The Metallothionein-Zinc Landscape: How It Shapes Antimicrobial Immunity

Abstract

Nutritional immunity refers to the ability of the host to sequester nutrients from pathogens during infection. Metal ions are important for microbial survival and pathogenesis as well as for host defenses. For example, while zinc (Zn²⁺) is crucial for microbial fitness within the host, immune cells deprive microbes of these metal ions and retain it for their own defense. However, excess Zn²⁺ may be toxic to both the host and the pathogen. Therefore, Zn²⁺ regulation is a central component of host-pathogen interactions and antimicrobial immunity. Metallothioneins (MTs) are a family of highly conserved cysteine-rich proteins that are ubiquitously expressed in most organisms. Immune cells express MTs in response to a variety of stimuli including cytokines, chemokines, and infectious agents. They regulate intracellular Zn²⁺ homeostasis, protect from oxidative stress, and modulate host immunity during infection. Although Zn²⁺ signals are well known to alter immunological processes, our knowledge of how the MTs-Zn²⁺ axis affects immune response to infections is relatively scarce. Emerging evidence points to a significant role for MTs in regulating host immunity. Thus, this chapter discusses immunomodulatory roles of MTs with a focus on Zn²⁺ regulation in response to pathogen attack.

4.1 Introduction

Nutritional immunity is a mechanism of defense by which the host restricts nutrient access to pathogens to inhibit their growth. Metal ions are micronutrients that are essential for life. They regulate important functions of immune cells as well as that of invading microbes. One form of nutritional immunity is limitation of metal ions by modulating their availability, concentration, and distribution inside the cell. Zn²⁺ is an important metal ion whose concentration in circulation (plasma and serum) rapidly declines during infection (Besecker et al. 2011; Uttra et al. 2011). This phenomenon is postulated to deprive invading pathogens of Zn²⁺, control their survival, and prevent dissemination (Hennigar and McClung 2016). Zn²⁺ deficiency or excess alters the number and activities of immune cells thereby modulating host susceptibility to infection. A decrease in dietary Zn²⁺ uptake increases the risk of infectious diseases such as tuberculosis, shigellosis, pneumonia, measles, human immunodeficiency virus (HIV), acute cutaneous leishmaniosis, and malaria (Maywald et al. 2017). On the other hand, dietary Zn²⁺ supplementation aids in improving immune defenses against some of the microbes that cause the aforementioned infections as well as diarrhea, leprosy, chronic hepatitis C, and acute lower respiratory infection (Overbeck et al. 2008). However, there may be a very narrow window within which Zn²⁺ exerts beneficial effects. Recent data show that excess dietary Zn²⁺ uptake increases susceptibility to Clostridioides difficile and intensifies disease severity suggesting that exogenous Zn²⁺ administration may adversely impact the clearance of some pathogens (Zackular et al. 2016).

Effects of Zn²⁺ on immunity are complex and have been under investigation for many years. Zn²⁺ deprivation or supplementation in vitro and in vivo affects the expression of several genes in immune cells associated with Zn²⁺ homeostasis, cytokine response, stress responses, reactive oxygen species and reactive nitrogen species (ROS and RNS) signaling, metabolism, and survival (Beck et al. 2006; Cousins 1998; Cousins et al. 2003; Haase et al. 2007). Zn²⁺ deficiency inhibits differentiation, proliferation, and survival of monocytes, polymorphonuclear leukocytes (PMN), natural killer cells, and T and B cells (Bonaventura et al. 2015). However, an excess of Zn²⁺ may lead to toxicity. Therefore, Zn²⁺ homeostasis is tightly regulated in immune cells by Zn²⁺ binding proteins such as metallothioneins (MTs), glutathione, the Zn²⁺-responsive transcription factor metal-response element-binding transcription factor-1 (MTF-1), Zn²⁺-transporters, Zn²⁺-permeable ion channels such as transient receptor potential mucolipin 1 (TRPML1), and Zn²⁺ storage organelles such as zincosomes (Andrews 2001; Crawford et al. 2018; Eide 2004; Inoue et al. 2015; Liu et al. 2012; Palmiter 2004; Palmiter and Huang 2004; Vallee 1995). Taken together, regulation of Zn²⁺ is necessary for adequate immune function and aberrant homeostasis of these metal ions may have adverse effects on the host’s ability to defend microbial invaders.

