# Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

Sian L Stafford, Nilesh J Bokil, Maud E S Achard, Ronan Kapetanovic, Mark

A Schembri, Alastair G Mcewan, Matthew J Sweet

► To cite this version:

Sian L Stafford, Nilesh J Bokil, Maud E S Achard, Ronan Kapetanovic, Mark A Schembri, et al. Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper. Bioscience Reports, 2013, 33 (4), 10.1042/bsr20130014. [https://doi.org/10.1007/s00094-013-0438-3](https://doi.org/10.1007/s00094-013-0438-3)

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# Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

Sian L Stafford, Nilesh J Bokil, Maud E S Achard, Ronan Kapetanovic, Mark

A Schembri, Alastair G Mcewan, Matthew J Sweet

► To cite this version:

Sian L Stafford, Nilesh J Bokil, Maud E S Achard, Ronan Kapetanovic, Mark A Schembri, et al. Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper. Bioscience Reports, 2013, 33 (4), 10.1042/bsr20130014. [https://doi.org/10.1038/bsr20130014](https://doi.org/10.1038/bsr20130014)

HAL Id: hal-04383333

https://hal.inrae.fr/hal-04383333v1

Submitted on 9 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

Sian L. STAFFORD*†<fnref n="1" />, Nilesh J. BOKIL†‡<fnref n="1" />, Maud E. S. ACHARD*†, Ronan KAPETANOVIC†‡,
Mark A. SCHEMBRI*†, Alastair G. McEWAN*† and Matthew J. SWEET†‡<fnref n="2" />

*School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia, †The Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland 4072, Australia, and ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia

## Synopsis

The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases *in vivo*, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells.

**Key words:** copper transporter (CTR), host defence, innate immunity, monocyte, Toll-like receptor, zinc transporter

Cite this article as: Stafford, S.L., Bokil, N.J., Achard, M.E.S., Kapetanovic, R., Schembri, M.A., McEwan, A.G. and Sweet, M.J. (2013) Metal ions in macrophage antimicrobial pathways: Emerging roles for zinc and copper. Biosci. Rep. **33**(4), art:e00049.doi:10.1042/BSR20130014

## INTRODUCTION

The immune system requires essential micronutrients and trace elements such as iron, zinc, copper and selenium for optimal function [1]. Deficiency in these elements leads to suppression in the activities of cells of the innate and adaptive immune systems, through impaired function and/or decreased cell number. Such defects can lead to increased morbidity and mortality to viral, microbial and parasitic infections, with immunocompetence being restored when the deficiency is reversed [1]. Despite the clear biological relevance of micronutrients to host defence, the cellular and molecular mechanisms by which they regulate host immune function are very much an emerging area of research. Here we review the literature on the beneficial effects of zinc and copper in host defence, as well as recent evidence linking copper and zinc directly to macrophage antimicrobial pathways. Certain parallels suggest that there may be common mechanisms by which they contribute to these responses. We do not extensively

cover strategies utilized by pathogens to defend against zinc- and copper-mediated host defence, since others have reviewed this literature in detail recently [2–4]. Similarly, we provide only a brief overview of the effects of zinc on signalling and gene expression in monocytes and macrophages; while clearly important for host defence, this area has been covered by other excellent reviews [5–7]. Rather, the major objective of this review is to summarize emerging evidence for direct contributions of zinc and copper to macrophage antimicrobial responses, to highlight the likely significance of this to host defence, and to identify the major knowledge gaps in this area, particularly relating to the roles of specific host zinc and copper transport genes.

### Zinc deficiency compromises immune function and host defence

Zinc is essential for the growth and development of most organisms [8], and in humans, it is the second most abundant transition metal ion [9]. Studies of zinc deficiency in humans, as well as

<footnote>Abbreviations used: Cβ ceruloplasmin; CTR, copper transporter; DC, dendritic cells; FPN1, ferroportin 1; GPI, glycosylphosphatidylinositol; IFNγ, interferon γ; IKKβ, IκB (inhibitor of nuclear factor κB) kinase β; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MMP matrix metalloproteinase; MT, metallothionein; NF-κB, nuclear factor κB; Nrf2, nuclear factor-erythroid 2-related factor 2; PDE, phosphodiesterase; ROS, reactive oxygen species; SOD, superoxide dismutase; TLR, Toll-like receptor; TNF, tumour necrosis factor; TRIF, TIR (Toll/interleukin-1 receptor) domain-containing adaptor protein inducing interferon β.</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>

<footnote n="2">² To whom correspondence should be addressed (email m.sweet@imb.uq.edu.au).</footnote>
<figure><img src="image_2.png" /></figure>

# Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

Sian L. STAFFORD*†<fnref n="1" />, Nilesh J. BOKIL†‡<fnref n="1" />, Maud E. S. ACHARD*†, Ronan KAPETANOVIC†‡,
Mark A. SCHEMBRI*†, Alastair G. McEWAN*† and Matthew J. SWEET†‡<fnref n="2" />

*School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia, †The Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland 4072, Australia, and ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia

## Synopsis

The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases *in vivo*, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells.

**Key words:** copper transporter (CTR), host defence, innate immunity, monocyte, Toll-like receptor, zinc transporter

Cite this article as: Stafford, S.L., Bokil, N.J., Achard, M.E.S., Kapetanovic, R., Schembri, M.A., McEwan, A.G. and Sweet, M.J. (2013) Metal ions in macrophage antimicrobial pathways: Emerging roles for zinc and copper. Biosci. Rep. **33**(4), art:e00049.doi:10.1042/BSR20130014

## INTRODUCTION

The immune system requires essential micronutrients and trace elements such as iron, zinc, copper and selenium for optimal function [1]. Deficiency in these elements leads to suppression in the activities of cells of the innate and adaptive immune systems, through impaired function and/or decreased cell number. Such defects can lead to increased morbidity and mortality to viral, microbial and parasitic infections, with immunocompetence being restored when the deficiency is reversed [1]. Despite the clear biological relevance of micronutrients to host defence, the cellular and molecular mechanisms by which they regulate host immune function are very much an emerging area of research. Here we review the literature on the beneficial effects of zinc and copper in host defence, as well as recent evidence linking copper and zinc directly to macrophage antimicrobial pathways. Certain parallels suggest that there may be common mechanisms by which they contribute to these responses. We do not extensively

cover strategies utilized by pathogens to defend against zinc- and copper-mediated host defence, since others have reviewed this literature in detail recently [2–4]. Similarly, we provide only a brief overview of the effects of zinc on signalling and gene expression in monocytes and macrophages; while clearly important for host defence, this area has been covered by other excellent reviews [5–7]. Rather, the major objective of this review is to summarize emerging evidence for direct contributions of zinc and copper to macrophage antimicrobial responses, to highlight the likely significance of this to host defence, and to identify the major knowledge gaps in this area, particularly relating to the roles of specific host zinc and copper transport genes.

### Zinc deficiency compromises immune function and host defence

Zinc is essential for the growth and development of most organisms [8], and in humans, it is the second most abundant transition metal ion [9]. Studies of zinc deficiency in humans, as well as

<footnote>**Abbreviations used:** Cβ ceruloplasmin; CTR, copper transporter; DC, dendritic cells; FPN1, ferroportin 1; GPI, glycosylphosphatidylinositol; IFNγ, interferon γ; IKKβ, IκB (inhibitor of nuclear factor κB) kinase β; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MMP matrix metalloproteinase; MT, metallothionein; NF-κB, nuclear factor κB; Nrf2, nuclear factor-erythroid 2-related factor 2; PDE, phosphodiesterase; ROS, reactive oxygen species; SOD, superoxide dismutase; TLR, Toll-like receptor; TNF, tumour necrosis factor; TRIF, TIR (Toll/interleukin-1 receptor) domain-containing adaptor protein inducing interferon β.</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>

<footnote n="2">² To whom correspondence should be addressed (email m.sweet@imb.uq.edu.au).</footnote>
<figure><img src="image_3.png" /></figure>

animal models, have conclusively demonstrated that zinc is es-
sential for normal immune function, but its precise cellular and
molecular role(s) remain enigmatic. Zinc deficiency was first
identified as a clinical syndrome in the Middle East approxim-
ately 50 years ago [10]. The first case was reported in Iran in
a male patient who presented with severe anaemia and growth
retardation [11]. This condition was associated with hepatosplen-
omegaly, hypogonadism, rough and dry skin, mental lethargy
and genophagia (clay eating), and was initially misdiagnosed as
iron-deficiency anaemia. Similar cases were reported in Egypt
[12], where a regimen of zinc supplementation reversed hypo-
gonadism and growth retardation [13]. In addition to develop-
mental anomalies and anaemia, the patients also suffered from
severe immune deficiency leading to intercurrent infection and
death before the age of 25 [8]. Furthermore, experimentally in-
duced mild zinc deficiency in human subjects [14] resulted in
a decrease in the ratio of CD4⁺ to CD8⁺ T-cells, and a de-
creased Th1 response as assessed by IFNγ (interferon γ), IL
(interleukin)-2 and TNFα (tumour necrosis factor α) secretion
[15]. It was proposed that defective Th1 responses compromised
host defence to intracellular parasitic infections, in particular.
In general, severe zinc deficiency is rare, but mild-to-moderate
deficiency is quite common throughout the world especially in
developing countries, lower socio-economic groups and in indi-
viduals with chronic alcoholism. The 2002 WHO world health
report attributed about 800 000 deaths (1.4% of global mortalit-
ies) to zinc deficiency, and estimated that it contributed to 16%
of lower respiratory tract infections, 18% of malaria and 10%
of diarrhoeal disease [16]. Animal models of zinc deficiency
have also been used to assess the role of zinc in regulating the
immune system and host defence. Early studies examined the
effect of zinc deficiency on T-cell function in young A/J mice
[17,18]. Thymic atrophy and diminished antigen-specific T-cell
responses were observed, which was reversed by dietary zinc
supplementation. In a mouse polymicrobial model of sepsis, zinc
deficiency resulted in increased bacterial burdens in the blood, a
heightened pro-inflammatory response, and increased mortality,
and these effects were reversed by zinc supplementation [19,20].
The enhanced inflammatory response correlated with increased
activity of NF-κB (nuclear factor κB) and expression of NF-κB-
dependent target genes [19], suggesting that zinc acts to constrain
this pathway during excessive inflammatory responses.

