3.2.2. Immunological Role of Zinc

Due to its crucial role in various physiological functions, zinc deficiency can have a significant impact on the immune system, which exhibits a high rate of cell division. The immune system relies on zinc for proper functioning, including the development and maturation of immune cells, the production of antibodies, and the regulation of immune responses. Zinc deficiency can impair immune cell proliferation and differentiation, compromise the production and activity of immune cells, and disrupt the balance between proinflammatory and anti-inflammatory responses. Consequently, individuals with zinc deficiency may experience increased susceptibility to infections, delayed wound healing, and impaired immune responses. Ensuring an adequate intake of zinc is vital for supporting optimal immune system function and maintaining overall health. Dietary sources rich in zinc include seafood, meat, legumes, nuts, and seeds [41]. The innate immune system and the adaptive immune system are the two main immune response subsystems [42]. The innate immune system's cells, particularly polymorphonuclear cells (PMNs), macrophages, and natural killer (NK) cells, are the first cells to function in pathogen identification and elimination when pathogens enter the body. The very first cells to actively infiltrate the infection site are PMNs. Through the process of phagocytosis, they capture pathogens and kill them by releasing reactive oxygen species (ROS) [35]. T and B lymphocytes, two highly specialized cells, are in charge of directing the adaptive immune response. T lymphocytes are involved in cell-mediated immune responses by activating other immune cells (T helper lymphocytes) and by producing toxic granules in cytotoxic T lymphocytes, whereas B lymphocytes are involved in the humoral immune response through the production of antibodies specifically targeted against an antigen. The innate and adaptive immune systems communicate with one another via dendritic cells (DCs). DCs circulate as immature cells, and when they come into contact with the antigen, they begin to express MHC molecules and coreceptors for the activation and stimulation of T cells.

(1) Zinc and the Innate Immune System. Proper functioning of both the innate and adaptive immune systems is essential for the optimal absorption and utilization of zinc. The innate immune system acts as the first line of defense against pathogens, providing immediate, nonspecific immune responses. Zinc plays a crucial role in supporting the functions of innate immune cells, such as neutrophils, macrophages, and NK cells, which are involved in phagocytosis, inflammation, and the elimination of pathogens. On the other hand, the adaptive immune system mounts specific immune responses by recognizing and targeting specific pathogens. Zinc is involved in the development and function of various adaptive immune cells, including T cells and B cells. It is required for T cell activation, proliferation, and differentiation, as well as for the production of antibodies by B cells. In addition to its direct effects on immune cell function, zinc is also involved in the regulation of cytokines, which are signaling molecules that coordinate immune responses. It helps maintain the balance between proinflammatory and anti-inflammatory cytokines, ensuring an appropriate immune response. Furthermore, zinc deficiency can impair the integrity and function of the gastrointestinal tract, which is critical for the absorption of nutrients, including zinc itself. This can create a vicious cycle, as zinc deficiency can further compromise immune function and lead to increased susceptibility to infections.

Overall, the proper functioning of both the innate and adaptive immune systems is essential for the optimal absorption and utilization of zinc, while zinc, in turn, plays a crucial role in supporting immune cell function and immune system regulation [43]. Zn is essential for the nicotinamide adenine dinucleotide phosphate oxidase activity of neutrophil granulocytes in the setting of the innate immunological response [44, 45]. Therefore, a Zn deficit may result in decreased production of ROS with a decreased capacity to kill [43]. In addition, in vivo research has demonstrated that Zn shortage impairs macrophage maturation and activity and reduces the adhesion and chemotaxis of monocytes and neutrophil granulocytes [46]. Zn has a crucial role in NK cells as well. Zn deficiency can result in a lower number of NK cells in the peripheral circulation and affect how well they function. In this respect, it has been shown that tumor cells or cells infected with viruses exhibit impaired chemotaxis and lysis [47, 48].

Through mechanisms that are still being fully elucidated, Zn controls a number of key procedures associated with the innate immune response. First, some immune cells are chemoattracted by Zn ions. Inversely, a superphysiological Zn concentration (500μM) increases PMN chemotaxis in vitro [49], and a Zn shortage reduces PMN chemotaxis [50]. Phagocytosis is reduced by Zn deficiency, but Zn supplementation has the opposite effect [51]. The early endosome antigen 1 (EEA1) and other Zn proteins involved in phagocytosis are likely how Zn affects this process. In order to facilitate membrane tethering and fusion, which are essential for phagosome and endosome maturation, EEA1 binds directly to the phospholipid phosphatidylinositol 3-phosphate (PI3K) at its C-terminal and binds to Rab5 via its N-terminal zinc finger domain [52, 53]. Zn is equally crucial for the neutralization of pathogens since both Zn excess and deficiency inhibit NAPDH, which controls the formation of superoxide anion that causes pathogen death following phagocytosis [54].

