Open access peer-reviewed chapter
Abstract
When the first line of defence—the integumentary system fails, the immune system protects us from infections by pathogens. Macrophages are crucial for mediating effects in the innate immune system by eliminating impaired cells and harmful micro-organisms through phagocytosis. Although other cells undergo phagocytosis, the cellular processes that regulate phagocytosis may vary from cell to cell. These include metabolic changes, signal transduction, and changes in molecular expression or post-translational modifications. This chapter will comprehensively review biological processes that regulate phagocytosis in macrophages, including; changes in metabolic processes, signal transduction, molecular expression, and post-translational modifications.
Keywords
- macrophage
- innate immunity
- phagocytosis
- regulation
- receptors
1. Introduction
There are millions of human pathogens grouped into about 1400 species [1]. The integumentary system serves as the first line of defence against infection; however, when the integumentary system fails, the immune system defends us against infectious pathogens [2, 3]. It consists of physical barriers such as the dermis, epidermis, and associated glands [4]. Innate immunity describes the initial reaction of the immune system to invasion by microbial pathogens by controlling tissue damage and coordinates the activation of the adaptive immune system [5, 6, 7, 8]. When the integuments fail, innate immune cells like macrophages recognise pathogen-associated molecular patterns (PAMP) through pathogen recognition receptors (PRR) and are activated [9]. Macrophage responses involve phagocytosis of the PAMP and the release of inflammatory cytokines resulting in inflammation [10]. Inflammation is a natural reaction that can prevent tissue injury and heal wounded tissues. The strength of inflammation is proportionate to the severity of tissue injury [8, 11]. A normal inflammatory response is structured and involves; vasodilation, higher permeability of blood capillaries, blood clotting, an influx of many granulocytes and monocytes, and tissue swelling [8].
This review chapter will discuss various biomolecules and biochemical processes that regulate phagocytosis in macrophages.
2. The macrophage: functions and phenotypes
Macrophages are crucial for mediating EFFECTS in the innate immune system [12]. Elie Metchnikoff first identified phagocytic cells in the 1900s and observed that macrophages effectively phagocytosed bacteria. Since then, there has been more research on macrophages—their types, function, polarisation, origin, and how they are regulated. Macrophage phagocytosis can be affected by its type, phenotype, and source. In addition, macrophages phagocytose other pathogens, such as viruses, fungi, and parasites [13]. They originate either from yolk-sac erythromyeloid progenitors or haematopoietic progenitors, thus generating monocyte-derived macrophages and tissue-resident macrophages. However, researchers have suggested heterogeneity in the origin of tissue-resident macrophages, as monocyte-derived macrophages can replace embryonic macrophages [8, 12, 14]. In addition, metabolic stimuli can regulate macrophage differentiation. For instance, haem and retinoic acid activate red pulp and peritoneal macrophage differentiation, respectively. Furthermore, tissue-resident macrophages contribute significantly to the heterogeneous functions of macrophages as they have specialised functions according to the tissue environment. Some examples of tissue-resident macrophages include alveolar macrophages, microglia, kupffer cells, and peritoneal macrophages [15].
2.1 Function of macrophages
The classical functions of macrophages include; cytokine secretion, the release of reactive oxygen species and reactive nitrogen species, removal of impaired cells and harmful micro-organisms, tissue surveillance, antigen presentation, T-cell activation, cytotoxicity and fibrosis [8, 16, 17, 18]. Tissue-resident macrophages carry out extra functions contingent upon the tissue requirements. For instance, alveolar macrophages clear away lung surfactants [19, 20]. Various stimuli coordinate macrophage fundamental functions and responses to tissue warning signals, including the presence of elements of microbial organisms [21].
In addition, macrophages participate in several pathologies that involve inflammation. For instance, macrophages regulate neuropathic and inflammatory pain by releasing cytokines and interacting with neurons [22]. In cancer, macrophages phagocytose tumour cells and participate in tumour immunosurveillance [23, 24]. In their research, Yang et al., 2021 [25] showed that macrophages could promote cartilage regeneration in mice where macrophage depletion hindered cartilage regeneration. Furthermore, macrophages encourage fibroblast proliferation. As a result, it regulates wound healing [26, 27]. Finally, poor differentiation of microglia during foetal development can cause neuropsychiatric disorders [28].
2.2 Macrophage phenotypes
There are three known macrophage phenotypes; M0 defines the macrophage in an inactive state, M1 defines a phenotype that promotes inflammation, and M2 defines a phenotype that resolves inflammation and promotes wound healing. In addition, M2 macrophages have four sub-phenotypes (M2a, M2b, M2c, M2d), which can affect the extent of phagocytosis [29, 30].