Zinc deficiency generally reflects insufficient dietary intake, but systemic inflammation also results in a reduction in the levels of circulating zinc (hypozincaemia). Indeed, it has long been appreciated that plasma zinc levels decline during systemic bacterial infections [21], as a physiological component of the acute phase response [22]. Hypozincaemia as a consequence of LPS (lipopolysaccharide) challenge has been experimentally demonstrated in humans. LPS administration to healthy human volunteers reduced serum zinc levels, without increasing zinc loss in the urine or decreasing levels of the major zinc-binding protein, serum albumin [23]. This suggests that the drop in circulating zinc reflects its redistribution elsewhere. Evidence from animal models suggests that increased uptake of zinc by hepatocytes in the liver is the mechanism responsible. Yasuno et al. [24]

showed that when rats were given an intravenous dose of zinc (50 µg/kg), the metal was rapidly distributed to the liver, spleen, intestine, kidney and pancreas. Moreover, systemic inflammation in mice resulted in IL-6-dependent up-regulation of the zinc importer *Slc39a14*, which enabled zinc uptake by hepatocytes in the liver [25,26]. Several studies have documented reduced serum zinc levels in various patient cohorts. For example, zinc levels in polymorphonuclear cells from hospitalized elderly patients were reduced by comparison with those of healthy elderly subjects [27]. A more recent study demonstrated that plasma zinc concentrations were decreased in critically ill patients, and were dramatically lower in patients with septic shock [28]. This suggests that hypozincaemia during systemic inflammation in humans may contribute to pathology. Thus it appears that, in both human and animal models, dietary zinc deficiency predisposes to infectious diseases, while hypozincaemia may also contribute to morbidity and mortality during severe infections.

## Benefits of zinc supplementation in host defence

Zinc supplementation has demonstrated beneficial effects in in-ectious disease, both clinically and in animal models. More than
three decades ago, Snyder and Walker [29] showed that ZnCl₂
administration to mice 1 h before LPS challenge offered almost
complete protection against an otherwise lethal dose. Improved
survival and reduced bacterial loads were also observed in a poly-
microbial sepsis model when C57Bl/6 mice received prophylactic
zinc gluconate treatment [30]. Similarly, zinc supplementation
decreased the parasite load in the blood in Wistar rats infected
with *Trypanosoma cruzi* [31], and restored the ability of alcohol-
fed animals to clear *Klebsiella pneumoniae* from the lung [32].
Beneficial effects of zinc administration in promoting pathogen
clearance and/or reducing pathology have also been reported in
a mouse *Candida albicans* infection model [33], a rat model of
*Escherichia coli*-mediated prostatitis [34], and a Rhesus monkey
model of severe diarrhoeal disease caused by enteropathogenic *E.
coli* [35]. More importantly, many studies have demonstrated that
zinc supplementation has beneficial effects in clinical settings,
particularly in severe diarrhoeal diseases and respiratory tract in-ections. Pooled analysis of randomized controlled trials of zinc
supplementation in children suffering from severe diarrhoea and
pneumonia showed that this treatment regime reduced the incid-
ence of pneumonia by 41% and the incidence of diarrhoea by
18% and its prevalence by 25% [36]. Similarly, a meta-analysis
of 22 independent studies demonstrated that oral zinc supple-
mentation reduced the frequency and duration of acute and per-
sistent diarrhoea in infants by up to 18% [37]. Indeed, a WHO
and UNICEF report recommended the inclusion of zinc in oral
rehydration solution to treat gastroenteritis in infants and children
[38]. Randomized placebo control trials on children with severe
pneumonia also showed that zinc supplementation (2 mg/kg per
day) reduced the length of hospital stay and the severity of infec-
tion [39]. Zinc supplementation has also been reported to show
beneficial effects for a range of other infectious diseases includ-
ing shigellosis, leprosy, tuberculosis and leishmaniasis [40]. Fi-
nally, Mocchegiani et al. [41] reported that zinc supplementation
<figure><img src="image_3.png" /></figure>

animal models, have conclusively demonstrated that zinc is es-
sential for normal immune function, but its precise cellular and
molecular role(s) remain enigmatic. Zinc deficiency was first
identified as a clinical syndrome in the Middle East approxim-
ately 50 years ago [10]. The first case was reported in Iran in
a male patient who presented with severe anaemia and growth
retardation [11]. This condition was associated with hepatosplen-
omegaly, hypogonadism, rough and dry skin, mental lethargy
and genophagia (clay eating), and was initially misdiagnosed as
iron-deficiency anaemia. Similar cases were reported in Egypt
[12], where a regimen of zinc supplementation reversed hypo-
gonadism and growth retardation [13]. In addition to develop-
mental anomalies and anaemia, the patients also suffered from
severe immune deficiency leading to intercurrent infection and
death before the age of 25 [8]. Furthermore, experimentally in-
duced mild zinc deficiency in human subjects [14] resulted in
a decrease in the ratio of CD4⁺ to CD8⁺ T-cells, and a de-
creased Th1 response as assessed by IFNγ (interferon γ), IL
(interleukin)-2 and TNFα (tumour necrosis factor α) secretion
[15]. It was proposed that defective Th1 responses compromised
host defence to intracellular parasitic infections, in particular.
In general, severe zinc deficiency is rare, but mild-to-moderate
deficiency is quite common throughout the world especially in
developing countries, lower socio-economic groups and in indi-
viduals with chronic alcoholism. The 2002 WHO world health
report attributed about 800 000 deaths (1.4% of global mortalit-
ies) to zinc deficiency, and estimated that it contributed to 16%
of lower respiratory tract infections, 18% of malaria and 10%
of diarrhoeal disease [16]. Animal models of zinc deficiency
have also been used to assess the role of zinc in regulating the
immune system and host defence. Early studies examined the
effect of zinc deficiency on T-cell function in young A/J mice
[17,18]. Thymic atrophy and diminished antigen-specific T-cell
responses were observed, which was reversed by dietary zinc
supplementation. In a mouse polymicrobial model of sepsis, zinc
deficiency resulted in increased bacterial burdens in the blood, a
heightened pro-inflammatory response, and increased mortality,
and these effects were reversed by zinc supplementation [19,20].
The enhanced inflammatory response correlated with increased
activity of NF-κB (nuclear factor κB) and expression of NF-κB-
dependent target genes [19], suggesting that zinc acts to constrain
this pathway during excessive inflammatory responses.

Zinc deficiency generally reflects insufficient dietary intake,
but systemic inflammation also results in a reduction in the levels
of circulating zinc (hypozincaemia). Indeed, it has long been
appreciated that plasma zinc levels decline during systemic bac-
terial infections [21], as a physiological component of the acute
phase response [22]. Hypozincaemia as a consequence of LPS
(lipopolysaccharide) challenge has been experimentally demon-
strated in humans. LPS administration to healthy human volun-
teers reduced serum zinc levels, without increasing zinc loss in
the urine or decreasing levels of the major zinc-binding protein,
serum albumin [23]. This suggests that the drop in circulating
zinc reflects its redistribution elsewhere. Evidence from animal
models suggests that increased uptake of zinc by hepatocytes
in the liver is the mechanism responsible. Yasuno et al. [24]

showed that when rats were given an intravenous dose of zinc
(50 µg/kg), the metal was rapidly distributed to the liver, spleen,
intestine, kidney and pancreas. Moreover, systemic inflamma-
tion in mice resulted in IL-6-dependent up-regulation of the zinc
importer Slc39a14, which enabled zinc uptake by hepatocytes
in the liver [25,26]. Several studies have documented reduced
serum zinc levels in various patient cohorts. For example, zinc
levels in polymorphonuclear cells from hospitalized elderly pa-
tients were reduced by comparison with those of healthy elderly
subjects [27]. A more recent study demonstrated that plasma
zinc concentrations were decreased in critically ill patients, and
were dramatically lower in patients with septic shock [28]. This
suggests that hypozincaemia during systemic inflammation in hu-
mans may contribute to pathology. Thus it appears that, in both
human and animal models, dietary zinc deficiency predisposes to
infectious diseases, while hypozincaemia may also contribute to
morbidity and mortality during severe infections.