Zinc is not only essential for the proper functioning of the immune system but also plays a crucial role in the generation of proinflammatory cytokines. Cytokines, such as interleukins IL-1β, IL-6, and tumor necrosis factor α (TNF-α), are key mediators of immune responses. Zinc is required for the production and release of these proinflammatory cytokines, which are involved in initiating and coordinating immune reactions against pathogens. Moreover, studies have demonstrated that zinc promotes the adherence of monocytes, a type of immune cell, to endothelial cells in laboratory settings. This interaction is significant as it facilitates the migration of monocytes to sites of infection or inflammation, allowing them to exert their protective functions. In addition to its role in immune response modulation, zinc has been shown to possess antimicrobial properties. It can directly inhibit the replication and proliferation of various pathogens, including viruses and bacteria, thus contributing to the defense against infections. It is important to note that maintaining an appropriate balance of zinc is crucial, as both zinc deficiency and excess can negatively impact immune function. While insufficient zinc levels can impair immune responses, excessive zinc can lead to immunosuppression and disrupt immune cell signaling [55]. Zn deficiency only impacts the generation of IL-6 and TNF-α in an in vitro culture of peripheral blood mononuclear cells, leaving out the phagocytosis and oxidative burst of monocytes. This shows that under Zn deficiency, basic innate immune processes take the place of intracellular communication [56]. In addition, Zn modifies the response of NK cells by enhancing their MHC-class I expression and decreasing recognition [57]. In addition, Zn supplementation improves CD34+ cells' capacity to differentiate into NK cells and their cytotoxicity [58].

DCs' ability to mature is similarly impacted by Zn. Dynamic changes in the expression of Zn transporters on the cell surface are seen during LPS-induced DC maturation: ZIP6 and ZIP10 are inhibited while numerous ZnTs are up-regulated [59]. In addition, LPS stimulation increases the expression of MHC-II and costimulatory molecules on DCs while decreasing free Zn in DCs. The up-regulation of MHC-II and costimulatory molecules is interestingly inhibited by Zn supplementation or ZIP6 overexpression, while the LPS impact is interestingly mimicked by Zn chelator therapy [59]. These findings imply that DC maturation and subsequent activation of the adaptive immune response depend on a decrease in Zn in DCs through ZIP-6 down-regulation.

(2) Zinc and Adaptive Immunity. Zn has a significant role in the development, maturity, and functionality of T cells, which are crucial aspects of the adaptive immune system [32, 60]. This is due to the fact that Zn plays a crucial structural role in the hormone thymulin, which is created by the epithelial cells of the thymus and facilitates the maturation of pre-T lymphocytes into T lymphocytes [61, 62].

Zn deficiency has a particularly negative impact on T lymphocytes' ability to grow and function. Thymic atrophy and consequent T-cell lymphopenia are brought on by zinc deficiency. In mice, a shortage of Zn during T-cell maturation causes a 50% reduction in the number of “potential” pre-T cells compared to “effective” T cells, which is accompanied by an increase in pre-T cell death [63].

Zn also plays a crucial role in the mechanisms involved in T-cell development [64]. The number of CD4+ T cells decreased in studies that caused a Zn shortage, which led to an imbalance in the CD4+/CD8+ ratio [65]. Clinically, it is believed that a significantly decreased CD4+/CD8+ ratio, such as one below 1.5, is a sign of immunological dysfunction or one of its causes and is hence predictive of the prognosis of a number of disorders. There may also be an imbalance between Th-1 and Th-2 cells within CD4+ cells. The Th-1 cells in this instance are in the foreground and exhibit a more pronounced decline. Consequences are, for example, reduced Th-1-mediated cytokine production of TNF-α, IL-2, or IFN-γ [57, 65].

Th-17 cells that promote inflammation are also harmed by a zinc shortage. IL-6-induced STAT3 activation during chronic inflammation is a key regulator of Th-17 cell formation, and Zn inhibits Th-17 cell development by reducing this activation [66]. For instance, Zn therapy decreases Th-17 growth in the animal model of RA known as collagen-induced arthritis [66]. A mild nutritional Zn shortage raises the ratio of memory T cells to naive T cells and cytotoxic T cells to total T cells [57], which may result in immune system malfunction and autoimmune responses [67].

The binding of zinc to metallothioneins (MTs) plays a pivotal role in controlling various cytokine-driven activities in T cells. Transcription factors STAT1 and STAT3, crucial for T cell survival and development, are regulated by MTs. The interaction between Zn and MTs can affect the phosphorylation process of these transcription factors. Consequently, this modulation leads to an increase in the production of interleukin-10 (IL-10) by Tr1 cells, a subset of non-FoxP3 regulatory T (Treg) cells. IL-10 is a potent immunosuppressive cytokine known for its ability to downregulate inflammatory responses. By promoting the production of IL-10, Zn–MT binding can modulate immune activity and contribute to the regulation of autoimmune processes. IL-10 acts by suppressing the production of proinflammatory cytokines and inhibiting the activation of immune cells involved in autoimmune responses. Furthermore, IL-10 plays a crucial role in maintaining immune homeostasis by balancing the immune system's reactivity. It helps prevent excessive immune responses that can lead to tissue damage and autoimmunity. By influencing the generation of IL-10 through Zn–MT interactions, zinc can impact the delicate balance between immune activation and regulation. Understanding the intricate relationship between Zn–MT binding, cytokine-driven T-cell activities, and IL-10 production provides insights into the complex mechanisms underlying immune regulation and the potential therapeutic implications. Further research in this area is essential to unravel the precise mechanisms and explore the therapeutic possibilities of modulating Zn–MT interactions for immune-related disorders and autoimmune diseases [68].