Microorganisms require Zn²⁺ for survival. Thus, restriction of Zn²⁺ during infection may be an effective strategy that immune cells utilize to inhibit microbial growth. Several recent studies have brought to light the importance of Zn²⁺ limitation by MTs in immune cells. Zn²⁺ is bound to MTs through seven binding sites with picomolar binding affinity. This attribute facilitates controlled Zn²⁺ exchange between proteins and promotes nutritional immunity in host cells, where accessibility of Zn²⁺ to pathogens must be restricted. Our work revealed that MT1 and MT2 inhibit fungal growth via sequestration of Zn²⁺ in infected macrophages (Subramanian Vignesh et al. 2013). The MT3 isoform has a very distinct role: it facilitates Zn²⁺ uptake by intracellular fungi (Subramanian Vignesh et al. 2016). Nonetheless, the finding that MTs are an important component of the antimicrobial defense arsenal raises interesting questions about their mechanisms of action. In sum, MTs are expressed in immune cells, regulate inflammatory responses, and control host-pathogen interactions indicating that they may be at the forefront of immunological fitness in the host. Given the role of Zn²⁺ in pathogen virulence and the emerging importance of MTs in immune responses, this chapter focuses on the roles of Zn²⁺ and the MT-Zn²⁺ landscape in antimicrobial immunity.

4.2 Low Zinc Spells a High Infection Risk

Zn²⁺ has vital roles in biochemical processes and is crucial for maintaining the structure, stability, and adequate activity of macromolecules, such as proteins and nucleic acids. About 10% of the human proteome consists of Zn²⁺ binding proteins which require this ion for proper physiological function (Andreini et al. 2006). Zn²⁺ is the second most abundant (total concentration in the human body 2–3 g) transition metal after Fe²⁺ in humans (Kehl-Fie and Skaar 2010).

Prasad et al. first discovered Zn²⁺ deficiency in human male dwarfs in the Middle East in 1963 (Prasad et al. 1963). Zn²⁺ deficiency is associated with susceptibility to infections, memory impairment, and growth retardation. Zn²⁺ deficient animals have decreased immunity to viral infections such as Herpes simplex (Feiler et al. 1982) and Semliki forest (Singh et al. 1992); bacterial infections such as Listeria monocytogenes (Carlomagno et al. 1986; Coghlan et al. 1988), Francisella tularensis (Pekarek et al. 1977), Mycobacterium tuberculosis (McMurray et al. 1990), and Salmonella enteritidis (Kidd et al. 1994); parasitic infections such as Trypanosoma cruzi (Fraker et al. 1982), T. musculi (Lee et al. 1983), Toxoplasma gondii (Tasci et al. 1995), and Plasmodium yoelii (Shankar et al. 1995); fungal infections such as Candida albicans (Salvin et al. 1987); and helminthic infections such as Fasciola hepatica (Flagstad et al. 1972), Heligmosomoides polygyrus (Minkus et al. 1992; Shi et al. 1994), Strongyloides ratti (Fenwick et al. 1990b), Schistosoma mansoni (Nawar et al. 1992), and Trichinella spiralis (Fenwick et al. 1990a). Therefore, deficiency of Zn²⁺ cripples the host’s ability to clear infections and has a considerable impact on human health.

4.3 The Zinc Pill: To Take or Not to Take?

Zn²⁺ supplementation in general has a beneficial role in antimicrobial immunity. Acrodermatitis enteropathica (caused by mutations in the Zn²⁺ importer, ZIP4) is a rare and severe genetic autosomal recessive disorder characterized by acral and periorificial dermatitis, alopecia, and diarrhea. Weakened resistance to fungi, bacteria, and viruses is observed in bovine acrodermatitis enteropathica during Zn²⁺ deficiency. In humans, oral Zn²⁺ supplementation ameliorates symptoms associated with this disorder (Ciampo et al. 2018; Hambidge et al. 1977). Moreover, Zn²⁺ supplementation reduces the incidence of acute and chronic persistent diarrhea, dysentery (DD), acute lower respiratory infections (Ruel et al. 1997; Sazawal et al. 1995, 1996, 1998), malaria (Bates et al. 1993; Shankar et al. 2000), recurrent furunculosis (Brody 1977), and infection caused by the parasite S. mansoni (Friis et al. 1997).