Lipopolysaccharide (LPS) and interferon-gamma (IFNγ), granulocyte-macrophage colony-stimulating factor (GMCSF), and PAMPs are conventional stimulators of the M1 macrophage phenotype. In contrast, macrophage colony-stimulating factors (MCSF), IL4, IL10, and IL13 are stimulators of the M2 macrophage phenotype [8, 31, 32]. Macrophages show phenotypic characteristics based on an environmental stimulus. Epigenetic factors, including non-coding RNAs, histone modifications, and DNA methylation, can reprogram macrophages to switch between M1 and M2 phenotypes [24, 33, 34, 35]. Likewise, macrophage metabolic pathways participate in polarisation into different phenotypes. Lipid metabolism plays a significant role in macrophage phenotype formation. There are metabolic pathways specific to the M1 and M2 macrophage phenotype [36, 37].
3. Regulation of phagocytosis In macrophages
3.1 Pathogen-associated molecular patterns
Various microbial pathogens exist; therefore, PAMPs vary accordingly [11, 38].
LPS is the toxin element of the exterior membrane of gram-negative bacteria. It primarily consists of three components: the variable O-antigen, the core oligosaccharide, covalently bound to the third component—a hydrophobic “anchor” termed lipid A, which commonly contains acyl tails attached to a phosphorylated β-1′, 6-linked glucosamine disaccharide head group. The lipid A component of LPS is highly potent; however, the structural variance of lipid A can influence its potency [8, 39]. In addition, some bacteria retain genetic mutation that hinders the expression of some components of LPS resulting in smooth, semi-rough and rough LPS chemotypes. Smooth LPS refers typically to the prevalent LPS containing the O-antigen. Smooth and Rough LPS may have differential mechanisms for regulating inflammation; rough LPS may be less CD14-dependent than smooth LPS [40]. In the same vein, rough LPS from B. abortus strains of bacteria are more potent in inducing the release of pro-inflammatory cytokines than smooth LPS [41]. Even amongst different species, there are dissimilarities in the strengths of LPS; for instance, the rough chemotype of E. coli LPS is more potent than the rough chemotype of B. abortus LPS [42]. LPS-induced activation of TLR4 activates signals that cause an increase in NFκβ and IRF3 activity hence the secretion of pro-inflammatory and pro-resolving cytokines [43].
Lipopeptides are on the cell walls of gram-positive bacteria, some species of gram-negative bacteria, and fungi. The structure of lipopeptides could be either cyclical peptides attached to an acyl chain, tri-palmitoyl peptides, or dipalmitoyl peptides. Tri-palmitoyl peptides activate TLR2/1 or TLR1/6 receptor heterodimers to induce inflammation. For example, Pam3CysK4 activates cytotoxic T lymphocytes against influenza-virus-infected cells [44, 45]. On the other hand, dipalmitoyl peptides activate TLR2/6 receptor heterodimers, activating the MyD88-dependent pathway and promoting the production of pro-inflammatory cytokines through NFκβ activation [46, 47].
Bacterial and viral DNA are potent macrophage stimulators. They have a repeated series of unmethylated CpG motifs that bind to TLR9 homodimers. Microbial DNA increases the synthesis and secretion of nitric oxide and pro-inflammatory cytokines. Unlike microbial DNA, mammalian DNA has low-frequency CpG dinucleotides, mostly methylated. Therefore, typical mammalian DNA would not cause inflammation [38, 48, 49, 50, 51, 52].
On the other hand, viral RNA exist in either a single-stranded or a double-stranded form resulting in differential inflammatory responses. For example, TLR7 and TLR8 commonly recognise single-stranded RNA [53, 54] and form homodimers after activation. However, some scientific evidence [55, 56] has suggested that TLR3, which commonly recognises double-stranded RNA, can also recognise single-stranded RNA.
Microbial RNA induces the secretion of type I interferons and tumoricidal activity in macrophages. They also activate the synthesis of NFκβ-dependent cytokines [57]. Although IRF3 is the primary transcription factor activated by the TRIF-dependent signalling pathway, a study showed that IFNβ could be significantly induced in the absence of detectable IRF3 activation by double-stranded RNA through an unknown mechanism. These studies indicate the necessity for a better understanding of microbial RNA’s interactions with its receptors [58, 59, 60, 61, 62, 63].