Zn alterations have a smaller impact on B cell growth and function than they do on T cells. Nevertheless, Zn shortage results in a decrease in B cells, which affects the maturation of immature and premature B cells [69] and the production of antibodies [70]. Zn addition has been tested as a supplement for immunization in people who are deficient, with contentious results [71, 72].

(3) Immunoregulating Effects of Zinc. In addition to its specific effects on immunological functions, the overall regulation of the immune system is closely associated with zinc status. Numerous studies have demonstrated a correlation between Zn deficiency and dysregulation of immune responses, leading to increased oxidative stress and systemic inflammation.

Zinc is involved in the regulation of various immune processes, including the activation and differentiation of immune cells, the production of cytokines, and the maintenance of immune cell functions. Insufficient levels of Zn can impair these essential immune functions, compromising the overall immune system's ability to mount an effective defense against pathogens, as shown in Table 3.

Overall, maintaining adequate Zn levels is crucial for the proper regulation of the immune system. It not only supports specific immunological functions but also helps prevent oxidative stress, control inflammation, and ensure the optimal activation of the adaptive immune response. Understanding the relationship between Zn status and immune regulation is vital for developing strategies to enhance immune health and prevent immune-related disorders associated with Zn deficiency [73–75]. Numerous inflammatory cytokines can be affected by zinc in terms of their signaling as well as their synthesis. A Zn shortage can also have a deleterious impact on the disease processes that are linked to chronic inflammation. Patients with systemic inflammatory disorders, such as rheumatoid arthritis, and a concurrent Zn shortage have been demonstrated to have higher levels of IL-1α, IL-1β, and IL-6 expression compared to those who consume more Zn [76, 77]. In addition, the chromatin architecture of the IL-1β and TNF-α promoters, which allow for the production of both genes, appears to be altered by long-term Zn deficiency [40]. Thus, Zn can be considered a trace element that inhibits the synthesis of proinflammatory cytokines and has a favorable impact on disease processes. Since T lymphocytes and macrophages are the primary producers of cytokines, the mechanisms indicated above may be the cause of this. The decrease in ROS also has a significant impact [78]. In addition, the evidence points to Zn's function as a contra-regulator of the NF-κB signaling pathway. The pathway controls the expression of genes involved in apoptosis, innate and adaptive immune responses, and inflammatory processes, which in turn affect the expression of proinflammatory cytokines like TNF-α, IL-1β, or IL-6 [79]. Based on how Zn impacts the expression of the protein A20, one of the most significant inhibitory mechanisms exists. A20 is a Zn finger protein that is well known for being anti-inflammatory and for negatively regulating the NF-κB signaling pathways initiated by the tumor necrosis factor receptor and TLR [73].

In addition, in vivo studies have demonstrated that Zn addition generates and stabilizes regulatory T cells (Treg) [80, 81]. Thus, Zn has a broad impact while still being unique to each type of cell.

3.2.3. Zinc and Infectious Diseases

Zinc plays a significant role in the context of viral infections, and emerging studies have consistently shown its positive impact on disease progression, and in some cases, its potential to prevent infections altogether. The mechanisms by which Zn exerts its antiviral effects involve various stages of the viral lifecycle, including viral particle entry, fusion, replication, translation of viral proteins, and release, encompassing a wide range of viruses. One crucial aspect of Zn's antiviral activity is its ability to inhibit viral entry into host cells. Zn can interfere with the attachment and internalization of viral particles, thereby preventing the initial steps of infection. By blocking viral entry, Zn reduces the viral load and limits the spread of the infection within the body. Moreover, Zn has been shown to inhibit viral fusion, which is the process by which the viral envelope fuses with the host cell membrane, allowing the release of viral genetic material into the cell. By disrupting this fusion process, Zn impedes viral replication and further propagation.

Zn also plays a role in regulating viral replication within infected cells. It can interfere with the translation of viral proteins, which are essential for the production of new viral particles. By inhibiting viral protein synthesis, Zn limits the replication capacity of the virus and hampers its ability to spread. In addition, Zn has been found to enhance the antiviral immune response. It can boost the activity of immune cells, such as NK cells and T cells, which play a crucial role in recognizing and eliminating infected cells. Zn supplementation has been shown to enhance the cytotoxic activity of these immune cells, enhancing the body's defense against viral infections. The specific mechanisms by which Zn exerts its antiviral effects may vary among different viruses. However, the cumulative evidence suggests that maintaining optimal Zn levels is crucial for mounting an effective antiviral defense. Zn supplementation has shown promise in mitigating the severity of viral illnesses and preventing their progression. Further research is ongoing to explore the detailed molecular mechanisms underlying Zn's antiviral properties and to identify specific targets within the viral lifecycle. Understanding the interplay between Zn and viral infections can provide valuable insights for the development of novel antiviral strategies and interventions [82, 83]. Regarding clinical effectiveness, a meta-analysis's findings revealed that Zn supplementation at a level >75mg/day considerably shortened the length of colds [84]. The elderly are a demographic that is particularly at risk in this situation. Increased susceptibility to infections and their severity are associated with immune system aging. In a sample of 55- to 87-year-old people, it was demonstrated that after 12 months of Zn supplementation (45mg elemental Zn-gluconate/day), the incidence of infections was much lower. This was accompanied by a rise in plasma Zn concentration and a decrease in the production of TNF-α and indicators of oxidative stress [85]. The fact that Zn cations, in particular, have been demonstrated to inhibit SARS coronavirus RNA polymerase (RNA-dependent RNA polymerase) by lowering viral replication in in vitro studies is more support [86]. These important findings show that Zn can be considered as an active agent in the treatment of COVID-19 [87].

