Introductory Chapter: Macrophages – More than Sentinel Innate Immune Cells

1. Introduction

Elia or Ilya Metchnikoff first described macrophages as phagocytes (eating cells) in 1882 in starfish larvae inserted with the tangerine tree thorns and later (1883–1884) in Daphnia magna or common water flea infected with fungal/yeast spores due to its transparent outer carapace [1, 2, 3, 4]. Metchnikoff considered macrophages/phagocytes as homeostatic agents of the body, which continuously vigil on foreign invaders and maintain the host’s internal environment (homeostasis) and tissue/organ remodeling [5]. However, the term phagocytosis or phagocytes (Greek derivative for devouring cells) was not introduced by Elia Metchnikoff, instead given by Carl or Karl Claus (a German Zoology professor, who invited Elia Metchnikoff to publish his findings in his journal in 1883) [6, 7]. Elia Metchnikoff won the Noble prize (Physiology and Medicine) for this discovery with Paul Ehrlich in 1908. Thus, macrophages were first discovered as phagocytes or devouring cells that engulf any foreign material (size ≥0.5 μm) entering the host almost 140 years ago [8, 9]. These comprise first innate immune cells responding in response to the invasion of the host by foreign particles (pathogens, allergens, and xenobiotics). Hence, macrophages are considered one of the sentinel innate immune cells of the immune system. They play a crucial role in immune homeostasis maintenance, host defense, inflammation, and different inflammatory and infectious diseases through different mechanisms (phagocytosis, secretion of different inflammatory mediators, and interacting directly with different immune (innate and adaptive) and non-immune cells) [10, 11, 12, 13, 14]. Hence, macrophages are potent innate immune cells with diverse immunological functions. This chapter discusses emerging non-immunological functions of macrophages.

2. Macrophages in the human reproduction and reproductive cycle

Macrophages are present in the male (testes) and female reproductive organs/tract (ovaries, fallopian tubes, uterus, and vagina) and serve as crucial accessory cells for reproduction (Figure 1) [15, 16, 17]. In the embryonic life of a male fetus, the yolk-sac macrophages regulate testes morphogenesis and vascularization [18]. Further studies have shown that the macrophage-specific genes/transcription factors Mafb and Maf are crucial mediators of morphogenesis and vascularization of testes during the embryonic development [19, 20]. The process involves distortion of the myeloid cell ratios (profound increase in gonadal macrophages in the absence of Mafb) as Mafb regulates macrophages’ differentiation. The Maf loss causes testicular hypervascularization, defective testis cord formation, deficiency of Leydig cells, and decreased germ cell number [19]. Mafb also regulates the cholesterol efflux in macrophages and can control the Leydig cell function by regulating the 2—hydroxycholesterol (25-HC) secretion [21]. However, Mafb or macrophages are not crucial for the maintenance of spermatogenesis in the adult mice [22, 23]. Notably, the resident macrophages of adult testes are the derivatives of embryonic precursors of macrophages (fetal macrophages) under normal conditions, and bone marrow-derived macrophages do not infiltrate testes [23]. On the other hand, in postnatal mice, testicular macrophages are crucial for steroidogenesis (sex hormone synthesis) by controlling Leydig cell proliferation and differentiation in the prepubertal stage [24, 25, 26, 27, 28, 29]. For example, depletion of interstitial macrophages in testes inhibits Leydig cell development, and pro-inflammatory activation of macrophages also inhibits steroidogenesis by Leydig cells by producing a lipophilic factor that controls steroidogenesis or testosterone production.

Figure 1.

Macrophages are crucial for different physiological process (sperm production, ovulation, fertilization, placentation, embryonic development, regeneration, neurogenesis, learning and memory) other than playing crucial role in immune function. Details are mentioned in the text.