## Benefits of zinc supplementation in host defence

Zinc supplementation has demonstrated beneficial effects in in-ectious disease, both clinically and in animal models. More than
three decades ago, Snyder and Walker [29] showed that ZnCl₂
administration to mice 1 h before LPS challenge offered almost
complete protection against an otherwise lethal dose. Improved
survival and reduced bacterial loads were also observed in a poly-
microbial sepsis model when C57Bl/6 mice received prophylactic
zinc gluconate treatment [30]. Similarly, zinc supplementation
decreased the parasite load in the blood in Wistar rats infected
with *Trypanosoma cruzi* [31], and restored the ability of alcohol-
fed animals to clear *Klebsiella pneumoniae* from the lung [32].
Beneficial effects of zinc administration in promoting pathogen
clearance and/or reducing pathology have also been reported in
a mouse *Candida albicans* infection model [33], a rat model of
*Escherichia coli*-mediated prostatitis [34], and a Rhesus monkey
model of severe diarrhoeal disease caused by enteropathogenic *E.
coli* [35]. More importantly, many studies have demonstrated that
zinc supplementation has beneficial effects in clinical settings,
particularly in severe diarrhoeal diseases and respiratory tract in-ections. Pooled analysis of randomized controlled trials of zinc
supplementation in children suffering from severe diarrhoea and
pneumonia showed that this treatment regime reduced the incid-
ence of pneumonia by 41% and the incidence of diarrhoea by
18% and its prevalence by 25% [36]. Similarly, a meta-analysis
of 22 independent studies demonstrated that oral zinc supple-
mentation reduced the frequency and duration of acute and per-
sistent diarrhoea in infants by up to 18% [37]. Indeed, a WHO
and UNICEF report recommended the inclusion of zinc in oral
rehydration solution to treat gastroenteritis in infants and children
[38]. Randomized placebo control trials on children with severe
pneumonia also showed that zinc supplementation (2 mg/kg per
day) reduced the length of hospital stay and the severity of infec-
tion [39]. Zinc supplementation has also been reported to show
beneficial effects for a range of other infectious diseases includ-
ing shigellosis, leprosy, tuberculosis and leishmaniasis [40]. Fi-
nally, Mocchegiani et al. [41] reported that zinc supplementation
reduced opportunistic infections in HIV-infected patients, al-
though the beneficial effect was restricted to certain pathogens
(*Pneumocystis carinii* and *C. albicans*) and not others [CMV
(cytomegalovirus), *Toxoplasma*]. Indeed, zinc supplementation
for the treatment of infectious diseases was not efficacious in
all infectious disease trials. For example, zinc supplementation
in a randomized placebo control trial among Polish children
suffering from acute gastroenteritis [42], and zinc and vitamin
A supplementation in pulmonary tuberculosis showed no thera-
peutic benefit [43]. Thus, zinc supplementation may be effective
in controlling specific infections only in individuals with not-
able zinc deficiency, or alternatively, other environmental and/or
genetic factors may impact on efficacy of this treatment. Fur-
thermore, zinc supplementation may actually exacerbate disease
severity for some pathogens. Vitamin A and zinc supplementa-
tion was assessed in gastrointestinal parasitic infections among
Mexican children [44]. Zinc reduced the incidence of *Giardia
lamblia* and *Entamoeba histolytica* infections, but increased As-
*caris lumbricoides*-associated diarrhoea. Animal model studies
also suggest that high-dose zinc supplementation may exert un-
desirable effects that are independent of immune function; for ex-
ample, leading to hippocampus-dependent memory impairment
[45]. Taken together, the above literature suggests that zinc has
an essential role in immune function and protecting against infec-
tious disease, but zinc supplementation is probably only effective
in conditions of zinc deficiency, and only for certain infections.
Thus, it is essential to understand the exact roles that zinc plays
during different infections, and the mechanism(s) by which it acts.

Zinc and macrophage antimicrobial responses

Zinc exerts a multitude of effects on numerous immune cell types [6]. Nonetheless, many of the studies reporting effects of zinc deficiency or supplementation on infectious disease outcomes also report effects on macrophage numbers or function. This suggests that macrophages may be an important cellular target of zinc action during infections. For example, beneficial effects of zinc supplementation in a mouse model of polymicrobial sepsis were associated with enhanced phagocytosis of *E. coli* and *Staphyl-ococcus aureus* by peritoneal macrophages [30]. Similarly, zinc supplementation increased peritoneal macrophage numbers in a *T. cruzi* infection model, while zinc deficiency impaired the ability of peritoneal macrophages to kill this parasite [31,46]. Several other studies have also reported that zinc promotes macrophage phagocytic capacity and/or pathogen clearance by these cells [47–49]. However, most of these studies have not addressed the molecular mechanisms responsible for such effects. Recent evidence suggests that regulated zinc trafficking within macrophages may play an active role in antimicrobial responses.

The macrophage activating cytokines TNFα and IFNγ pro-
moted the phagosomal accumulation of zinc in *Mycobacterium
avium*-infected mouse macrophages, and phagosomal zinc also
accumulated over time in response to infection with *Mycobac-
terium tuberculosis* [50]. Thus, this metal ion can concentrate
within the macrophage phagolysosome, where it presumably may
contribute to antimicrobial responses. A recent study supported

this concept by showing that upon infection of human macro-
phages, *M. tuberculosis* expressed *P*, which encodes a zinc
efflux pump [51]. This would likely be required to cope with
a high zinc environment. The same study also reported that *M.
tuberculosis* infection triggered the accumulation of free zinc
within macrophage phagosomes at 4 h post-infection, and that
this zinc co-localized with intracellular bacteria [51]. Such evid-
ence suggests that high levels of zinc may exert direct bactericidal
effects within macrophages. The specific mechanisms by which
this might occur are unknown, but are most likely to involve es-
sential proteins required for bacterial survival being inactivated,
for example by destruction of Fe–S clusters [52]. Competition
with other metal ions may also be involved. For example, high
concentrations of zinc can starve *Streptococcus pneumoniae* of
essential manganese, by competing for binding to the manganese
solute binding protein PsaA [53]. Whether similar mechanisms
operate for the professional intramacrophage pathogens such as
*M. tuberculosis* is unknown. It is also possible that the positive
effects of zinc on macrophage responses to pathogen challenge
relate to the numerous zinc-containing proteins with roles in
host defence. For example, MMPs (matrix metalloproteinases)
are zinc-dependent proteases [54], some of which have functions
in antimicrobial responses. MMP12, also referred to as macro-
phage elastase, has direct antimicrobial effects against bacteria
within the macrophage phagolysosome. It adheres to bacterial
cell walls and disrupts the cell membrane leading to cell death,
and this effect was reportedly independent of enzymatic activity
[55]. MMP7 is involved in the activation of defensins by cleaving
the pro form of α- and β-defensins to the active form [56], which
then can have direct antimicrobial effects.

In contrast to the above studies, zinc starvation may also be em-
ployed as part of the macrophage response to *Histoplasma cap-
sulatum* [57], a fungal pathogen that can survive intracellularly
within these cells. This study showed that zinc chelation restricted
*H. capsulatum* growth, and that infection of GM-CSF (granulo-
cyte/macrophage colony stimulating factor)-derived murine peri-
toneal and bone marrow macrophages with *H. capsulatum* de-
creased the intracellular zinc concentration. The TLR (Toll-like
receptor) 4 agonist LPS from Gram-negative bacteria also re-
duced the intracellular zinc concentration within mouse DC
(dendritic cells) [58], suggesting that zinc export may occur
in response to infection by some micro-organisms. Thus, zinc
restriction may also be utilized as a macrophage antimicrobial
mechanism, somewhat analogous to the antimicrobial effect of
zinc chelation by neutrophil-derived calprotectin in the extra-
cellular space [59–61]. Not surprisingly, some pathogens have
evolved mechanisms to thwart zinc starvation by the host. For
example, *Salmonella enterica* serovar Typhimurium (S. Typh-
imurium) thrives in the inflamed gut by expressing ZnuABC,
a high-affinity zinc transporter that overcomes calprotectin-
mediated zinc chelation [62]. The utilization of zinc in mac-
rophage antimicrobial pathways versus zinc sequestration as
a nutrient starvation strategy to limit microbial growth high-
lights the complexity of zinc involvement in macrophage func-
tions. Zinc trafficking may have distinct functions in these cells,
depending on the specific infectious agent encountered and/or the
reduced opportunistic infections in HIV-infected patients, al-
though the beneficial effect was restricted to certain pathogens
(*Pneumocystis carinii* and *C. albicans*) and not others [CMV
(cytomegalovirus), *Toxoplasma*]. Indeed, zinc supplementation
for the treatment of infectious diseases was not efficacious in
all infectious disease trials. For example, zinc supplementation
in a randomized placebo control trial among Polish children
suffering from acute gastroenteritis [42], and zinc and vitamin
A supplementation in pulmonary tuberculosis showed no thera-
peutic benefit [43]. Thus, zinc supplementation may be effective
in controlling specific infections only in individuals with not-
able zinc deficiency, or alternatively, other environmental and/or
genetic factors may impact on efficacy of this treatment. Fur-
thermore, zinc supplementation may actually exacerbate disease
severity for some pathogens. Vitamin A and zinc supplementa-
tion was assessed in gastrointestinal parasitic infections among
Mexican children [44]. Zinc reduced the incidence of *Giardia
lamblia* and *Entamoeba histolytica* infections, but increased As-
*caris lumbricoides*-associated diarrhoea. Animal model studies
also suggest that high-dose zinc supplementation may exert un-
desirable effects that are independent of immune function; for ex-
ample, leading to hippocampus-dependent memory impairment
[45]. Taken together, the above literature suggests that zinc has
an essential role in immune function and protecting against infec-
tious disease, but zinc supplementation is probably only effective
in conditions of zinc deficiency, and only for certain infections.
Thus, it is essential to understand the exact roles that zinc plays
during different infections, and the mechanism(s) by which it acts.