These studies highlight the importance of zinc in maintaining immunological health. However, other research indicates that excessive zinc intake, ranging from 100 to 300mg/day, can have detrimental effects on immune function and potentially contribute to various health problems. In line with this, Deuster recommends a maximum daily zinc intake of 40mg as a tolerable limit. These findings underscore the challenges faced in conducting research in this field, as it can be complex to determine the optimal dosage and balance of zinc for overall health and immune function [88].

3.3.2. Immunological Role of Copper

While the precise mechanisms by which copper contributes to the development and functioning of the immune system remain unclear, its importance in these processes is well recognized. Copper is necessary for the normal development and optimal functioning of the immune system. Deficiencies in copper levels have been associated with reduced effectiveness of cellular and/or humoral immune system aspects. This deficiency can increase the risk of infections, as copper plays a role in immune response modulation. Further research is needed to fully elucidate the specific involvement of copper in immune system processes and its impact on infection susceptibility [101]. Naturally, newborns are particularly susceptible to the detrimental effects of low copper levels on immune system function. Infants with Menkes disease (MD), a condition characterized by severe copper deficiency, experience frequent, and severe infections. The compromised immune system in these individuals leaves them more vulnerable to microbial pathogens, leading to a higher incidence of infections. Neonates with MD require special attention and care to address their copper deficiency and mitigate the associated risks to their immune system [102, 103]. In addition, giving underweight, copper-deficient children a copper supplement improved their ability to phagocytize germs [104]. Another sign of a copper shortage in humans is neutropenia, and even mild copper deficiency may impair the function of various immune cells (such as macrophages and lymphocytes). For instance, after an in vitro immunological challenge, the proliferation of peripheral blood mononuclear cells isolated from adult males fed a low-copper diet (0.38mg/day) for 42 days was inhibited [105]. In addition, individuals' plasma copper levels were low, and some cuproenzymes' activity was suppressed. The innate immune response to bacterial infection may also require copper [106]. Collectively, these studies demonstrate significant roles for copper in immune system function, but conclusive evidence is absent, in part because marginal or subclinical copper deficiency could not be identified in human intervention trial participants.

On the other hand, diminished humoral and cellular immunological function results from a Cu shortage. Cu-deficient mice had a smaller thymus and a larger spleen, according to research. Lower T cell counts and neutropenia are also present. B cell and NK cell function, as well as mitogen-induced T-cell proliferation, are all compromised [105, 107]. As a result, raising plasma Cu levels may improve both innate and adaptive immunity in people. Cu is referred to in this context as a trace element with antiviral properties that can treat and prevent COVID-19 [108]. The body can prevent the growth of pathogens by limiting their access to Cu, yet Cu is also a necessary nutrient for microbial pathogens. The human body's intake of Cu should be balanced from the standpoint of the immune system because too much can simultaneously have negative effects while too little is necessary for proper immunological function [109].

(1) Copper and the Innate Immune System. While the effects of copper deficiency on neutrophils and macrophages are well understood, there is limited knowledge regarding its impact on eosinophils and basophils. Due to their low abundance, studying these two cell types presents challenges in research. It is worth noting that mast cells originate from basophils within tissues. In the cremaster muscle of mice with copper deficiency, further investigation is required to fully comprehend the specific consequences on eosinophils, basophils, and the development of mast cells. Schuschke et al. [110] discovered an increase in the mast cell population, indicating that copper shortage might affect the maturation patterns of the leukocyte population or the distribution of blood cells into tissues.

In humans, neutropenia is a clinical indicator of copper insufficiency [111, 112]. The 1960s were when it was first noticed [113]. Humans with copper deficiency had more promyelocytes and fewer metamyelocytes and banded cells in their bone marrow aspirates. This was understood to be a granulocyte maturation arrest brought on by a copper deficit [114–116]. Inadequate secretion from the bone marrow, early death of progenitor cells, a shorter life expectancy of the circulating peripheral cells, and redistribution into tissues or organs are some other processes that could cause neutropenia [117]. Antineutrophil antibodies were found by Higuchi et al. [118] in the serum of people with copper deficiency, which may point to a mechanism of neutrophil depletion. In addition to having fewer circulating neutrophils, the copper shortage also affected how well those neutrophils functioned. A copper shortage was observed to cause decreased superoxide anion generation and decreased candidacidal activity without altering phagocytosis [119, 120]. In macrophages, comparable modifications were seen [121]. Neutrophil dysfunction was also impacted by a minor copper shortage. Neutrophils drawn from the peritoneal cavities of rats with borderline copper deficiency in the perinatal model described earlier produced 60% less superoxide anion than did control rats' neutrophils [122]. This demonstrates once again that immunity is compromised even when copper status indices are within the normal range. However, despite our best efforts, we have not been able to identify whether the general population is marginally deficient in copper, despite the potential importance of the influence of marginal copper status on human health. The ideal quantity of copper one should eat has not been determined, although a recent study utilizing low-copper diets in young males revealed that the requirement for copper fell somewhere in the range of 0.4–0.8mg/day [123].