Zn²⁺ lozenges reduce the duration of common cold (Mossad et al. 1996) by blocking the binding of HRV14 on the viral surface to the adhesion molecule ICAM-1 on the nasal mucosal surface, ultimately leading to reduced viral uptake (Novick et al. 1996). Low Zn²⁺ levels commonly detected in the plasma and serum of human HIV patients is associated with disease progression (Bogden et al. 1990; Khalili et al. 2008). Zn²⁺ supplementation partially reverses these effects (Falutz et al. 1988; Shankar and Prasad 1998), not by curtailing viral load but by dampening the frequency of diarrheal episodes and delaying immunological failure. This evidence suggests that adequate Zn²⁺ supplementation may be used as an adjunct therapy in HIV-infected adults (Baum et al. 2010). However, poor survival has also been reported in HIV-infected patients with high Zn²⁺ intake (Tang et al. 1996). In addition, as noted above, excess dietary Zn²⁺ intake may adversely affect C. difficile clearance. Clearly, determining accurate Zn²⁺ dosage is an indispensable step in the success of Zn²⁺ therapy for infection control. While optimal Zn²⁺ availability promotes resistance to infection, Zn²⁺ excess may be an Achilles’ heel in maximizing the therapeutic potential of this metal ion.

4.4 Zinc: A Prominent Driver on the Road to Innate Defense

Innate immunity delivers a rapid first line of defense against invading pathogens. Activation of myeloid cells with pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) triggers phagocytosis, cytokine release, antigen presentation to T cells, and bolsters antimicrobial defenses. In the following sections, we discuss the molecular mechanisms of Zn²⁺ regulation in innate immune cells and counter-defense mechanisms utilized by pathogens.

Polymorphonuclear leucocytes (PMNs) or granulocytes such as neutrophils, eosinophils, and basophils exert robust antimicrobial functions. Neutrophils are the most abundant PMNs. Shortly after phagocytosis of microbes, PMNs migrate into the infected tissue via adhesion and chemotaxis. They generate reactive oxygen species (ROS) through nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) and kill the invading pathogens. Oxidative burst is an important defense mechanism of activated PMNs that is impaired by Zn²⁺ chelation. Conversely, Zn²⁺ is redox-inert, it acts as an antioxidant via catalytic action of the Cu²⁺ (copper)/Zn²⁺-superoxide dismutase. Moreover, Zn²⁺ dampens inflammatory responses that would otherwise augment oxidative stress (Lee 2018).

Zn²⁺ has an important role in the regulation of number and activities of myeloid cells. Rats fed a Zn²⁺ deficient diet have increased total white blood cells and granulocytes (neutrophils, eosinophils, and basophils) in blood (Someya et al. 2009). Decreased dietary Zn²⁺ intake or absorption diminishes eosinophil numbers in blood, liver, and lungs of mice and impairs their ability to clear H. polygyrus or Ascaris suum, a parasitic nematode (large roundworm) that causes ascariasis in pigs (Laubach 1990; Scott and Koski 2000). In contrast, Zn²⁺ deficiency augments eosinophilic allergic inflammation, and dietary Zn²⁺ supplementation reduces its intensity (Richter et al. 2003). Eosinophil cationic protein (ECP) is a potent secretory cytotoxic granule that has bactericidal and antiviral activities. Zn²⁺ inhibits the release of ECP from eosinophils in culture (Winqvist et al. 1985). Likewise, Zn²⁺ inhibits the release of granular protein histamine from basophils and mast cells in the human lung (Marone et al. 1986).

Zn²⁺ regulates important processes in immune cells that are crucial to infection control. Physiological Zn²⁺ concentration (10⁻³–10⁻² mmol/L) in culture medium facilitates serum opsonic activity and ROS-generating capability in human neutrophils whereas excess Zn²⁺ (10 mmol/L) subdues it (Hasegawa et al. 2000). Reduced Zn²⁺ levels in the serum and in neutrophils is associated with impaired phagocytosis and diminished T cell-mediated immunity (Karzakova 2005). Voltage-gated proton (Hv1) channels transport H⁺ across the phagosomal membrane and regulate NADPH oxidase function. Hv1-mediated proton efflux balances the negative charge translocated by NADPH oxidase and provides substrate H⁺ for the formation of hydrogen peroxide, hypochlorous acid, and ROS crucial to killing pathogens (Decoursey 2012). Zn²⁺ inhibits Hv1 via two plausible mechanisms: it binds at low concentrations to one site on the channel which prevents the opening of the Hv1 pore, thereby inhibiting proton conduction. At high concentrations, Zn²⁺ binds to a second site and thwarts the outward movement of Hv1 voltage sensor (Qiu et al. 2016). In sum, Zn²⁺ exerts antioxidant functions by mitigating superoxide burst (Maret 2006; Prasad 2014), which may explain increased superoxide stress in Zn²⁺-deficient cells.

Antimicrobial peptides produced by some myeloid cells can trigger nutritional immunity through Zn²⁺ restriction. Neutrophils release calprotectin, a heterodimer of S100A8 and S100A9, and S100A12 (calgranulin C) peptides that sequester Zn²⁺ from pathogens to impair their growth. Calprotectin and calgranulin C restrict Zn²⁺ access to Staphylococcus aureus, C. albicans, and Helicobacter pylori thus stalling their growth (Besold et al. 2018; Corbin et al. 2008; Kehl-Fie and Skaar 2010). Likewise, S100A7 (psoriasin) released by keratinocytes kills Escherichia coli by withholding Zn²⁺ (Gläser et al. 2005).