Zinc and macrophage antimicrobial responses

Zinc exerts a multitude of effects on numerous immune cell types [6]. Nonetheless, many of the studies reporting effects of zinc deficiency or supplementation on infectious disease outcomes also report effects on macrophage numbers or function. This suggests that macrophages may be an important cellular target of zinc action during infections. For example, beneficial effects of zinc supplementation in a mouse model of polymicrobial sepsis were associated with enhanced phagocytosis of *E. coli* and *Staphyl-ococcus aureus* by peritoneal macrophages [30]. Similarly, zinc supplementation increased peritoneal macrophage numbers in a *T. cruzi* infection model, while zinc deficiency impaired the ability of peritoneal macrophages to kill this parasite [31,46]. Several other studies have also reported that zinc promotes macrophage phagocytic capacity and/or pathogen clearance by these cells [47–49]. However, most of these studies have not addressed the molecular mechanisms responsible for such effects. Recent evidence suggests that regulated zinc trafficking within macrophages may play an active role in antimicrobial responses.

The macrophage activating cytokines TNFα and IFNγ pro-
moted the phagosomal accumulation of zinc in *Mycobacterium
avium*-infected mouse macrophages, and phagosomal zinc also
accumulated over time in response to infection with *Mycobac-
terium tuberculosis* [50]. Thus, this metal ion can concentrate
within the macrophage phagolysosome, where it presumably may
contribute to antimicrobial responses. A recent study supported

this concept by showing that upon infection of human macro-
phages, *M. tuberculosis* expressed *P*, which encodes a zinc
efflux pump [51]. This would likely be required to cope with
a high zinc environment. The same study also reported that *M.
tuberculosis* infection triggered the accumulation of free zinc
within macrophage phagosomes at 4 h post-infection, and that
this zinc co-localized with intracellular bacteria [51]. Such evid-
ence suggests that high levels of zinc may exert direct bactericidal
effects within macrophages. The specific mechanisms by which
this might occur are unknown, but are most likely to involve es-
sential proteins required for bacterial survival being inactivated,
for example by destruction of Fe–S clusters [52]. Competition
with other metal ions may also be involved. For example, high
concentrations of zinc can starve *Streptococcus pneumoniae* of
essential manganese, by competing for binding to the manganese
solute binding protein PsaA [53]. Whether similar mechanisms
operate for the professional intramacrophage pathogens such as
*M. tuberculosis* is unknown. It is also possible that the positive
effects of zinc on macrophage responses to pathogen challenge
relate to the numerous zinc-containing proteins with roles in
host defence. For example, MMPs (matrix metalloproteinases)
are zinc-dependent proteases [54], some of which have functions
in antimicrobial responses. MMP12, also referred to as macro-
phage elastase, has direct antimicrobial effects against bacteria
within the macrophage phagolysosome. It adheres to bacterial
cell walls and disrupts the cell membrane leading to cell death,
and this effect was reportedly independent of enzymatic activity
[55]. MMP7 is involved in the activation of defensins by cleaving
the pro form of α- and β-defensins to the active form [56], which
then can have direct antimicrobial effects.

In contrast to the above studies, zinc starvation may also be em-
ployed as part of the macrophage response to *Histoplasma cap-
sulatum* [57], a fungal pathogen that can survive intracellularly
within these cells. This study showed that zinc chelation restricted
*H. capsulatum* growth, and that infection of GM-CSF (granulo-
cyte/macrophage colony stimulating factor)-derived murine peri-
toneal and bone marrow macrophages with *H. capsulatum* de-
creased the intracellular zinc concentration. The TLR (Toll-like
receptor) 4 agonist LPS from Gram-negative bacteria also re-
duced the intracellular zinc concentration within mouse DC
(dendritic cells) [58], suggesting that zinc export may occur
in response to infection by some micro-organisms. Thus, zinc
restriction may also be utilized as a macrophage antimicrobial
mechanism, somewhat analogous to the antimicrobial effect of
zinc chelation by neutrophil-derived calprotectin in the extra-
cellular space [59–61]. Not surprisingly, some pathogens have
evolved mechanisms to thwart zinc starvation by the host. For
example, *Salmonella enterica* serovar Typhimurium (S. Typh-
imurium) thrives in the inflamed gut by expressing ZnuABC,
a high-affinity zinc transporter that overcomes calprotectin-
mediated zinc chelation [62]. The utilization of zinc in mac-
rophage antimicrobial pathways versus zinc sequestration as
a nutrient starvation strategy to limit microbial growth high-
lights the complexity of zinc involvement in macrophage func-
tions. Zinc trafficking may have distinct functions in these cells,
depending on the specific infectious agent encountered and/or the
<figure><img src="image_4.png" /></figure>

zinc concentrations that are present. Indeed, the effects of zinc on macrophage inflammatory responses vary in a concentration-dependent manner [5].

## Zinc effects on macrophage signalling and gene expression and links to host defence

While the above emerging literature implicates regulated zinc trafficking in macrophages in direct antimicrobial responses and/or nutrient starvation, a much more substantial literature has documented effects of zinc on signalling and inflammatory outputs in immune cells, including monocytes and macrophages [5,6]. Indeed, approximately 5% of genes were reported to be zinc-responsive in a human monocytic cell line [63]. Such effects are likely to be an important component of zinc-mediated host defence. Zinc regulates inflammatory gene expression through multiple pathways including protein tyrosine phosphorylation, MAPKs (mitogen-activated protein kinases), PKC (protein kinase C), PDEs (phosphodiesterases) and NF-κB [5,6], many of which lie downstream of TLRs. Indeed, much of the focus relating to zinc signalling in monocytes and macrophages has been on TLR signalling. Relatively few studies have assessed other pathways such as Nod-like receptor-mediated activation of the inflammasome, a large intracellular signalling platform that processes caspase-1 leading to maturation of caspase-1-dependent cytokines such as IL-1β [64]. However, one study has reported that IL-1β release downstream of the NLRP3 inflammasome was dependent on zinc [65]. Hence, zinc may contribute to macrophage-mediated host defence through promoting inflammasome activation, in addition to regulating TLR responses. A reoccurring theme in zinc signalling is that zinc concentrations are critical; while low concentrations may be required for the activation of a specific pro-inflammatory signalling pathway, high concentrations can suppress the same pathway [5]. How regulated zinc trafficking in macrophages (introduced above) intersects with effects on inflammatory signalling and gene expression in these cells requires more detailed investigation, but existing evidence suggests that distinct temporal changes may be important. LPS triggers a rapid and transient accumulation of free zinc in human and mouse monocytes/macrophages (within minutes), and this effect was required for the activation of pro-inflammatory signalling pathways in these cells [66]. However, LPS decreased the intracellular zinc levels in DC in a TRIF [TIR (Toll/interleukin-1 receptor) domain-containing adaptor protein inducing interferon β]-dependent manner over a longer time course (several hours), and this was required for efficient DC maturation, and thus antigen presentation [58]. Thus, time is also likely to be an important factor in dictating intracellular zinc concentrations and how this affects inflammatory responses. The complex interplay between zinc trafficking and macrophage functions is summarized in Figure 1, and below, we briefly outline the existing literature linking zinc to pro- and anti-inflammatory signalling and gene expression in macrophages.

Zinc regulates the pro-inflammatory transcription factor NF-κB. Modest zinc supplementation (15 mg/day) of healthy young men substantially enhanced LPS-induced mRNA expression of

the NF-κB-dependent gene TNF in monocytes [67], whereas dietary zinc depletion suppressed LPS-inducible TNF production from whole blood [68]. Given the crucial role of this cytokine in control of infectious diseases, such effects are likely to be important for zinc-mediated host defence. The direct requirement for zinc in activating NF-κB has been demonstrated in T-cells [69], and in response to LPS in human and mouse monocytes/macrophages [66]. However, an extensive literature has also documented inhibitory effects of zinc on NF-κB activation, both *in vitro* [70] and *in vivo* [71]. In the latter case, this also correlated with reduced TNF expression and liver injury, an effect that was MT (metallothionein)-independent. Others have also reported that zinc exerts protective effects *in vivo* by limiting NF-κB activation during innate immune responses [19]. Multiple mechanisms of NF-κB inhibition have been identified. Very recently, direct inhibition of IKKβ [IκB (inhibitor of NF-κB) kinase β] by zinc was reported [72], while others have also shown that zinc indirectly inhibits IKKβ via cGMP-dependent activation of PKA (protein kinase A) [73]. Zinc also up-regulates the expression of A20, a negative regulator of inflammatory responses [74]. Divergent effects of zinc have also been noted with respect to PDEs, which require zinc for activity but are inhibited by high concentrations of zinc [5,6]. Various other primary TLR-activated signalling pathways including MAPK p38 and ERK (extracellular-signal-regulated kinase) are also zinc-dependent [66]. Thus, it would seem likely that zinc acts as a key component of many primary TLR signalling events, but also as an important mechanism of feedback control. This basic premise may partly underlie the seemingly opposing pro- and anti-inflammatory effects of zinc on macrophage signalling and gene expression. At least part of the mechanism by which zinc exerts anti-inflammatory effects may also relate to its indirect antioxidant proprieties. Zinc can protect essential thiol-containing proteins by stabilizing them, and can also reduce the formation of ROS (reactive oxygen species) by competition with redox-active transition metals such as Cu⁺ and Fe²⁺ [75]. Zn²⁺ is also an important co-factor of SOD (superoxide dismutase) [76], which plays an important role in the degradation of pro-inflammatory ROS. There are certainly some studies linking protective effects of zinc during inflammation to reduced oxidative stress. Zinc supplementation to alcohol-fed rats restored expression of Nrf2 (nuclear factor-erythroid 2-related factor 2) [32], a transcription factor that protects against oxidative stress and limits host pathology during sepsis [77]. Similarly, the zinc-mediated reduction in LPS-induced liver injury in mice was associated with reduced oxidative stress [71]. Such mechanisms may contribute to the reduced immunopathology associated with zinc supplementation during severe infectious diseases.