(2) Immunoregulating Effects of Copper. The balance of Cu in the body is also influenced by nutrition. Balance studies have shown that daily intakes below 0.8mg/day result in a net Cu loss, but net increases are seen beyond 2.4mg/day [99]. It is worth mentioning that there seems to be an inverse relationship between the intake of zinc and copper. When significant amounts of zinc are consumed, lower levels of copper enter the body through the colon. This effect is utilized in the treatment of Wilson's disease, a condition characterized by copper overload, to regulate copper levels [100]. Contrarily, Cu shortages are extremely rare, occurring, for instance, in severe intestinal disorders. However, this frequently results in severe deficiency symptoms, like anemia and bone deterioration [109]. Cu is not a standard parameter since a high blood level of Cu is not necessarily harmful. Elevated levels can be seen in a number of illnesses, including cancer, infections, and diabetes mellitus; however, they are believed to have little impact on the course of the illness or the effectiveness of treatment [99].

3.4.2. Immunological Role of Iron

(1) Iron and the Innate Immune System. The handling of iron by macrophages is influenced by immune activation. In turn, the effectiveness of macrophages' antimicrobial immune response is directly influenced by their iron levels. Many of these interactions are attributed to the effects of iron on the binding activity of transcription factors that promote inflammation. Iron regulates the activity of various transcription factors, including NF-B, NF-IL6, HIF-1, STAT1, and Nrf2, although not consistently. Iron also stimulates the production of reactive oxygen intermediates (ROIs), which subsequently enhances NF-B activity [130]. A variety of proinflammatory cytokines, chemokines, antimicrobial enzymes, peptides, and adhesion molecules are transactivated by NF-B, one of the key transcription factors in the beginning and intensification of the immune response [131]. Because labile iron may catalyze the Fenton reaction, which produces oxygen radicals, it has strong proinflammatory effects. However, because NF-B activation also transactivates the Fth and Ftl genes, it also aims to reduce iron-induced cytotoxicity [132]. TNF has the ability to trigger apoptosis, while TNF-induced ROI promotes Ft expression via NF-B, starting the iron storage and antiapoptotic pathways [133].

NF-IL6, HIF-1, and STAT1 are frequently suppressed by cellular iron excess, in stark contrast to NF-B [134–136]. Iron lowers NF-IL6 activation and STAT1 phosphorylation, which inhibit Nos2 transcription and NO generation in macrophages treated with IFN and LPS to imitate TH1 immunity [134]. Similar to this, iron chelation with DFO stimulates HIF-1, which increases the transactivation of the Nos2 gene [135]. Bmp6 increases Nos2 expression in macrophages, which is intriguing and suggests that it is a key coordinator of iron homeostasis and the immune response [137].

However, there is limited understanding of the interplay between the SMAD1/5/8 signaling cascades and their relationship with iron. By contrast, there is substantial crosstalk between the NF-B and HIF-1 pathways. HIF-1 has the ability to inhibit gene expression induced by NF-B, while NF-B enhances the transcription of HIF-1 [138, 139]. HIF-1 and NF-B are both activated during infections, and they work together to increase the expression of target genes that are shared and crucial for host defense [140]. How iron might impact the interaction between these two important pathways is not yet known. Since the NF-B component p65 uses pirin as an interaction partner and redox sensor, it appears that pirin is the basis for the specificity with which iron drives NF-B activity. When under oxidative stress, pirin preferentially takes on its ferric form, which binds to p65 and increases its DNA-binding activity [141]. It's interesting to note that iron is required for HIF-1's stability and that the von Hippel Lindau complex and prolyl hydroxylases are involved in iron-mediated regulation of HIF-1's degradation [142]. NF-B-dependent Ft transcription is induced by TLR4 signaling, and Ft then integrates iron, lowering the metal's availability in the cytoplasm. Since iron serves as a cofactor for prolyl hydroxylases that degrade HIF-1, the decrease in free intracellular iron in DC induced by LPS results in HIF-1 stability [143]. It is plausible to suppose that this route influences the course of leishmaniasis because HIF-1 is crucial for host defense against Leishmania; however, it is unclear how much HIF-1 may influence the immunological response of macrophages that contain parasites [144]. HIF-1, however, is crucial for limiting bacterial reproduction in macrophages infected with Streptococcus pyogenes or Pseudomonas aeruginosa because it promotes the development of TNF, Nos2, and other antibacterial effectors. S. pyogenes can infect mice's skin more easily when HIF-1 is missing from myeloid cells [145].