The testicular macrophages secrete 25-HC, converted to testosterone by the neighboring Leydig cells [30]. However, the overproduction of 25-HC by testicular macrophages during infection or other inflammatory conditions impacting the testes inhibits the steroidogenic function of Leydig cells. The macrophage and Leydig cell interaction details are mentioned elsewhere [31]. The macrophage ubiquitin-specific protease 2 (USP2) contributes to the sperm motility, hyperactivation, capacitation, and fertilization capacity of the murine sperm, and myeloid-selective USP2 knockout (msUSP2KO) mice sperm has a decreased mitochondrial membrane potential for ovum fertilization [32]. The treatment of sperms derived from msUSP2KO mice with granulocyte macrophage-colony stimulating factor (GM-CSF) rescued their potential for fertilizing the ovum. Thus, testicular macrophages regulate steroidogenesis, spermatogenesis (maintenance of spermatogonial niche in adult testes), sperm motility, capacitation, and in vitro fertilization of mouse sperm that are crucial for sexual reproduction in mammals, including humans (Figure 1) [33, 34, 35]. Hence, testicular macrophages (peritubular macrophages) are close to peritubular myoid cells in seminiferous tubules where spermatogenesis takes place, and interstitial macrophages are located within the testicular interstitium in close contact with Leydig cells crucial for male reproductive function.

Macrophages are abundant in the mesenchymal and connective tissue stroma of the gravid and non-gravid uterus, comprising the necessary amount of villous or labyrinthine mesenchymal cells of human and mouse placenta (Figure 1) [36, 37]. The large (10–30 μm) human placental macrophages present in the fetal villi of the placenta (from the first trimester until birth) are called Hofbauer cells and maintain placental and fetal development and homeostasis (Figure 1) [38]. Thus, placental macrophages are composed of two different populations: (1) decidual macrophages and (2) Hofbauer cells [39]. Detailed ontogeny and function of placental macrophages have been discussed elsewhere [40, 41]. Recently identified, three different subsets of macrophages (CCR2⁻CD11c^low, CCR2⁻CD11c^hi, and CCR2⁺CD11c^hi) express CD45 and CD14 differently in the human uterus during early pregnancy maintain maternal-fetal homeostasis [42, 43]. In the absence of pregnancy, these uterine or endometrial macrophages play a crucial role in menstruation and the maintenance of tissue integrity to prepare the uterus for reproductive events, including fertilization and implantation (Figure 1) [44]. However, the macrophage number varies (1–15%) in the endometrium depending on the stage of the menstrual cycle [45]. The number of macrophages also increases in the uterus and cervix preceding the parturition, indicating their involvement in childbirth through releasing several biological mediators [46]. A recent human study has indicated that women with recurrent pregnancy loss have alternatively activated CD45⁺CD14⁺ICAM3⁻ macrophages expressing low levels of CD209 and CD206 expression and have high expression of TNF-α in their proinflammatory CD45⁺CD14⁺CD80⁺HLA-DR⁺ macrophages in their uterus at the maternal-fetal interface [47]. Hence, any imbalance in the uterine/decidual macrophages in human females and in experimental mammals prevents pregnancy/blastocyst implantation and induces recurrent pregnancy loss [48, 49].

In addition to the uterus and placenta, macrophages are also present in the ovary and regulate the ovarian cycle (ovulation, corpus luteum formation, and luteolysis) through different processes, including the phagocytosis of the apoptotic cells and the release of different mediators (Figure 1) [50, 51]. The details are beyond the scope of this introductory chapter and have been discussed elsewhere [50, 51]. The macrophage-derived multinucleated giant cells increase in the ovaries of aged mice indicating ovarian aging and a decline in its reproductive potential or ovum production and release [52]. Of note, in aged male mice, the testicular macrophages show lipofuscin granule accumulation and altered morphology that are absent in testicular macrophages of young adult mice [53]. The lipofuscin granule accumulation in the testicular macrophages of aged mice occurs due to altered metabolism but not due to its phagocytosis.

Further studies are required to indicate the role of testicular macrophages in the event of andropause. However, the expression of transient receptor potential vanilloid 2 (TRPV2, a cation channel) increased in CD206⁺MHC II⁺ testicular macrophages of aged mice due to inflammaging in the testes microenvironment may play a crucial role in andropause [54]. The TRPV2 expression affects the phagocytic potential of macrophages by controlling cytosolic Ca2+ levels and their motility [55, 56, 57]. In addition, aging impacts Leydig cell functions, including steroidogenesis (testosterone synthesis), and testicular interstitial macrophages are crucial in affecting these cells [58]. Hence, macrophages are crucial for sexual reproduction and developmental process.