## What zinc transporters regulate macrophage functions?

Zinc homeostasis is maintained through MT, a family of zinc-binding proteins controlling cellular zinc distribution in a redox-dependent manner [78]. LPS rapidly up-regulates MT expression in human macrophages [79]; presumably this may act to sequester
<figure><img src="image_4.png" /></figure>

zinc concentrations that are present. Indeed, the effects of zinc on macrophage inflammatory responses vary in a concentration-dependent manner [5].

## Zinc effects on macrophage signalling and gene expression and links to host defence

While the above emerging literature implicates regulated zinc trafficking in macrophages in direct antimicrobial responses and/or nutrient starvation, a much more substantial literature has documented effects of zinc on signalling and inflammatory outputs in immune cells, including monocytes and macrophages [5,6]. Indeed, approximately 5% of genes were reported to be zinc-responsive in a human monocytic cell line [63]. Such effects are likely to be an important component of zinc-mediated host defence. Zinc regulates inflammatory gene expression through multiple pathways including protein tyrosine phosphorylation, MAPKs (mitogen-activated protein kinases), PKC (protein kinase C), PDEs (phosphodiesterases) and NF-κB [5,6], many of which lie downstream of TLRs. Indeed, much of the focus relating to zinc signalling in monocytes and macrophages has been on TLR signalling. Relatively few studies have assessed other pathways such as Nod-like receptor-mediated activation of the inflammasome, a large intracellular signalling platform that processes caspase-1 leading to maturation of caspase-1-dependent cytokines such as IL-1β [64]. However, one study has reported that IL-1β release downstream of the NLRP3 inflammasome was dependent on zinc [65]. Hence, zinc may contribute to macrophage-mediated host defence through promoting inflammasome activation, in addition to regulating TLR responses. A reoccurring theme in zinc signalling is that zinc concentrations are critical; while low concentrations may be required for the activation of a specific pro-inflammatory signalling pathway, high concentrations can suppress the same pathway [5]. How regulated zinc trafficking in macrophages (introduced above) intersects with effects on inflammatory signalling and gene expression in these cells requires more detailed investigation, but existing evidence suggests that distinct temporal changes may be important. LPS triggers a rapid and transient accumulation of free zinc in human and mouse monocytes/macrophages (within minutes), and this effect was required for the activation of pro-inflammatory signalling pathways in these cells [66]. However, LPS decreased the intracellular zinc levels in DC in a TRIF [TIR (Toll/interleukin-1 receptor) domain-containing adaptor protein inducing interferon β]-dependent manner over a longer time course (several hours), and this was required for efficient DC maturation, and thus antigen presentation [58]. Thus, time is also likely to be an important factor in dictating intracellular zinc concentrations and how this affects inflammatory responses. The complex interplay between zinc trafficking and macrophage functions is summarized in Figure 1, and below, we briefly outline the existing literature linking zinc to pro- and anti-inflammatory signalling and gene expression in macrophages.

Zinc regulates the pro-inflammatory transcription factor NF-κB. Modest zinc supplementation (15 mg/day) of healthy young men substantially enhanced LPS-induced mRNA expression of

the NF-κB-dependent gene TNF in monocytes [67], whereas dietary zinc depletion suppressed LPS-inducible TNF production from whole blood [68]. Given the crucial role of this cytokine in control of infectious diseases, such effects are likely to be important for zinc-mediated host defence. The direct requirement for zinc in activating NF-κB has been demonstrated in T-cells [69], and in response to LPS in human and mouse monocytes/macrophages [66]. However, an extensive literature has also documented inhibitory effects of zinc on NF-κB activation, both *in vitro* [70] and *in vivo* [71]. In the latter case, this also correlated with reduced TNF expression and liver injury, an effect that was MT (metallothionein)-independent. Others have also reported that zinc exerts protective effects *in vivo* by limiting NF-κB activation during innate immune responses [19]. Multiple mechanisms of NF-κB inhibition have been identified. Very recently, direct inhibition of IKKβ [IκB (inhibitor of NF-κB) kinase β] by zinc was reported [72], while others have also shown that zinc indirectly inhibits IKKβ via cGMP-dependent activation of PKA (protein kinase A) [73]. Zinc also up-regulates the expression of A20, a negative regulator of inflammatory responses [74]. Divergent effects of zinc have also been noted with respect to PDEs, which require zinc for activity but are inhibited by high concentrations of zinc [5,6]. Various other primary TLR-activated signalling pathways including MAPK p38 and ERK (extracellular-signal-regulated kinase) are also zinc-dependent [66]. Thus, it would seem likely that zinc acts as a key component of many primary TLR signalling events, but also as an important mechanism of feedback control. This basic premise may partly underlie the seemingly opposing pro- and anti-inflammatory effects of zinc on macrophage signalling and gene expression. At least part of the mechanism by which zinc exerts anti-inflammatory effects may also relate to its indirect antioxidant proprieties. Zinc can protect essential thiol-containing proteins by stabilizing them, and can also reduce the formation of ROS (reactive oxygen species) by competition with redox-active transition metals such as Cu⁺ and Fe²⁺ [75]. Zn²⁺ is also an important co-factor of SOD (superoxide dismutase) [76], which plays an important role in the degradation of pro-inflammatory ROS. There are certainly some studies linking protective effects of zinc during inflammation to reduced oxidative stress. Zinc supplementation to alcohol-fed rats restored expression of Nrf2 (nuclear factor-erythroid 2-related factor 2) [32], a transcription factor that protects against oxidative stress and limits host pathology during sepsis [77]. Similarly, the zinc-mediated reduction in LPS-induced liver injury in mice was associated with reduced oxidative stress [71]. Such mechanisms may contribute to the reduced immunopathology associated with zinc supplementation during severe infectious diseases.

## What zinc transporters regulate macrophage functions?

Zinc homeostasis is maintained through MT, a family of zinc-binding proteins controlling cellular zinc distribution in a redox-dependent manner [78]. LPS rapidly up-regulates MT expression in human macrophages [79]; presumably this may act to sequester
<figure><img src="image_5.png" /><figcaption>**Figure 1** Interplay between zinc trafficking, inflammatory signalling and antimicrobial responses in macrophages</figcaption></figure>

TLR4 signalling promotes the rapid accumulation of free zinc (within minutes) within macrophages, an effect that may be mediated by the zinc importer SLC39A8 (1) and/or redistribution of intracellular zinc pools (2). Zinc is required for the activation of many TLR signalling responses and for pannexin-1-dependent inflammasome activation (3). However, high levels of zinc can also inhibit inflammatory signalling pathways in macrophages, for example by inhibiting IKKβ or promoting the expression or activity of Nrf2 and A20 (4). TLR signalling modulates the expression of many zinc transport genes (5) and mobilizes the intracellular zinc pool. TLR-induced SLC39A family members (e.g. SLC39A1, 4 and/or 6) may promote zinc efflux to starve pathogens of zinc (6) and/or deliver zinc to the phagosome and other intracellular vesicles (7) to activate antimicrobial responses (including direct zinc toxicity). Inhibition of expression of some SLC39A family members (e.g. SLC39A6, 10) by TLR signalling may contribute to net zinc export, whereas up-regulation of others (e.g. SLC39A8, 14) may contribute to zinc uptake under some conditions (8) and/or vesicular export of zinc and/or other metal ions to the cytoplasm (9).

the free intracellular zinc that is generated immediately after TLR activation [66], and would be broadly consistent with the anti-inflammatory roles of MT, as identified by gene knock-out studies [80–82]. Zinc homeostasis is additionally controlled by two distinct families of zinc transporters, the SLC30A family (ZnT; vertebrate cation diffusion facilitator family) and the SLC39A family [Zip (Zrt/Irt-like proteins)]. SLC30A family members are predicted to have six transmembrane domains, while members of the SLC39A family are predicted to have eight transmembrane domains [83]. Human and mouse orthologues of the ten identified members of the SLC30A family and the 14 identified members of the SLC39A family are closely related as assessed by phylogenetic analysis of their encoded proteins (Figure 2). A neighbour-joining phylogenetic tree of protein sequences for the SLC30A family

shows that human and mouse SLC30A9 diverge from the rest of the family, possibly indicating a disparate function for this family member. Members of the SLC39A family can be divided into three groups with SLC39A11 in one group, SLC39A1, 2 and 3 in another group and the rest of the family (SLC39A4–10, 12 and 13) making up the last group. Within the latter group, SLC39A8 and 14 are closely related, which is interesting in light of studies suggesting roles for both of these genes in innate immune pathways (discussed below). The SLC30A family have generally been linked to zinc efflux from cells, whereas members of the SLC39A family promote zinc influx [83]. Importantly, several SLC39A proteins have been shown to transport other metal ions, so the specificity of individual transporters is likely to be highly dependent on the specific cell-type, as well as the
<figure><img src="image_5.png" /><figcaption>**Figure 1** Interplay between zinc trafficking, inflammatory signalling and antimicrobial responses in macrophages</figcaption></figure>

