycle and cellular respiration. The resultant effect is the formation of metabolites such as itaconate with microbicidal effect and immediate provision of energy. In addition, M1 polarization results in the formation of reactive oxygen species due to inhibition of cellular respiration. These species get integrated into the phagosome to aid the phagocytic process. M2 macrophages on the other hand utilize fatty acid oxidation and cellular respiration to carry out their functions. Macrophages also have a role to play in the induction of pathological conditions. Primary defects in macrophage pathways result in the development of disorders such as increased susceptibility to tuberculosis, idiopathic pulmonary fibrosis, and macrophage activation syndrome. Secondarily, macrophages also aid in the pathogenesis of inflammatory diseases, infections, and metabolic syndromes.

10. Conflict of interest

The authors declare no conflict of interest.