In the case of HFE-associated HH, an autosomal-recessive illness, many of the impacts that iron has on macrophage effector functions have been examined. Caucasians make up 5% of the heterozygous population for the causative HFE gene deficiency, which is most frequently the C282Y mutation, and 0.225% of the homozygous population [146]. Hfe's ability to interact with Tfr1 on the cell surface, wherein binding to Tfr1 decreases its affinity for holo-Tf, is its primary function, according to research [147]. As a result, when Hfe is altered, TBI is taken up by hepatocytes and other parenchymal cell types at a higher rate. As a result, there is an increase in parenchymal iron deposition, which may be made worse by the relative lack of hepcidin caused by improper detection of blood iron in the absence of Hfe. The iron deficiency of monocytes and macrophages in HFE-associated HH appears paradoxical. While hepcidin levels in HH may have decreased in part as a result of this observation [146], Hfe itself may have adverse effects on iron release from monocytes and macrophages [148]. Because macrophage iron overload suppresses Lcn2 transcription, Hfe-deficient macrophages export more iron and develop an iron-depleted phenotype, which makes it easier to produce Lcn2 [149]. Based on Hfe's requirement for cytokine translation, iron in general and Hfe in particular regulate immune function on yet another level. Because the ribosome needs sufficient amounts of iron to translate these particular mRNAs, it has been demonstrated that Hfe/macrophages produce less TNF and IL-6 proteins [150].

In addition, macrophages are both hepcidin's targets and producers, offering a putative autoregulation mechanism and a further connection between iron homeostasis and immunity. Proinflammatory signals like IL-6 or PAMP cause the synthesis of macrophage hepcidin, which appears to be resistant to iron status [151–154]. Hepcidin affects macrophages in a paracrine and autocrine manner after being secreted [155]. Hepcidin produced by macrophages promotes Fpn1 internalization, which results in mononuclear iron retention in the AI [156]. In addition, it has been demonstrated that the connection between hepcidin and Fpn1 inhibits the production of TNF and IL-6 through Janus kinase-2 and STAT3 signaling, which activate suppressor of cytokine signaling-3 [157]. Hepcidin has anti-inflammatory effects on macrophages by regulating these signaling pathways and raising cellular iron concentration, which may be important for the shutdown of the innate immune response [158]. This impact can be partially attributed to iron's direct inhibitory effects on immune effector pathways triggered by IFN and LPS, such as the production of TNF, IL-6, or Nos2 [136, 159–161].

Another transcription factor that links innate immunity and iron homeostasis is Nrf2. The role of Nrf2 in the cellular response to oxidative stress has long been understood. Therefore, Nrf2 binds to the promoter region of genes having an antioxidant response element (ARE) and is activated by stressors such as iron-induced ROI and NO [162–164]. It's significant that some iron-related genes also have AREs in their regulatory regions. In order to control oxidative stress and maintain iron homeostasis, Nrf2 stimulates the expression of Hmox1, Fth, and Fpn1 [165, 166]. As a result [167], Nrf2 serves as a molecular bridge between these two processes. In conclusion, macrophages control the system's iron homeostasis, and iron has an impact on various aspects of the effector activities of macrophages, including the transcriptional and translational control of substances that cause inflammation.

3.4.3. Iron and Infectious Diseases

(1) Extracellular Infections. An important factor in determining the course of infectious disorders is the regulation of iron homeostasis on both the mammalian and microbial sides [168]. Both adversaries work to meet their iron needs when the host and pathogen interact. Because iron is necessary for almost all infections, its availability alters immune effector pathways as well as microbial proliferation [169–174]. The only bacteria that apparently do not require any iron at all but instead use other divalent metals are those belonging to the genus Lactobacillus, which are commensals, and Borrelia burgdorferi, which causes Lyme disease [175, 176]. Therefore, one effective mammalian defense approach to prevent the development and pathogenicity of almost all microorganisms is iron sequestration in areas that are inaccessible to invasive bacteria. It's important to note that the mechanisms of iron withdrawal differ fundamentally depending on whether infections are located extracellularly or intracellularly.

An excess of iron worsens the course of many infectious disorders and raises the risk of bacterial infections. Patients with genetic and acquired hemologic diseases who require frequent packed RBC transfusions have been shown to have a substantial positive correlation between infections and iron overload. For instance, blood transfusions, inefficient erythropoiesis, and increased iron absorption all contribute to iron overload in thalassemia syndromes [177, 178]. Although excess iron is hazardous to parenchymal organs like the heart and liver, it also increases the risk of infection. In fact, patients with β-thalassemia have been reported to have invasive infections with Streptococcus pneumoniae, P. aeruginosa, Klebsiella species, Yersinia species, Escherichia coli, and Vibrio vulnificus, and their risk is directly correlated with the degree of iron overload and a delay in therapeutic iron chelation [179]. The pathophysiology of inborn hemolytic anemias, such as thalassemias, is complex, and there may be a number of factors contributing to the higher incidence of infections, such as functional problems with the enlarged spleen and immunological dysregulation at many levels. In addition, hemolysis, tissue iron overload, and a rise in serum iron above Tf's binding capacity make it simple for bacteria to reach iron sources. The latter hypothesis is supported by the observation that V. vulnificus induces lethal septicemia in Hamp/Mice, a mouse model of extreme iron overload, showing that iron overload predisposes to a catastrophic outcome of normally uncommon infections [180]. Importantly, infections are a major contributing cause of iron overload's detrimental effects on leukemia and myelodysplastic syndrome patients' survival after stem cell transplantation [181, 182].