The cell walls of bacteria [64] and fungi [65] contain microbial polysaccharides such as glucans, mannans, and peptidoglycans. A broad variety of receptors, including; toll-like receptors TLR4, TLR2, and TLR6 [11], mannose receptors, DC-SIGN, complement receptors, and dectin receptors recognise microbial polysaccharides and peptidoglycans [66]. Nonetheless, they have differential mechanisms for mediating inflammation [67, 68].
Flagellin from gram-negative bacteria, profilin from T. gondii, and hemozoin from P. Falciparum are examples of microbial proteins that cause inflammation. Knockout of TLR5 weakens flagellin-induced inflammation, implying that TLR5 is crucial for recognising flagellin [69, 70]. Flagellin also binds to the inflammasome receptor NLRC4 resulting in the cleaving of pro-IL1β by caspase 1 to IL1β [71]. Moreso, TLR11 recognises profilin; however, this is limited to mice as human TLR11 is nonfunctional due to a stop codon in its gene [72]. Finally, hemozoin indirectly induces an inflammatory response by enhancing TLR9 responses to DNA from malaria parasites [73, 74].
3.2 Opsonins
Immunoglobulins are well-characterised molecules that recognise foreign micro-organisms or bodies [75]. The basic structure of immunoglobulin comprises two heavy chains and two light chains. The Fab fragment, known to bind and crosslink antigens, and the Fc fragment, which binds to pathogen recognition receptors on phagocytes, are also sub-structures of immunoglobulins [76]. In addition, Immunoglobulin G (IgG) plays a crucial role in immunity by binding invading pathogens and consequently activating the classical pathway of the complement system in macrophages [77]. Furthermore, the interaction of immunoglobulin A (IgA) with Fc alpha receptors (FcαRs) mediates macrophage phagocytosis [49].
Pentraxins refer to a group of serum proteins with a pentameric structure that binds and opsonises microbial pathogens or cellular debris during infection and inflammation. Their pentameric design allows high stability and resistance to enzymatic activity [78]. Both complement receptors and Fc receptors recognise pentraxins. Serum amyloid P (SAP) and C-reactive protein (CRP) are notable pentraxins. SAP recognises phosphoethanolamine, DNA, chromatin, heparin, apoptotic cells and amyloid fibrils in a calcium-dependent manner. On the other hand, CRP recognises phosphocholine, snRNP, histones, apoptotic cells, and oxidised low-density lipoproteins (LDL) [78, 79].
The recognition of microbial pathogens initiates the complement system. Complement proteins involved in recognising microbial pathogens also function as opsonins. Such complement proteins include C1q, mannose-binding lectin (MBL), ficolins, C3b, and C4b [80]. As the cell requires, C3 is cleaved to produce C3a, an anaphylatoxin and C3b, an opsonin [81]. The complement system has three pathways; C1q is involved with the classical pathway, MBLs and ficolins participate in the lectin pathway, and C3b and C4b are concerned with the alternative pathway [80]. In addition, complement proteins tend to promote the secretion of anti-inflammatory cytokines [80, 82].
3.3 Pathogen recognition receptors
Non-opsonic pathogen recognition receptors consist of Toll-Like receptors, RIG-I-Like receptors, Nod-Like receptors, and C-Type Lectin receptors.
Nod-Like and RIG-I-Like receptors localise in the cell cytoplasm. RIG-I, MDA5, and LGP2 helicases recognise single- and double-stranded microbial RNA in the cytosol. They cause a substantial secretion of type I interferons to fight viral infection [83]. On the other hand, over 20 subtypes of Nod-Like receptors exist. Nod-like receptors have four categories according to their functions: autophagy, inflammasome assembly, transcription activation, and signal transduction. They recognise a variety of pathogens, including flagellin, viral RNA, and peptidoglycan. Activation of Nod-Like receptors results in the secretion of IL1β through the inflammasome pathway, and it activates other transcription factors such as NFκβ and CREBBP [84, 85, 86].
C-type lectin receptors bind to mannans and peptidoglycans from microbes and primarily facilitate phagocytosis [87, 88, 89].
At least nine subtypes of TLRs exist, and they have LRR motifs and TIR domains. TLRs bind to components of microbial pathogens and interact with TIR-containing adapter proteins such as MyD88, Mal, TRIF, and TRAM. The signalling cascade interacts with transcription factors, producing inflammatory cytokines [90, 91, 92, 93, 94, 95].
Macrophages have Fc receptors (FcR) and complement receptors that recognise opsonins such as immunoglobulins, CRP, SAP, and complement proteins.