NETosis, defined as the development and secretion of neutrophil extracellular traps (NETs), is a cell death mechanism of neutrophils in response to infections. NETs are composed of DNA, chromatin, and granular proteins which entrap and subsequently kill extracellular bacteria (Brinkmann et al. 2004). Zn²⁺ acts as a signaling molecule to facilitate NETosis (Hasan et al. 2013). Activated neutrophils elevate intracellular free Zn²⁺ via a protein-kinase C-ROS-dependent mechanism. While the precise source of this Zn²⁺ pool in neutrophils is not known, ROS may trigger the release of Zn²⁺ bound to sulfur on proteins such as MTs or glutathione (Maret 1994, 2000). Perhaps, Zn²⁺ is released from multiple reservoirs that may include Zn²⁺-bound proteins, Zn²⁺ storage organelles, or may be imported from the extracellular milieu. Deciphering the origin of the Zn²⁺ pool may provide clues to the mechanisms by which this ion prepares the neutrophil defense armor that is rapidly deployed during infection. Zn²⁺ also regulates neutrophil chemotaxis. These cells migrate into the host infected tissues in response to chemoattractants such as the bacterial product, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF). In vitro, Zn²⁺ promotes chemotaxis of neutrophils to fMLF (Hasan et al. 2016; Hujanen et al. 1995; Vruwink et al. 1991). In sum, Zn²⁺ alters several functions of neutrophils such as chemotaxis, phagocytosis, NETosis, and pathogen killing.

Monocytes and macrophages phagocytose microbes, present antigen, and secrete cytokines to shape the immune response. Rats fed a Zn²⁺-deficient diet have increased number of total monocytes in blood (Someya et al. 2009). A lack of Zn²⁺ promotes differentiation and maturation of monocytes into macrophages by augmenting cAMP production by adenylate cyclase in vitro (Dubben et al. 2010). Zn²⁺ depletion in human monocytes improves the clearance of E. coli, S. aureus, and Streptococcus pneumoniae via phagocytosis and oxidative burst (Mayer et al. 2014).

Studies investigating the role of Zn²⁺ on cytokine expression and secretion have produced varying results depending on Zn²⁺ concentration, duration of Zn²⁺ depletion, experimental conditions, and model system. Supplementation of Zn²⁺ in serum-free media enhances the expression of interleukin 1 beta (IL-1β) and TNF-α in human peripheral blood mononuclear cells (PBMC) (Wellinghausen et al. 1996). Increasing amounts of Zn²⁺ dose-dependently inhibit monocyte activation caused by the superantigens, staphylococcal enterotoxins A and E (SEA, SEE), the Mycoplasma arthritidis–derived superantigen (MAS), but not toxic shock syndrome toxin-1 (TSST-1). Zn²⁺ interferes with the interactions between SEA, SEE, and MAS and their major histocompatibility complex class II (MHC-II)-binding sites. These data demonstrate that Zn²⁺ levels control the secretion of cytokines and response to superantigen challenge (Driessen et al. 1995).

Macrophages deploy two divergent Zn²⁺-associated defense mechanisms against intracellular pathogens. On the one hand, these cells may intoxicate M. tuberculosis or E. coli with excess Zn²⁺ to kill it (Botella et al. 2011). On the other hand, macrophages sequester Zn²⁺ from Histoplasma capsulatum residing within phagosomes (Subramanian Vignesh et al. 2013). Thus, one may speculate that Zn²⁺ acts as a double-edged sword: inadequate amounts arrest microbial growth, while an excess intoxicates them. What mechanisms influence the immune system’s decision to “withhold” versus “intoxicate” to overcome microbial pathogenesis remains a conundrum.

T cell–derived IFNγ is important for macrophage activation (Prasad 2014). Insufficient dietary Zn²⁺ intake compromises IFNγ production by T helper type 1 (Th1) cells resulting in impaired activation of monocytes/macrophages (Agnello et al. 2003; Prasad 2000). Moreover, Zn²⁺ deficiency reduces the production of IL-12 by monocytes/macrophages, yielding poor Th1 differentiation (Bao et al. 2011; Langrish et al. 2004). Thus, changes in the Zn²⁺ status not only affect differentiation and activation of monocytes/macrophages but may also compromise adaptive immunity.