Numerous ways have been developed by microbes to exploit host iron supplies. Numerous extracellular bacteria contain Hb, heme, Ft, or Tf receptors. As a result, haptoglobin and Hpx, two acute phase proteins that bind free Hb and heme for later uptake into the MPS via CD163 and CD91, respectively, reduce the amount of iron that is available to bacteria [183, 184]. To reduce the pool of TBI during bacteremic episodes, Tf itself is downregulated during infection and is also eliminated from the blood via Tfr1. Although serum Ft is iron-poor in and of itself, it can also be removed from the bloodstream by Ft receptors on macrophages [185]. As a result, the Ft receptors on macrophages and microbes appear to be in direct competition for the same ligand that carries iron [186].

By employing iron-binding siderophores, which they either generate themselves or receive from other microbes but use as an iron supply, many bacteria, including mycobacteria and enterobacteriaceae like E. coli and Klebsiella species, kidnap iron from TBI [187–190]. Siderophores are various chemically structured, tiny molecular-weight iron chelators that share two characteristics. They exhibit an incredibly high affinity for ferric iron and are enzymatically produced outside of the ribosome [191–193]. In fact, siderophores can remove iron from host molecules like Tf which have a high yet low affinity for iron. Bacteria then absorb iron-rich siderophores through certain receptors found in their outer membrane. Certain fungi as well as bacteria can create siderophores. For example, Aspergillus fumigatus has both external siderophores that are associated with its pathogenicity and an internal siderophore for storing iron [194–196].

Given the significance of siderophore-mediated iron uptake for bacterial pathogenicity, mammals have a unique defense system called lipocalin-2 (Lcn2) that can reduce part of the threat provided by siderophores. Neutrophils, macrophages, and other immunological and parenchymal cell types all produce the peptide Lcn2, which has antibacterial and immune-regulating properties [197–204]. Its primary purpose is to bind siderophores of the catechol type, like enterobactin, to prevent iron from moving to bacteria [187, 205]. Due to the high susceptibility of Lcn2 mice to enterobacteriaceae like E. coli, this pathway appears to be particularly significant in these infections [206]. Salmonella species alter the structure of enterobactin to evade Lcn2's effects. Enterobactin is modified by enzymes and receptors produced by the iroA gene cluster to create salmochelins, which are too large to be neutralized by Lcn2 but still have the ability to bind iron [207]. Salmochelins do, however, need particular efflux pumps and membrane receptors [208, 209]. Lcn2 is particularly resistant to other siderophores made by Pseudomonas pyoverdin, Yersiniabactin generated by Yersinia and Klebsiella species, and triacetylfusarinine C produced by A. fumigatus [210–212]. In addition, Lcn2 guards against possible pathogens colonizing or infecting the gut mucosa [213, 214]. In addition, Lcn2 may be important for host defense against a wider variety of extracellular microorganisms because it attracts neutrophils to the site of infection [198]. Due to reduced iron availability in the macrophages' milieu, Hfe/macrophages and mice were protected from Salmonella typhimurium infection in a mouse model of HFE-associated HH by enhanced Lcn2 production [215]. However, when Yersinia pestis, the plague-causing extracellular bacterium, infected an individual with HFE-associated HH, the clinical course was unfavorable because elevated serum iron levels and parenchymal iron overload promote pathogen proliferation [216]. It is unknown how much the relative hepcidin shortage that characterizes HFE-associated HH may be responsible for this behavior. However, Yersinia enterocolitica, a closely related disease, is extremely virulent in Hamp/mice, indicating that Hfe and Hamp are crucial for host defense against Yersinia species [217, 218].

(2) Intracellular Infections. Intracellular infections are a major worry for world health. Serious infections can be brought on by Mycobacterium species, Listeria monocytogenes, Legionella pneumophila, Chlamydia, Salmonella, and Leishmania species. In addition, a variety of parasites, notably Trypanosoma and Plasmodium, have intricate, partially intracellular life cycles [219]. Some of these infections are becoming more resistant to antibiotics, and there aren't yet effective vaccinations available for all of them. Therefore, it's crucial to have a better understanding of immunity, particularly dietary immunological techniques like iron withdrawal, to increase the range of available treatments for illnesses like malaria, typhoid fever, and tuberculosis. One innovative method for treating infections caused by intracellular microorganisms is the pharmacological manipulation of iron homeostasis in host cells [220].