TLR4 signalling promotes the rapid accumulation of free zinc (within minutes) within macrophages, an effect that may be mediated by the zinc importer SLC39A8 (1) and/or redistribution of intracellular zinc pools (2). Zinc is required for the activation of many TLR signalling responses and for pannexin-1-dependent inflammasome activation (3). However, high levels of zinc can also inhibit inflammatory signalling pathways in macrophages, for example by inhibiting IKKβ or promoting the expression or activity of Nrf2 and A20 (4). TLR signalling modulates the expression of many zinc transport genes (5) and mobilizes the intracellular zinc pool. TLR-induced SLC39A family members (e.g. SLC39A1, 4 and/or 6) may promote zinc efflux to starve pathogens of zinc (6) and/or deliver zinc to the phagosome and other intracellular vesicles (7) to activate antimicrobial responses (including direct zinc toxicity). Inhibition of expression of some SLC39A family members (e.g. SLC39A6, 10) by TLR signalling may contribute to net zinc export, whereas up-regulation of others (e.g. SLC39A8, 14) may contribute to zinc uptake under some conditions (8) and/or vesicular export of zinc and/or other metal ions to the cytoplasm (9).

the free intracellular zinc that is generated immediately after TLR activation [66], and would be broadly consistent with the anti-inflammatory roles of MT, as identified by gene knock-out studies [80–82]. Zinc homeostasis is additionally controlled by two distinct families of zinc transporters, the SLC30A family (ZnT; vertebrate cation diffusion facilitator family) and the SLC39A family [Zip (Zrt/Irt-like proteins)]. SLC30A family members are predicted to have six transmembrane domains, while members of the SLC39A family are predicted to have eight transmembrane domains [83]. Human and mouse orthologues of the ten identified members of the SLC30A family and the 14 identified members of the SLC39A family are closely related as assessed by phylogenetic analysis of their encoded proteins (Figure 2). A neighbour-joining phylogenetic tree of protein sequences for the SLC30A family

shows that human and mouse SLC30A9 diverge from the rest of the family, possibly indicating a disparate function for this family member. Members of the SLC39A family can be divided into three groups with SLC39A11 in one group, SLC39A1, 2 and 3 in another group and the rest of the family (SLC39A4–10, 12 and 13) making up the last group. Within the latter group, SLC39A8 and 14 are closely related, which is interesting in light of studies suggesting roles for both of these genes in innate immune pathways (discussed below). The SLC30A family have generally been linked to zinc efflux from cells, whereas members of the SLC39A family promote zinc influx [83]. Importantly, several SLC39A proteins have been shown to transport other metal ions, so the specificity of individual transporters is likely to be highly dependent on the specific cell-type, as well as the
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 2 Nearest-neighbour-joining generated phylogenetic tree of human and mouse SLC30A and SLC39A family members**</figcaption></figure>

Protein sequences for all the family members were obtained from the Ensembl genome browser website (http://www.ensembl.org/) and aligned using clustal omega from EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalo/). The alignment was imported into MEGA5.1 software [152] and a phylogenetic tree was generated.

intracellular and extracellular environments. Expression analyses and genetic studies provide some evidence for roles for individual zinc transporters in host defence, but very few functional studies in macrophages are yet to emerge.

## The SLC30A family

The decrease in intracellular zinc concentrations in innate immune cells in response to *H. capsulatum* [57] and LPS [58]

implicate specific SLC30A family members in zinc efflux from macrophages during pathogen challenge. Most of the SLC30A family are expressed in various immune cell populations [84], and it is unknown which specific members are involved in zinc efflux from macrophages. Nevertheless, LPS up-regulated the mRNA expression of *Slc30a1*, *Slc30a4* and *Slc30a6* in murine DC [58], thus presenting these as obvious candidates. Interestingly, this effect was dependent on the TLR adaptor protein TRIF, as was the reduction in intracellular zinc levels. This provides circumstantial evidence that one or more of these transporters may contribute to LPS-triggered zinc efflux from macrophages. *SLC30A1* expression was also up-regulated by *M. tuberculosis* infection in human macrophages [51]. As metal ion exporters, it is possible that one or more of these transporters may also contribute to the accumulation of zinc within macrophage phagosomes in response to *M. tuberculosis* [50,51] and perhaps other infectious agents. Indeed, several members of this family including SLC30A2 [85,86], SLC30A6 [87], SLC30A7 [88] and SLC30A8 have been localized to acidic endosomal/lysosomal vesicles, the *trans*-Golgi network, the Golgi apparatus and intracellular vesicles, respectively, in non-macrophage cell types.

## The SLC39A family

The SLC39A family are thought to transport metal ions to the cytoplasm from either the extracellular environment or intracellular vesicular compartments. In keeping with the LPS-mediated reduction in intracellular zinc levels in mouse DC, LPS down-regulated mRNA levels of *Slc39a6* and *Slc39a10* in these cells [58]. Furthermore, Slc39a6 overexpression blocked the LPS-triggered reduction in intracellular zinc levels, implying that the down-regulation of these transporters may be required for regulated zinc export in response to Gram-negative bacterial pathogens. Slc39a6 was also recently reported to have functions in T-cells during immunological synapse formation with DC. Knock down of *Slc39a6* expression diminished zinc uptake by activated T-cells, which was required for signalling downstream of the T-cell receptor [89]. Although no studies have yet assessed Slc39a6 function in macrophages, the above reports hint that this transporter may have a role in regulating zinc trafficking, and potentially antimicrobial responses, in these cells.

SLC39A8 has been linked to inflammatory responses in various cell types. It was reported to have a cytoprotective function in respiratory epithelial cells, where it protected these cells from TNFα-induced cytotoxicity [90]. TNFα promoted the translocation of SLC39A8 to the plasma membrane in these cells, probably leading to enhanced zinc uptake and cytoprotection. SLC39A8 function has also been investigated in human T-cells [91]. In these cells, it accumulated within the lysosomal compartment and enhanced zinc-inducible IFNγ production. Importantly, elevated SLC39A8 mRNA expression in monocytes has also been associated with sepsis severity [28], and more recently SLC39A8 was identified as a gene affecting the course of malaria infection in West African children [92]. These links to infectious disease outcomes and to inflammatory pathways suggest that SLC39A8 may regulate macrophage antimicrobial pathways, although this
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 2 Nearest-neighbour-joining generated phylogenetic tree of human and mouse SLC30A and SLC39A family members**</figcaption></figure>

Protein sequences for all the family members were obtained from the Ensembl genome browser website (http://www.ensembl.org/) and aligned using clustal omega from EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalo/). The alignment was imported into MEGA5.1 software [152] and a phylogenetic tree was generated.

intracellular and extracellular environments. Expression analyses and genetic studies provide some evidence for roles for individual zinc transporters in host defence, but very few functional studies in macrophages are yet to emerge.

## The SLC30A family

The decrease in intracellular zinc concentrations in innate immune cells in response to *H. capsulatum* [57] and LPS [58]

implicate specific SLC30A family members in zinc efflux from macrophages during pathogen challenge. Most of the SLC30A family are expressed in various immune cell populations [84], and it is unknown which specific members are involved in zinc efflux from macrophages. Nevertheless, LPS up-regulated the mRNA expression of *Slc30a1*, *Slc30a4* and *Slc30a6* in murine DC [58], thus presenting these as obvious candidates. Interestingly, this effect was dependent on the TLR adaptor protein TRIF, as was the reduction in intracellular zinc levels. This provides circumstantial evidence that one or more of these transporters may contribute to LPS-triggered zinc efflux from macrophages. *SLC30A1* expression was also up-regulated by *M. tuberculosis* infection in human macrophages [51]. As metal ion exporters, it is possible that one or more of these transporters may also contribute to the accumulation of zinc within macrophage phagosomes in response to *M. tuberculosis* [50,51] and perhaps other infectious agents. Indeed, several members of this family including SLC30A2 [85,86], SLC30A6 [87], SLC30A7 [88] and SLC30A8 have been localized to acidic endosomal/lysosomal vesicles, the *trans*-Golgi network, the Golgi apparatus and intracellular vesicles, respectively, in non-macrophage cell types.

## The SLC39A family

The SLC39A family are thought to transport metal ions to the cytoplasm from either the extracellular environment or intracellular vesicular compartments. In keeping with the LPS-mediated reduction in intracellular zinc levels in mouse DC, LPS down-regulated mRNA levels of *Slc39a6* and *Slc39a10* in these cells [58]. Furthermore, Slc39a6 overexpression blocked the LPS-triggered reduction in intracellular zinc levels, implying that the down-regulation of these transporters may be required for regulated zinc export in response to Gram-negative bacterial pathogens. Slc39a6 was also recently reported to have functions in T-cells during immunological synapse formation with DC. Knock down of *Slc39a6* expression diminished zinc uptake by activated T-cells, which was required for signalling downstream of the T-cell receptor [89]. Although no studies have yet assessed Slc39a6 function in macrophages, the above reports hint that this transporter may have a role in regulating zinc trafficking, and potentially antimicrobial responses, in these cells.