4.5 The MT-Zinc Immune-Landscape: An Old Axis with a New Tale

In 1957, Margoshe and Vallee discovered MTs as Cd²⁺ (cadmium)-binding proteins from the horse renal cortex (Margoshes and Vallee 1957). MTs sense intracellular as well as environmental cues and regulate cellular Zn²⁺ homeostasis through sequestration, mobilization, and release (Subramanian Vignesh and Deepe Jr 2017). MTs have a higher Zn²⁺-binding constant (K_Zn = 3.2 × 10¹³ M⁻¹ at pH 7.4) than most Zn²⁺-binding proteins. Despite this property, they facilitate controlled Zn²⁺ release to proteins with a lower stability constant for Zn²⁺ (Jacob et al. 1998). This metal is readily released from only one of the sites on MT1 and MT2 through interactions with adenosine triphosphate (ATP), guanosine triphosphate (GTP), or glutathione (Maret 2000).

There are 4 MT isoforms in mice and over 16 in humans (Quaife et al. 1994; Uchida et al. 1991). MTs are present in immune cells including those in the bone marrow (Liu et al. 2004), axillary lymph nodes (Haerslev et al. 1994), spleen (Huang et al. 2019; Mita et al. 2002), and thymus (Savino et al. 1984). In immune cells, MTs are induced by Zn²⁺, Cu²⁺, and Cd²⁺ (Aydemir et al. 2006; Huber and Cousins 1993; Makhijani 1998; Thorvaldsen et al. 1995); cytokines such as GM-CSF, TNFα, IFNγ, IL-1, IL-4, IL-6, and IL-27 (Cousins and Leinart 1988; Schroeder and Cousins 1990; Sciavolino and Vilček 1995; Ullio et al. 2015; Subramanian Vignesh et al. 2013, 2016; Chuan Wu et al. 2013a, b); and microbial ligands such as lipopolysaccharide (LPS) (Arizono et al. 1995; Leibbrandt and Koropatnick 1994), ROS (Dalton et al. 1994; Nourani et al. 2011; Tate et al. 1995), and nitric oxide (NO) (Arizono et al. 1995). In turn, MTs may regulate the activity of some of these immune modulators. For example, MTs scavenge ROS and regulate the function of GM-CSF and IL-4-polarized macrophages (Li et al. 2004; Subramanian Vignesh et al. 2013, 2016). The interrelationship between immune modulators and MTs is schematically represented in Fig. 4.1.

Fig. 4.1

The association between MTs and immune mediators. MTs are induced by metal ions (Zn²⁺, Cu²⁺), infection, pathogen associate molecular patterns (PAMPs), cytokines, and superoxide radicals. The protein family in turn controls responses to each of these stimuli, thereby establishing a feedback loop between immune mediators and MTs

MTs influence a variety of immune responses in vivo and in vitro. For example, IL-27 induces MT1 and MT2 that prevent type 1 regulatory T (Tr1) cell development. This effect of MTs is due to negative feedback inhibition of signal transducer and activator of transcription (STAT)1 and STAT3 phosphorylation, resulting in diminished Tr1 differentiation and IL-10 production. Thus, the dynamic balance between STATs and MTs calibrates the development and suppressive function of Tr1 cells. The control of Zn²⁺ within the intracellular milieu may arm MTs with the ability to control STAT phosphorylation. This is plausible because Zn²⁺ inhibits the function of phosphatases that downmodulate STAT signaling. By sequestering the ion, MTs may render phosphatases active, leading to increased STAT dephosphorylation (Supasai et al. 2017; Chuan Wu et al. 2013a, b). From an antimicrobial immunity standpoint, this attribute of MTs in Tr1 cells may benefit the host in clearing infection rapidly, before suppressive immunity emerges to subdue inflammation and promote tissue repair.

Mt1^−/−Mt2^−/− mice exhibit stronger humoral responses through the elevation of nuclear factor-kappaB (NF-κB) transcription factor activity in splenocytes. These knockout mice display higher circulatory immunoglobulin levels, enhanced B cell differentiation upon OVA challenge, and lymphoproliferative responses to mitogenic stimulation (Crowthers et al. 2000). Exogenous administration of MTs into these mice dampens humoral immunity (Lynes et al. 1993). Thus, MTs temper antibody production by B cells. MTs also influence cytokine production by basophils. Stimulation of the Fc epsilon receptor 1 (FcɛRI) induces MT1 and MT2 in mouse basophils to regulate intracellular Zn²⁺. Lack of MTs increases intracellular free Zn²⁺ which inhibits calcineurin (CaN) phosphatase activity and thus impacts FcɛRI-induced nuclear factor of activated T-cell (NFAT)-dependent IL-4 production (Ugajin et al. 2015).