As the name implies, Fc receptors are 60kD glycoproteins that recognise and bind to immunoglobulins to mediate phagocytosis [96]. FcγR recognises and binds to IgG, whereas FcαR recognises and binds to IgA [78]. FcR also recognises and binds to other opsonins, such as SAP and CRP. FcR-mediated phagocytosis leads to internalisation in clathrin-coated pits and vesicles, delivery to endosomes and acid hydrolase-rich lysosomes [97]. Not all FcR transmit signals; however, signalling FcR require either ITAM or ITIM domains for signal transduction. The ITAM pathway is pro-inflammatory, and the ITIM pathway is anti-inflammatory [98]. FcR also requires ubiquitination to mediate phagocytosis [99]. Research has shown that the FcR-ITAM-Syk signalling pathway is similar to the Dectin-1 signalling pathway [100], and there is a crosstalk with the TLR-MyD88 pathway [101].
On the other hand, complement receptors are members of the integral family that primarily recognise and bind to complement proteins [102]. Although there are several complement receptors, scientific research has only shown CR3, CR4, and CRIg on macrophages. CR3 and CR4 are involved in phagocytosis, leukocyte trafficking and migration, synapse formation and co-stimulation. Furthermore, CRIg is part of the immunoglobulin superfamily [103]. There are species-specific differences in complement receptor activation [104]. Although early phagocytosis studies concluded that complement receptor-mediated phagocytosis was less pro-inflammatory in macrophages, recent research found significant up-regulation of pro-inflammatory mediators during complement receptor-mediated phagocytosis [105]. As is the case for many receptors, other biomolecules can affect the expression or function of complement receptors. For example, Pyk2 is essential for CR3-mediated phagocytosis as it significantly contributes to the coordination of phagocytosis-promoting signals downstream of CR3 [102]. Likewise, Vitamin D upregulates the expression of CRIg and its phagocytic activity [106].
3.4 Biochemical processes that regulate receptor function
Ubiquitination describes post-translational modification with small conserved peptides known as ubiquitin. Ubiquitin covalently attaches to the amino group of lysine residues of target proteins. Amongst other functions, protein ubiquitination enables the internalisation and formation of early endosomes [99].
Three major classes of ubiquitinating enzymes mediate ubiquitination: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin ligases. Two genes encode for the E1 ubiquitin-activating enzymes, about 100 genes encode the E2 ubiquitin-conjugating proteins, and over 1000 genes encode for the E3 ubiquitin ligases. E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases work together to create high specificity of protein ubiquitination [107, 108]. E3 ubiquitin ligases regulate TLR signalling; Nrdp1 ubiquitylates MyD88 and targets it for degradation [109]; TRAF6 is also essential for MyD88-dependent, and TRIF-dependent TLR signalling [110], Triad3A and Pelle-interacting proteins also participate in TLR signalling [111, 112]. In addition, the translocation of NFκβ to the nucleus in response to TLR activation highly depends on the ubiquitination of IKK proteins bound to NFκβ to keep it in the cytosol [107, 113]. Monoubiquitylation may indirectly influence PRR function by; initiating the internalisation of cell surface receptors by phagocytosis, sourcing amino acids for protein synthesis, negatively regulating RIG-I helicases and affecting antigen presentation by MHC class I molecules [107, 114, 115].
Phosphorylation describes the attachment of phosphate groups to amino acid residues such as tyrosine, serine, and threonine by protein kinases. TLR Phosphorylation occurs on tyrosine residues and activates interaction with adapter proteins. LPS causes IRAK1-mediated phosphorylation; consequently, IRAK1 phosphorylates Tollip–a negative regulator of TLR-MyD88 signalling, enabling TRAF6 activity essential for the downstream TLR-MyD88 signalling. Moreso, IRAKs interact with the MyD88 death domain [116, 117]. The Serine/Threonine kinase PI3 is vital for activating transcription factors downstream of the TLR signalling pathway [116, 118]. Furthermore, knockout of MyD88 enhanced phosphorylation of IRF3, resulting in significant secretion of IFNβ. Finally, inhibition of MNK kinases decreased macrophage TNFα secretion [119, 120].
The phospholipid remodelling pathway describes the release and esterification of fatty acids in phospholipid pools. Phospholipid remodelling is an efficient energy source, generates membrane diversity and asymmetry, regulates protein lipidation, and the synthesis of PAF, leukotrienes, and eicosanoids [121, 122]. The quantity of arachidonic acid during inflammation in macrophages relies on the reacylation and deacylation of phospholipids. Macrophage TLR activation also alters the phospholipid composition of the macrophage membrane by activating phospholipid remodelling enzymes [123, 124, 125].