SLC39A8 has been linked to inflammatory responses in various cell types. It was reported to have a cytoprotective function in respiratory epithelial cells, where it protected these cells from TNFα-induced cytotoxicity [90]. TNFα promoted the translocation of SLC39A8 to the plasma membrane in these cells, probably leading to enhanced zinc uptake and cytoprotection. SLC39A8 function has also been investigated in human T-cells [91]. In these cells, it accumulated within the lysosomal compartment and enhanced zinc-inducible IFNγ production. Importantly, elevated SLC39A8 mRNA expression in monocytes has also been associated with sepsis severity [28], and more recently SLC39A8 was identified as a gene affecting the course of malaria infection in West African children [92]. These links to infectious disease outcomes and to inflammatory pathways suggest that SLC39A8 may regulate macrophage antimicrobial pathways, although this
awaits functional evidence. Given that this transporter can reside
at both the cell surface and in lysosomal compartments, it is dif-
ficult to predict how it might exactly function in antimicrobial
pathways. However, recent work from Liu *et al* did identify a role
for this gene in acting as a feedback controller of macrophage in-
flammatory responses [72]. The authors confirmed that LPS and
TNF up-regulated *SLC39A8* expression, and that the mechan-
ism involved direct regulation by the transcription factor NF-κB.
The authors also showed that this up-regulation of SLC39A8 en-
abled enhanced zinc uptake, which acted to directly inhibit IKKβ
function. Very recently, evidence for SLC39A14 in macrophage
function has emerged. LPS up-regulated *SLC39A14* mRNA in
primary human macrophages and this acted to constrain inflam-
matory responses [93]. *Slc39a14* mRNA and protein was also
LPS-inducible in mice, and *Slc39a14*⁻¹⁻⁻ mice have impaired
zinc uptake, altered plasma zinc and IL-6 levels after LPS admin-
istration, as well as dysregulated metabolism [25]. Furthermore,
the up-regulation of *Slc39a14* was previously reported to be IL-6
dependent [26], suggesting that there may be some level of in-
terdependence between *Slc39a14* and *Il-6* expression. Given this
literature, one would anticipate a likely role for *Slc39a14* in host
responses to infection. Thus, while functional studies on zinc im-
porters in infectious diseases are limited, SLC39A6, SLC39A8
and/or SLC39A14 represent obvious candidates for regulating
zinc trafficking within macrophages, which would likely impact
on zinc-regulated signalling, inflammatory responses and mi-
crobicidal pathways.

Copper deficiency compromises immune function
and host defence

Copper is required for fundamental metabolic processes, but can
be toxic when present in excess [94]. It can exist in two oxidation
states in biological systems, cycling between Cu(I) (reduced) and
Cu(II) (oxidized) forms, which is harnessed by redox-active en-
zymes that use copper to accept and donate electrons [95]. Like
zinc, copper is required for the development and maintenance of
immune function. For example, it is an essential component of
the SOD enzyme, which catalyses the production of H₂O₂ from
superoxide in neutrophils and monocytes [96]. Several lines of
evidence indicate that copper deficiency perturbs immune func-
tion. Firstly, copper deficiency, in addition to causing neurolo-
gical dysfunctions, also results in haematological abnormalities,
most commonly neutropenia and anaemia [97]. Copper-deficient
patients also displayed decreased numbers of myeloid precurs-
ors in the bone marrow, as well as vacuolization of these cells
[97]. Susceptibility to infections, such as recurrent pulmonary
and urinary tract infections and septicaemia has also been repor-
ted in Menkes disease [98–101], an X-linked neurodegenerative
disease caused by dysregulated copper trafficking [102].

Numerous studies have monitored effects of copper deficiency
on immune function in animal models. One of the first reports
demonstrated compromised humoral immunity in mice fed on
a low copper diet [98]. Subsequent studies have reported in-
creased susceptibility to infectious diseases in copper-deficient
animals. For example, mortality rates in response to infection with

*Pasteurella haemolytica* were enhanced in mice fed on a copper-
deficient diet [103]. Similarly, copper-deficient rats had enhanced
mortality rates upon infection with *S. Typhimurium* [104] or *C.
albicans* [105], as well as elevated parasitaemia after infection
with *Trypanosoma lewisi* [106]. The capacity of leucocytes isol-
ated from copper-deficient livestock to kill *C. albicans* was also
significantly reduced [107], which may relate to defects in the
functions of neutrophils [108] or macrophages (discussed below).

Copper as a regulator of macrophage function

Copper deficiency impacts innate and acquired immune re-
sponses, suggesting that copper is likely to regulate the func-
tions of multiple immune cell types [109]. As with the zinc lit-
erature, several studies have demonstrated that copper regulates
macrophage antimicrobial functions. Macrophages from copper-
deficient rats were unimpaired in their ability to phagocytose
erythrocytes, but had a defective respiratory burst and were com-
promised in their ability to kill *C. albicans* [105]. *In vitro* stud-
ies also show that copper regulates macrophage antimicrobial
pathways. Some of the first evidence for this again came from
elemental analysis of macrophage phagosomes; the macrophage
activating cytokines IFNγ and TNFα promoted the accumulation
of copper within the phagosomes of *M. avium*-infected macro-
phages [50], suggesting that this metal ion may have some role in
macrophage antimicrobial responses. Consistent with this, exo-
genous copper promoted the bactericidal activity of IFNγ-treated
RAW264.7 mouse macrophages against *E. coli*, and this effect
was inhibited by the anti-oxidant ebselen [110]. This suggests
that copper may contribute to ROS-dependent killing in mac-
rophages, in keeping with the well-known capacity of Cu(I) to
catalyse the generation of hydroxyl radical from H₂O₂. This study
also showed that both IFNγ and LPS regulated copper-trafficking
pathways in macrophages. Studies on the intramacrophage patho-
gen *S. Typhimurium* also provide functional evidence for a role
in macrophage antimicrobial pathways. Infection of mouse mac-
rophages with *S. Typhimurium*, as well as treatment with LPS,
promoted the accumulation of copper within intracellular ves-
icles. This response peaked at about 14 h post-stimulation. Fur-
thermore, a cell impermeable copper chelator (bathocuproinedi-
sulfonic acid) reduced vesicular copper accumulation in macro-
phages, and this impaired the ability of primary mouse macro-
phages to kill *S. Typhimurium* [111]. The use of a *Salmonella*
copper-responsive promoter reporter strain also provided evid-
ence that *S. Typhimurium* within macrophage phagosomes were
subjected to an increase in copper levels [112]. Collectively, these
studies suggest that copper may regulate both immediate and
delayed macrophage antimicrobial pathways. Notably, whereas
phagocytosis-induced ROS is an immediate response occurring
within the first 30 min of particle uptake, TLR signalling also pro-
motes a slower accumulation of ROS, which is derived from the
mitochondria [113]. Hence, it is conceivable that the delayed vesi-
cular accumulation of copper within macrophages in response to
LPS could also contribute to this pathway of oxidative stress. It
is also possible that inducible copper redistribution contributes
to macrophage-mediated host defence by promoting the export
awaits functional evidence. Given that this transporter can reside
at both the cell surface and in lysosomal compartments, it is dif-
ficult to predict how it might exactly function in antimicrobial
pathways. However, recent work from Liu *et al* did identify a role
for this gene in acting as a feedback controller of macrophage in-
flammatory responses [72]. The authors confirmed that LPS and
TNF up-regulated *SLC39A8* expression, and that the mechan-
ism involved direct regulation by the transcription factor NF-κB.
The authors also showed that this up-regulation of SLC39A8 en-
abled enhanced zinc uptake, which acted to directly inhibit IKKβ
function. Very recently, evidence for SLC39A14 in macrophage
function has emerged. LPS up-regulated *SLC39A14* mRNA in
primary human macrophages and this acted to constrain inflam-
matory responses [93]. *Slc39a14* mRNA and protein was also
LPS-inducible in mice, and *Slc39a14*⁻¹⁻² mice have impaired
zinc uptake, altered plasma zinc and IL-6 levels after LPS admin-
istration, as well as dysregulated metabolism [25]. Furthermore,
the up-regulation of *Slc39a14* was previously reported to be IL-6
dependent [26], suggesting that there may be some level of in-
terdependence between *Slc39a14* and *Il-6* expression. Given this
literature, one would anticipate a likely role for *Slc39a14* in host
responses to infection. Thus, while functional studies on zinc im-
porters in infectious diseases are limited, SLC39A6, SLC39A8
and/or SLC39A14 represent obvious candidates for regulating
zinc trafficking within macrophages, which would likely impact
on zinc-regulated signalling, inflammatory responses and mi-
crobicidal pathways.

Copper deficiency compromises immune function
and host defence

Copper is required for fundamental metabolic processes, but can
be toxic when present in excess [94]. It can exist in two oxidation
states in biological systems, cycling between Cu(I) (reduced) and
Cu(II) (oxidized) forms, which is harnessed by redox-active en-
zymes that use copper to accept and donate electrons [95]. Like
zinc, copper is required for the development and maintenance of
immune function. For example, it is an essential component of
the SOD enzyme, which catalyses the production of H₂O₂ from
superoxide in neutrophils and monocytes [96]. Several lines of
evidence indicate that copper deficiency perturbs immune func-
tion. Firstly, copper deficiency, in addition to causing neurolo-
gical dysfunctions, also results in haematological abnormalities,
most commonly neutropenia and anaemia [97]. Copper-deficient
patients also displayed decreased numbers of myeloid precurs-
ors in the bone marrow, as well as vacuolization of these cells
[97]. Susceptibility to infections, such as recurrent pulmonary
and urinary tract infections and septicaemia has also been repor-
ted in Menkes disease [98–101], an X-linked neurodegenerative
disease caused by dysregulated copper trafficking [102].

Numerous studies have monitored effects of copper deficiency
on immune function in animal models. One of the first reports
demonstrated compromised humoral immunity in mice fed on
a low copper diet [98]. Subsequent studies have reported in-
creased susceptibility to infectious diseases in copper-deficient
animals. For example, mortality rates in response to infection with

*Pasteurella haemolytica* were enhanced in mice fed on a copper-
deficient diet [103]. Similarly, copper-deficient rats had enhanced
mortality rates upon infection with *S. Typhimurium* [104] or *C.
albicans* [105], as well as elevated parasitaemia after infection
with *Trypanosoma lewisi* [106]. The capacity of leucocytes isol-
ated from copper-deficient livestock to kill *C. albicans* was also
significantly reduced [107], which may relate to defects in the
functions of neutrophils [108] or macrophages (discussed below).