4.6 The MT-Zinc Axis in Infection

Several studies have brought to light the complex functions of MTs and the MT-Zn²⁺ axis in antimicrobial defense. Below, we discuss how the control of signaling pathways, Zn²⁺ homeostasis, and inflammatory responses by different MTs converge to dictate the outcome of host–pathogen interactions.

4.6.1 Bacterial Infection

NF-κB is essential for adequate innate immunity to infection. Zn²⁺ is an important negative regulator of NF-κB, while ROS is a positive regulator. MTs subvert the action of Zn²⁺, possibly by sequestering the intracellular free Zn²⁺ pool indicating that MT is an important intracellular modulator of NF-κB activation (Kim et al. 2003).

In polymicrobial sepsis in mice, deficiency of Zn²⁺ promotes systemic infection and NF-κB activation leading to elevated inflammation, lung injury, and mortality. Zn²⁺ supplementation prior to initiation of sepsis effectively reverses these effects (Bao et al. 2010). NF-κB induces the expression of the Zn²⁺ importer SLC39A8 (ZIP8) that imports Zn²⁺ to inhibit IκB kinase (Iκκ) activity. These findings identify a negative feedback loop that directly regulates a master transcription factor via coordination of Zn²⁺ metabolism (Liu et al. 2013). Salmonella typhimurium is a causative agent for inflamed gut. Macrophages infected with this pathogen exhibit elevated levels of free cytoplasmic Zn²⁺ that downmodulates NF-κB activity, as a result affecting the expression of reactive species (ROS and RNS) -forming enzymes phos47 (an NADPH oxidase subunit), inducible NO synthase (iNOS), and proinflammatory cytokines. Macrophages counter this change in Zn²⁺ homeostasis by augmenting MT1 and MT2 that scavenge free Zn²⁺ and restore ROS and RNS production to kill the pathogen. Thus, the limitation of free Zn²⁺ by MTs facilitates the control of intestinal colonization by S. typhimurium (Wu et al. 2017). In contrast, M. tuberculosis–infected macrophages rapidly increase free Zn²⁺ to poison this intracellular pathogen. This phenomenon is associated with an increase in MTs, MTF-1, and ZnT1, an exporter of Zn²⁺. MTF-1 translocates to the nucleus upon infection to induce MTs and ZnT1, suggesting that the host mounts a direct and quick response to protect itself from Zn²⁺ intoxication (Botella et al. 2011). Together, these studies suggest that macrophages possess two opposing mechanisms to exert antimicrobial immunity: Zn²⁺ depletion and Zn²⁺ poisoning. The effect of MTs on NF-κB activation is paradoxical depending on whether MTs scavenge Zn²⁺ or ROS. By scavenging ROS, MTs stabilize Iκκ, an inhibitor of NF-κB, ultimately downmodulating activation of this transcription factor. In gastric cells of Mt1^−/−Mt2^−/− mice with Helicobacter pylori infection, NF-кB activation and downstream production of macrophage inflammatory protein (MIP)-1α and monocyte chemoattractant protein (MCP)-1 is increased. The absence of MT1 and MT2 leads to erosive lesions and elevates infiltration of inflammatory leukocytes in the gastric mucosa. Thus, MTs protect against gastric ulceration during H. pylori infection by negatively regulating NF-кB activation (Mita et al. 2008).

4.6.2 Fungal Infection

How macrophages utilize the Zn²⁺ pool to resolve mycobacterial versus fungal infection presents an interesting paradox. Granulocyte macrophage-colony stimulating factor (GM-CSF) augments antimicrobial defenses against the intracellular fungus H. capsulatum. GM-CSF activated, infected macrophages increase MT1 and MT2 expression via activation of STAT3 and STAT5 transcriptional factors. These MTs bind to the macrophage free Zn²⁺ pool, denying Zn²⁺ access to the pathogen residing within phagosomes. In fact, Zn²⁺ is mobilized into the Golgi apparatus in association with an increase in the Zn²⁺ exporters, ZnT4 and ZnT7 that are expressed on the Golgi membrane. The Zn²⁺ sequestration “feat” by MTs simultaneously boosts ROS to stall fungal growth. Intriguingly, GM-CSF also elevates Zn²⁺ import via the importer Zip2, perhaps, to support an increased demand for Zn²⁺-dependent host processes during pathogen insult (Subramanian Vignesh et al. 2013).