The iron exporter Fpn1 is a good candidate for an augmentation treatment in infections with intracellular pathogens. This theory is supported by the observation that Fpn1 regulates the reproduction of iron-dependent pathogens within host cells as well as macrophage effector functions [221]. For instance, overexpression of Fpn1 decreases the amount of iron available to intramacrophage bacteria and consequently the microbial burden in infections with Chlamydia psittaci, L. pneumophila, Mycobacterium tuberculosis, or S. typhimurium [222–224]. Exogenous hepcidin, on the other hand, promotes the growth of infected cells [222, 224, 225]. It is significant to note that several intracellular pathogens, including Chlamydia pneumoniae, L. monocytogenes, S. typhimurium, and Trypanosoma brucei, have been shown to induce intrinsic Fpn1 in infected macrophages [226–229]. This suggests that intracellular PRR senses microbial structures or metabolites and induces iron export to deprive the pathogenic invader of the micronutrient iron. Nos2's output, NO, promotes Nrf2 to transactivate Fpn1 transcription in S. typhimurium-infected macrophages, whereby Fpn1 is induced [230]. This does not rule out the possibility that different Fpn1 regulatory pathways could predominate in infections with other intracellular bacteria. For instance, L. monocytogenes replicates in the cytosol of the macrophage, while S. typhimurium infects murine macrophages via intraphagolysosomal infection. Therefore, it is plausible to believe that a separate molecular mechanism may be involved in the induction of Fpn1 transcription in response to L. monocytogenes. In addition, Fpn1 expression may be differentially regulated over the course of infection or between different organs that harbor pathogens, potentially allowing for systemic iron redistribution to maintain vital processes like erythropoiesis without interfering with nutritional immune strategies [231–234].

The phagolysosomal membrane of macrophages infected with Mycobacterium TB has also been shown to contain Fpn1. Its role there and how it interacts with Nramp1, also known as Slc11a1 (for natural resistance-associated macrophage protein-1), with which it colocalizes, are still unknown. Fpn1 may transfer iron from the cytoplasm into the phagosome, assuming normal orientation and potentially enhancing ROI formation. Accordingly, moderate dietary iron supplementation promotes ROI production, which lowers mycobacterial burden [235]. Contrarily, the bulk of papers on Nramp1's function contend that it serves as a mechanism for nutritional withdrawal by transporting iron out of the phagosome. Given that hepcidin was also discovered in the phagosomal area [236], the picture of Fpn1's probable function in the phagolysosome becomes even more complicated. However, it is still unclear whether it interacts with Fpn1 there or if its defensin-like structure enables it to perform direct antimicrobial actions.

In mice, Nramp1 was initially identified as a protein necessary for the management of intracellular pathogen infections, particularly those caused by Leishmania species, Mycobacterium species, and S. typhimurium. It was presumptively believed that Nramp1 must target a metabolic pathway important to both bacteria and protozoa because both pathogens are taxonomically unrelated. Because Nramp1 functions as an antiporter of protons and divalent metal ions, including iron, zinc, and manganese, across the phagolysosomal membrane, iron was found to be exactly at the center of that pathway [237, 238]. To specifically remove these ions as vital nutrients from absorbed iron-sensitive bacteria, Nramp1 transfers them from the phagolysosomal compartment into the cytoplasm. Nramp1 performs immune-regulatory tasks concurrently. A single nucleotide polymorphism (Nramp1s) within the coding region has been demonstrated to impair Nramp1's transport activity in mice. By contrast, the functional Nramp1r allele defends against infection with S. typhimurium, Mycobacterium species, and Leishmania species. Nramp1r macrophages produce more NO, TNF, IL-6, and Lcn2 and less of the anti-inflammatory cytokine IL-10 when S. typhimurium is present [239–242]. These findings support the hypothesis that Nramp1 promotes a proinflammatory macrophage phenotype, regulates the immune system, and helps in the eradication of intraphagolysosomal pathogens.

However, Nramp1's immune-regulatory effects seem to have a price. To date, no mutation in the NRAMP1 coding area in humans has been reported. Instead, polymorphisms in the NRAMP1 promoter's regulatory region control whether the gene is transcribed at higher or lower levels. An increased risk of autoimmune diseases such as rheumatoid arthritis, type 1 diabetes, and inflammatory bowel disease was linked to higher expression levels of NRAMP1. Leishmania, Mycobacterium leprae, and Mycobacterium TB infections, on the other hand, rose in frequency when promoter variations that induce low levels of NRAMP1 expression were present [243, 244]. It is tempting to hypothesize that these effects are related to different iron availability, which influences the balance of pro- and anti-inflammatory cytokines and, in turn, regulates tissue damage and the host's response to intracellular infections. In addition, Nramp1 is necessary for effective iron recycling after erythrophagocytosis, adding yet another crucial mechanism that may be able to manage both hemolysis and host defense simultaneously [245].

LIT1, a ferrous iron ZIP (for ZRT- and IRT-like protein) transporter discovered in Leishmania species, may directly compete for Nramp1's substrate in Leishmaniasis. When Leishmania is present in the phagolysosome, where it may obtain iron, LIT1 is specially produced. Accordingly, Nramp1r macrophages express more LIT1 [246]. In addition, as shown by the elevated IRP-binding activity of infected macrophages, Leishmania takes up hemoglobin and targets the labile iron pool that is present in the cytoplasm of macrophages [218, 247, 248].