3. Macrophages in organ and tissue regeneration

Abraham Trembley, a naturalist in the eighteen century, gave the idea of regeneration and wanted to know about the process responsible for regressing the heads of hydra and earthworms [59]. Hence, the regeneration process involves regressing the lost tissue or organ in animals when they lose it. However, some animals (planarians (a class of flatworms considered masters of regeneration and can rebuild any body part once it is lost or cut) and cnidarians) are considered immortal under the edge of a knife [60, 61, 62]. Amphibians, including salamanders and axolotls, also regenerate their body parts [63]. In reptiles, regeneration is proper for lizards, geckos, and iguanas as they regenerate their lost tails, but it is not valid for snakes and crocodiles [64]. Birds can regenerate their lost mechanoreceptive sensory (auditory) hair cells in the inner ear responsible for hearing that is absent in mammals [65, 66]. Hence, the loss of auditory hair cells in humans leads to permanent deafness. In humans, liver and cardiac muscle cells have the power of regeneration only. Hence, the loss of auditory hair cells in humans leads to permanent deafness. In humans, liver and cardiac muscle cells only have the power of regeneration. The biology of regeneration is beyond the scope of this chapter.

Macrophages also play a crucial role in the process of tissue or organ regeneration (Figure 1). For example, macrophages have been shown to play a crucial role in the regeneration process in Salamanders (Figure 1) [67]. In these organisms, their recruitment at the site of limb amputation peaks around 6 days after the amputation in response to the profound release of macrophage chemoattractants [68]. Even heart regeneration in salamanders requires macrophage infiltration, which is essential for fibroblast activation and the extracellular landscape without affecting cardiomyocyte proliferation [69]. Macrophage depletion prevents heart regeneration and induces fibroblast activation, and alters collagenase deposition and arrangement process that may lead to fibrosis, a condition that may lead to organ malfunction and mortality [69, 70]. The details of macrophage function in tissue and organ (skin, heart, and liver) regeneration in mammals, including humans, have been described elsewhere [71, 72, 73, 74, 75].

4. Microglia or brain macrophages in neurogenesis, learning, and memory

Microglia are resident brain macrophages of the central nervous system (CNS), which solely originate from the embryonic yolk sac erythromyeloid precursors under normal conditions, which also give rise to macrophages in other tissues and organs [76, 77]. Of note, adult hematopoietic progenitors do not significantly contribute to the maintenance of microglia in the adult brain. In addition to their primary function as local innate immune cells of the brain and the maintenance of brain homeostasis, microglia also play a crucial role in neurogenesis, learning, and memory [78, 79, 80]. Recent studies have suggested the role of microglia in brain development or neurogenesis (Figure 1), and their dysregulation induces different neurodevelopmental diseases and other neurological diseases later in life [81, 82, 83]. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B or AKT (PI3K-AKT) signaling pathway in microglia is crucial for inducing neurogenic protein expression in primary cortical cells [84]. For example, microglia in the CNS control synaptic pruning, which is crucial to promote synapse formation and the regulation of neuronal and synaptic plasticity via different signals involving complement proteins and CX3CR1 signaling [85, 86]. The microglia-based regulation of synaptic pruning, plasticity, and neuronal activity is beyond the current chapter’s scope and has been discussed in detail elsewhere [87].

Deleting the circadian clock gene Bmal1, specifically in mice’s microglia (the circadian clock regulates their function), increases their learning potential and memory in mice fed a high-fat diet [88]. On the other hand, the microglia-specific brain-derived neurotrophic factor (BDNF) depletion promotes working memory and stimulates neural precursor proliferation following traumatic brain injury (TBI) [89]. However, under normal conditions, BDNF depletion, specifically from microglia, impairs memory and reduces motor-learning-dependent synapse formation [90]. Thus, microglia’s memory and synaptic plasticity function vary with the brain’s homeostatic alteration. Further studies have shown that the repopulation of microglia in the rat brain after their depletion significantly improves their learning and memory performance [91]. Of note, microglia depletion does not impact short-term memory in rats acutely.