IL-4 and IL-13 are cytokines that shape macrophage polarization to the M(IL-4) and M(IL-13) phenotypes, respectively. Studies on how Zn²⁺ levels influence macrophage polarization in rodents and human cell lines have produced distinct results. Zn²⁺ deficiency in rodents diminishes IL-4 production by Th2 cells and the proportion of M(IL-4) polarized macrophages in the spleen (Kido et al. 2019). In contrast, in human THP1 monocyte-derived macrophages, Zn²⁺ deficiency inhibits M1 polarization by IFNγ and LPS but does not affect M2 polarization by IL-4. Exogenous addition of Zn²⁺ suppresses the emergence of M(IL-4) macrophages in vitro (Dierichs et al. 2018). The use of different experimental models (rodents versus human cell line and dietary Zn²⁺ deficiency versus Zn²⁺ depletion/supplementation in culture media) may explain some of these findings. Nonetheless, these studies suggest that Zn²⁺ impacts macrophage polarization and can regulate the balance between M1 and M2 polarization states.

M2 macrophages aid in parasite clearance but harbor a permissive milieu for persistence of intracellular pathogens. Recent literature has demonstrated that IL-4 augments intracellular free Zn²⁺ in bone marrow–derived macrophages, microglia, and human monocyte-derived macrophages (Aratake et al. 2018; Subramanian Vignesh et al. 2016). M(IL-4) macrophages from the bone marrow specifically upregulate the MT3 isoform via STAT6 and interferon regulatory factor (IRF)4 signaling. The relationship between MT1/MT2 and MT3 is dichotomous, in that the latter expands the free Zn²⁺ pool while the former shrinks it in macrophages (Subramanian Vignesh et al. 2013, 2016). The action of cathepsin proteases enhances Zn²⁺ release from MT3 (Subramanian Vignesh et al. 2016). This is notable because such an increase in the free Zn²⁺ reservoir places the intracellular pathogen at an advantage: it assimilates a pool of Zn²⁺ that was once a part of the host. The finding establishes a link between MT-Zn²⁺ metabolism and the permissive nature of M(IL-4) macrophages to intracellular microbes. Figure 4.2 schematically outlines the distinct functions of MT1, MT2, and MT3 in macrophage defenses.

Fig. 4.2

MT isoforms have distinct roles in host–pathogen interactions. GM-CSF and IL-6 elevate MT1 and MT2 that promote intracellular Zn²⁺ sequestration. Limitation of free Zn²⁺ in the host inhibits intracellular microbial growth via the induction of oxidative burst and restriction of Zn²⁺ access to the microbes. In contrast, IL-4 and IL-13 augment MT3 expression that increases the intracellular free Zn²⁺ pool and may dampen superoxide defenses. Moreover, intracellular pathogens may exploit this mechanism for acquisition of the metal ion for survival

4.6.3 Viral and Parasitic Infections

Our knowledge of how the MT-Zn²⁺ axis controls immune responses to viruses and parasites is limited. Infection with viruses including coxsackievirus B type 3 and influenza A/PR8 upregulates MTs in the liver, lung, kidney, and spleen. The mechanism of induction involves MTF-1, STAT3 signaling, and glucocorticoids (Ghoshal et al. 2001; Ilbäck et al. 2004). In contrast, infection with the hepatitis C virus is associated with a reduction in MT expression in the liver. Increasing MT levels through Zn²⁺ supplementation decreases viral load pointing at a protective function of the MT-Zn²⁺ axis in viral clearance (Carrera et al. 2003; Read et al. 2018). Further studies are required to elucidate the precise mechanisms by which MTs influence the immune systems’ ability to curtail viral uptake, replication, and shedding.

The parasite T. cruzi, the causative agent of Chagas disease, leads to cardiomyopathy and gastrointestinal inflammation. Infection with this pathogen reduces the expression of MT1 in the liver, while augmenting NO levels and oxidative stress. Whether the benefits of reducing NO are conferred by restoration of MTs is unknown, but chemically scavenging NO in animals infected with T. cruzi restores MT1 expression and arrests the growth of this parasite (Gonzalez-Mejia et al. 2014).