Lipid rafts function as platforms for internalisation and early endosomal sorting functions. They are nano-sized dynamic liquid-ordered plasma membrane domains enriched with cholesterol and sphingolipids and resistant to extraction with non-ionic detergents [126, 127, 128, 129, 130]. Lipid rafts participate in membrane transport [130] and signal transduction. They are also essential for receptor-mediated endocytosis [128] and control signal transduction by averting protein-protein interactions and inherent protein activities [129].
3.5 Regulators of phagosomes, and lysosomes
The cellular mechanism of phagocytosis involves the formation of phagosomes, phagosome maturation and the fusion of phagosomes with lysosomes [18, 131]. Phagosomes are cellular vesicles formed to contain the ingested pathogen [132]. There are early and late phagosomes; early phagosomes fuse with early endosomes, whereas phagosome maturation results in late phagosomes. Profound rearrangements of the actin cytoskeleton occur to extend the plasma membrane into a phagocytic cup that internalises the pathogen [133]. Several biomolecules influence this process. For example, dynamin-2 participates in phagosome closure in macrophages. It co-localises with actin during phagosome formation [134].
Furthermore, converting PIP2 to PIP3 is essential for pseudopod extension and phagosome closure. Although PIP2 participates in clathrin-mediated endocytosis, research has shown that clathrin-mediated endocytosis does not influence phagosome formation or maturation [134, 135]. Phagosomal development occurs when phagosomes acquire microbicidal and lytic enzymes after fusion with various endolysosomal compartments. During phagosomal maturation, the phagosome lumen increases its acidification levels [136].
The Nod-like receptor (NLRP3), critical for inflammasome activation, also affects phagosome maturation. Knockout of NLRP3 from macrophages impaired phagosome acidification and phagolysosome formation [137].
SNAP23, a membrane SNARE protein, caused a significant delay in phagosome maturation after its knockdown. On the hand, overexpression of SNAP23 enhances phagosome acidification in J774 macrophages [138].
During FcγR-mediated phagocytosis, actin polymerisation and reorganisation occur, which drives the formation of a phagocytic cup. Rho GTPases promote the polymerisation of F-Actin, thereby regulating cytoskeletal dynamics and affecting cell polarity and motility. As phagolysosome formation requires the disappearance of the F-Actin structure surrounding the phagosome, Rho GTPases participate in this process. Scientific evidence shows that RhoC modulates phagosome formation by modifying actin cytoskeletal remodelling [133]. Furthermore, Syk, which mediates FcgammaR signalling, interrupts the reconstruction of F-Actin around phagosomes, thereby accelerating the fusion of phagosomes with lysosomes [132].
Rab GTPases are proteins that play crucial roles in phagosome maturation [136, 139]. They constitute the most prominent family of small monomeric GTPases that function as molecular switches by cycling between their GDP and GTP-bound forms and regulating membrane trafficking [140]. Rab5 participates in early phagosome maturation by regulating fusion with sorting endosomes, and Rab 7 allows late phagosomes leading to the formation of phagolysosomes [136, 140]. Rab20 regulates phagosome maturation during FcγR-mediated phagocytosis [140].
Lysosomes are membrane-bound acidic compartments formed by lipid bilayers containing proteins such as LAMPs, Rab GTPases, LIMP, CD63, and over 60 hydrolases [141, 142, 143]. Lysosome function is heavily dependent on its fusogenic and acidic properties. The cytosolic tails of LAMP proteins interact with microtubules, thus having an essential role in lysosome function. Moreso, the lack of Rab14 slowed the addition of LAMP1 and lysosomal cathepsin, implying a slower formation of completely bioactive lysosomes [136].
In conclusion, the complex process of phagocytosis is crucial in macrophages as they are professional phagocytes. Numerous biomolecules participate directly or indirectly in macrophage phagocytosis, hence the complexity. This chapter has described some of these biomolecules and biochemical processes that regulate macrophage phagocytosis.
3.6 Conclusion
In conclusion, macrophages play an important role as early responders to infection through their primary phagocytic function. This primary function is upheld by the synergy of pathogen associated molecular patterns and macrophage recognition molecules (opsonins and pattern recognition receptors) leads to downstream effects such as phagosome formation, lysosome formation, ubiquitination, phosphorylation, and phospholipid remodelling. Macrophage regulation is still being studied and there are recent discoveries of how macrophages can be regulated. Therefore, in spite of ample information about the regulation of phagocytosis in macrophages, there is more to learn. A better understanding of the regulation of phagocytosis can aid the use macrophages for therapeutic purposes (Figure 1).
Figure 1.
Graphical summary.
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