The microglia also regulate learning and memory through IL-33 (expressed in adult hippocampal neurons and also secreted by astrocytes) as they express IL-33 receptor (IL-33R) along with controlling spine plasticity, synapse homeostasis during CNS development, newborn neuron integration, and remote fear memories [92, 93]. The IL-33 binding to the microglial IL-33R induces uptake of extracellular matrix (ECM), and its loss causes over-accumulation of ECM in contact with synapses that affect synaptic plasticity and memory. Microglia inhibition in rodents with neurodegenerative diseases, including Alzheimer’s disease (AD) and other neuroinflammatory conditions with minocycline (a centrally penetrant tetracycline antibiotic), affects long-term potentiation (LTP), synaptic plasticity, neurogenesis, and hippocampal-dependent spatial memory [94, 95, 96, 97]. However, the treatment with minocycline impairs human spatial and temporal memory by disrupting striatal processing [98]. Hence, modulating the microglial function in humans is not a straightforward approach and is a two-edge sword to restoring memory and learning associated with different neurodegenerative diseases. The details of microglia in neurogenesis, learning, and memory are mentioned elsewhere [87, 99, 100, 101].

5. Macrophages regulating systemic metabolism

Metabolism plays a crucial role in the organism’s well-being and homeostasis. The disruption in the metabolic pathways through different factors (including intake of high-calorie or high-fat diet, lack of exercise, insulin tolerance, and different genetic factors) in individuals predispose them to type 1 or type 2 diabetes mellitus (T1DM or T2DM), obesity, hypertension, and atherosclerosis. However, the potential pro-inflammatory action of macrophages in the pathogenesis of metabolic syndrome (obesity, insulin resistance, T2DM, atherosclerosis) has been well studied and established due to their direct involvement in the pathogenesis of inflammation and inflammatory diseases [102, 103, 104, 105]. This section is not intended to describe the immunological function of macrophages in metabolism but to discuss their non-immune role in regulating systemic metabolism.

The anti-inflammatory drugs show a modest effect on metabolism, as shown by many clinical trials that indicate the limited impact of inflammation on the metabolism [106, 107]. However, the depletion of liver macrophages (LMs) or Kupffer cells in obese mice affects their metabolism and improves fatty liver, indicating their role in insulin sensitivity [108, 109, 110]. Notably, the pro-inflammatory phenotype of LMs does not play a significant role in obesity and insulin resistance in flies, mice, and humans [111]. Instead, LMs produce non-inflammatory insulin-like growth factor-binding protein 7 (IGFBP7) that directly controls liver metabolism. The IGFBP7 released from LMs binds to the insulin receptor, which induces lipogenesis and gluconeogenesis by activating extracellular-signal-related kinase (ERK) signaling [111]. Hence, LMs contribute to systemic metabolism and associated diseases independent of their immune functions. Thus, the discovery of other non-immune factors in other macrophages, including adipose-tissue macrophages (ATMs) in obese people controlling systemic metabolism, may serve as a better therapeutic approach where their inflammatory role is secondary in the pathogenesis. Further studies are required in this direction.

6. Conclusion

Macrophages are first discovered as phagocytic innate immune cells. After 140 years of their discovery, they are ruling the world of immunology as a potent innate immune cell in almost every organ. They are present in even organs considered previously devoid of immune cells (brain, testes, and retina). Along with potent innate immune cells, which protect the host from foreign invasion, they also control the adaptive immune response by serving as potent antigen-presenting cells (APC). In addition, they also serve as potent immune cells, which control different non-immune functions (reproduction and embryonic development, regeneration, neurogenesis, and neurological functions, including learning and memory and systemic metabolism) that I have described in this chapter. Thus, macrophages are not only the body’s guards against pathogens but also maintain homeostasis through different mechanisms via controlling different biological functions varying from reproduction to regeneration and from metabolism to learning and memory. Thus, after 140 years of their discovery, macrophages sit over the top in immunology and biology.