4.7 Survival Edge: Microbes (Aim to) Get the Upper Hand

Several pathogens have developed counter-defense mechanisms to thrive within the host (Fig. 4.3). For example, to circumvent Zn intoxication by macrophages, M. tuberculosis induces heavy metal efflux P-type ATPases. CtpC, a P-type ATPase, is upregulated rapidly to expel Zn²⁺ from the microbe. A lack of CtpC causes Zn²⁺ retention within the mycobacterial cytoplasm, thereby poisoning it. Therefore, P1-type ATPases contribute to the defense armor of M. tuberculosis by dampening the toxic effects of Zn²⁺ (Botella et al. 2011). Group A Streptococcus growth is restricted by Zn²⁺ limitation caused by neutrophil-derived calprotectin. Streptococcus pyogenes encodes the Zn²⁺ importer AdcA and a Zn²⁺ sensor AdcR to compete with Zn²⁺ sequestration by calprotectin (Makthal et al. 2017). ZrfC, a plasma membrane Zn²⁺ transporter of Aspergillus fumigatus, has the ability to scavenge Zn²⁺ efficiently from lungs enabling it to grow even in the presence of calprotectin (Amich et al. 2014). Neisseria meningitides uses ZnuD, a high-affinity Zn²⁺ transporter, to circumvent Zn²⁺ deprivation (Lappann et al. 2013). This pathogen also responds to low Zn²⁺ by expression of curved DNA binding protein A (CbpA), which is a calprotectin receptor, on its outer membrane. This molecule facilitates the acquisition of Zn²⁺ bound to calprotectin by N. meningitides. Thus, the microbe defies a vital host defense mechanism and exploits it for its benefit (Stork et al. 2013). Yersinia pestis utilizes a zincophore, yersiniabactin (Ybt) synthetase, and the high-affinity Zn²⁺ transporter, ZnuABC, to obtain Zn²⁺. These are crucial in the progression of lethal septicemic plague in mice (Bobrov et al. 2014). S. typhimurium can also express ZnuABC and thrives by subduing the host’s Zn²⁺ deprivation strategies. These studies indicate that Zn²⁺ acquisition may be a “virulence determinant” in some pathogens (Liu et al. 2012).

Fig. 4.3

Zn²⁺-associated defense mechanisms. Immune cells employ various pathways (phagocytosis, oxidative burst, NETosis, Zn²⁺ depletion, and Zn²⁺ intoxication) to defend microbial invaders, whereas microbes also utilize counter-defense mechanisms including P1-type ATPases, Zn²⁺ acquisition transporters/proteins, and biofilm formation

Biofilms contain microbial communities associated with a polymeric matrix structure composed of factors such as extracellular DNA, polysaccharides, and proteins. Micromolar (100–250 μmol l⁻¹) concentrations of Zn²⁺ block biofilm formation by Actinobacillus pleuropneumoniae, Haemophilus parasuis, S. typhimurium, Escherichia coli, S. aureus, Streptococcus suis, and Klebsiella pneumoniae strains. Mechanistically, Zn²⁺ may interfere with the stability of extracellular DNA and polymers contained within the biofilm matrix and impede critical microbial processes such as iron homeostasis and energy metabolism of associated microbes (Hancock et al. 2010; Polyudova et al. 2018; Chan Wu et al. 2013a, b). Of note, in the context of biofilms, an excess of Zn²⁺ or a deficiency of it may exert the same effect: inhibition of biofilm formation. For example, biofilm formation by Staphylococcus epidermidis, S. aureus, and S. pneumoniae is inhibited by Zn²⁺ chelation or Zn²⁺ excess (Brown et al. 2017; Conrady et al. 2008; Formosa-Dague et al. 2016). It is plausible that Zn²⁺ concentrations within a narrow range are necessary to maintain structural integrity of the biofilm, while intoxicating amounts of the metal ion adversely impact the growth of microbes that facilitate biofilm development.

4.8 Concluding Remarks

The value of dietary Zn²⁺ intake to maintain immunological robustness has long been appreciated. The highly conserved nature of MTs and their ability to bind Zn²⁺ across prokaryotes and eukaryotes has prompted scientists to query their importance in cellular functions. Immune cells are no exception. As it turns out, this class of proteins fiercely guards the Zn²⁺ reservoir in immune cells and can dictate the ions’ spatiotemporal presence both intracellularly and in the extracellular milieu. This attribute of MTs is notable, because the versatility of Zn²⁺ ions in biochemical processes demands that adequate amounts of Zn²⁺ be available for immune cells when and where they need it. Recent years have illuminated our knowledge of how the host taps into the MT-Zn²⁺ landscape to challenge microbial intrusion and overcome inflammatory damage caused by pathogen insult. These findings have opened newer apertures to explore the extent to which MTs orchestrate immunological responses. An important possibility to consider is that MT may function independent of Zn²⁺, perhaps in its apo-form or by binding to another metal ion. Of note, the protein also interacts with Cu²⁺ ions to regulate Cu²⁺ homeostasis. Whether an MT-Cu²⁺ axis impacts immunological performance or the triad (MT- Zn²⁺-Cu²⁺ axis) prevails over the two is unanswered. Nonetheless, MTs have surfaced prominently in the host-pathogen realm and will pave the path to a galvanizing story that continues to be told.