Copper as a regulator of macrophage function

Copper deficiency impacts innate and acquired immune re-
sponses, suggesting that copper is likely to regulate the func-
tions of multiple immune cell types [109]. As with the zinc lit-
erature, several studies have demonstrated that copper regulates
macrophage antimicrobial functions. Macrophages from copper-
deficient rats were unimpaired in their ability to phagocytose
erythrocytes, but had a defective respiratory burst and were com-
promised in their ability to kill *C. albicans* [105]. *In vitro* stud-
ies also show that copper regulates macrophage antimicrobial
pathways. Some of the first evidence for this again came from
elemental analysis of macrophage phagosomes; the macrophage
activating cytokines IFNγ and TNFα promoted the accumulation
of copper within the phagosomes of *M. avium*-infected macro-
phages [50], suggesting that this metal ion may have some role in
macrophage antimicrobial responses. Consistent with this, exo-
genous copper promoted the bactericidal activity of IFNγ-treated
RAW264.7 mouse macrophages against *E. coli*, and this effect
was inhibited by the anti-oxidant ebselen [110]. This suggests
that copper may contribute to ROS-dependent killing in mac-
rophages, in keeping with the well-known capacity of Cu(I) to
catalyse the generation of hydroxyl radical from H₂O₂. This study
also showed that both IFNγ and LPS regulated copper-trafficking
pathways in macrophages. Studies on the intramacrophage patho-
gen *S. Typhimurium* also provide functional evidence for a role
in macrophage antimicrobial pathways. Infection of mouse mac-
rophages with *S. Typhimurium*, as well as treatment with LPS,
promoted the accumulation of copper within intracellular ves-
icles. This response peaked at about 14 h post-stimulation. Fur-
thermore, a cell impermeable copper chelator (bathocuproinedi-
sulfonic acid) reduced vesicular copper accumulation in macro-
phages, and this impaired the ability of primary mouse macro-
phages to kill *S. Typhimurium* [111]. The use of a *Salmonella*
copper-responsive promoter reporter strain also provided evid-
ence that *S. Typhimurium* within macrophage phagosomes were
subjected to an increase in copper levels [112]. Collectively, these
studies suggest that copper may regulate both immediate and
delayed macrophage antimicrobial pathways. Notably, whereas
phagocytosis-induced ROS is an immediate response occurring
within the first 30 min of particle uptake, TLR signalling also pro-
motes a slower accumulation of ROS, which is derived from the
mitochondria [113]. Hence, it is conceivable that the delayed vesi-
cular accumulation of copper within macrophages in response to
LPS could also contribute to this pathway of oxidative stress. It
is also possible that inducible copper redistribution contributes
to macrophage-mediated host defence by promoting the export
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 3** Possible mechanisms by which copper may contribute to macrophage antimicrobial responses</figcaption></figure>

Copper is imported into the macrophage by CTR1 and/or CTR2 (most likely CTR1). It is then bound to chaperones (COX17, CCS and ATOX1) or metal-binding proteins such as MT. Copper is transported to the mitochondria for energy production, Cu, Zn-containing SOD1 for cytoprotection or to Atp7a for protein synthesis. IFNγ and/or LPS up-regulate the expression of several copper transport genes in mouse macrophages (e.g. *Ctr1*, *Ctr2*, *Atp7a*) and promote copper uptake into these cells. Possible mechanisms for antimicrobial effects of copper within macrophages include: (1) direct toxicity through Fenton chemistry within phagosomes; (2) vesicular accumulation which may contribute to oxidative stress over a slow time course (e.g. mitochondrial ROS production); and (3) indirect effect via the GPI-anchored form of the copper-containing ferroxidase CP which promotes FPN1-dependent iron export and thus starves intracellular bacteria of this essential element.

of iron (discussed below). The potential mechanisms by which copper could contribute to pathogen clearance by macrophages are outlined in Figure 3.

**What copper transport proteins regulate macrophage functions?**

Several of the above studies provide evidence for regulated copper transport in macrophages in response to infectious agents [50,110,111]. Due to the highly toxic nature of Cu(I), copper trafficking must be very tightly regulated. This is achieved through a series of high-affinity copper transport proteins and copper chaperones. Although we have detected moderate increases (<10 fold) in the mRNA expression of the copper chaperones, *Atox1*, *Ccs* and *Cox17* in primary mouse macrophages in response to LPS and *Salmonella* (S. L. Stafford, unpublished work), functional studies of these proteins in macrophage responses to pathogen challenge are lacking. However, emerging evidence implicates some copper transport proteins in macrophage-mediated host defence.

*CTR (copper transporter) copper importers*

The CTR proteins are a family of dedicated high-affinity transport proteins which were first identified in yeast [114], but are conserved through to humans. Mammals have two CTR proteins, CTR1/SLC31A1 and CTR2/SLC31A2 [114], which are thought to form trimeric oligomers in membranes [115]. In cell lines and mouse tissue, CTR1 is found at both the plasma membrane and intracellular vesicles. CTR1 has been shown to traffic from the plasma membrane to endosomal compartments when extracellular copper levels are increased [116,117], which may enable regulatory control of copper uptake. CTR2 shares sequence similarity with CTR1 [114] and is primarily expressed on intracellular vesicles, including late endosomes and lysosomes [118,119]. However, it has also been reported to partially localize to the plasma membrane and facilitate copper uptake [118]. The localization of endogenous CTR1 and CTR2 in unstimulated or activated macrophages has not yet been reported, nor have direct functional studies been performed. Nonetheless, both LPS and IFNγ promoted copper uptake into mouse macrophages [110], and
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 3** Possible mechanisms by which copper may contribute to macrophage antimicrobial responses</figcaption></figure>

Copper is imported into the macrophage by CTR1 and/or CTR2 (most likely CTR1). It is then bound to chaperones (COX17, CCS and ATOX1) or metal-binding proteins such as MT. Copper is transported to the mitochondria for energy production, Cu, Zn-containing SOD1 for cytoprotection or to Atp7a for protein synthesis. IFNγ and/or LPS up-regulate the expression of several copper transport genes in mouse macrophages (e.g. *Ctr1*, *Ctr2*, *Atp7a*) and promote copper uptake into these cells. Possible mechanisms for antimicrobial effects of copper within macrophages include: (1) direct toxicity through Fenton chemistry within phagosomes; (2) vesicular accumulation which may contribute to oxidative stress over a slow time course (e.g. mitochondrial ROS production); and (3) indirect effect via the GPI-anchored form of the copper-containing ferroxidase CP which promotes FPN1-dependent iron export and thus starves intracellular bacteria of this essential element.

of iron (discussed below). The potential mechanisms by which copper could contribute to pathogen clearance by macrophages are outlined in Figure 3.

**What copper transport proteins regulate macrophage functions?**

Several of the above studies provide evidence for regulated copper transport in macrophages in response to infectious agents [50,110,111]. Due to the highly toxic nature of Cu(I), copper trafficking must be very tightly regulated. This is achieved through a series of high-affinity copper transport proteins and copper chaperones. Although we have detected moderate increases (<10 fold) in the mRNA expression of the copper chaperones, *Atox1*, *Ccs* and *Cox17* in primary mouse macrophages in response to LPS and *Salmonella* (S. L. Stafford, unpublished work), functional studies of these proteins in macrophage responses to pathogen challenge are lacking. However, emerging evidence implicates some copper transport proteins in macrophage-mediated host defence.

*CTR (copper transporter) copper importers*

The CTR proteins are a family of dedicated high-affinity transport proteins which were first identified in yeast [114], but are conserved through to humans. Mammals have two CTR proteins, CTR1/SLC31A1 and CTR2/SLC31A2 [114], which are thought to form trimeric oligomers in membranes [115]. In cell lines and mouse tissue, CTR1 is found at both the plasma membrane and intracellular vesicles. CTR1 has been shown to traffic from the plasma membrane to endosomal compartments when extracellular copper levels are increased [116,117], which may enable regulatory control of copper uptake. CTR2 shares sequence similarity with CTR1 [114] and is primarily expressed on intracellular vesicles, including late endosomes and lysosomes [118,119]. However, it has also been reported to partially localize to the plasma membrane and facilitate copper uptake [118]. The localization of endogenous CTR1 and CTR2 in unstimulated or activated macrophages has not yet been reported, nor have direct functional studies been performed. Nonetheless, both LPS and IFNγ promoted copper uptake into mouse macrophages [110], and
similarly, a cell impermeable copper chelator inhibited the form-
ation of LPS- and *Salmonella*-inducible copper-containing ves-
icles in macrophages [111]. These stimuli also up-regulated
*Ctrl*1 and *Ctrl*2 expression in primary mouse macrophages and
RAW264.7 cells [110,111], implicating one or both of these
transporters in copper uptake by macrophages.

ATP7A and ATP7B

ATP7A is a 178 kDa, Cu(I)-transporting P<sub>1B</sub>-type ATPase [120-122], and defects in *ATP7A* are responsible for Menkes disease. ATP7A is expressed in the majority of tissues except for the liver, and has roles in biosynthesis and homoeostasis [123,124]. It is essential for dietary uptake of copper by releasing copper at the basolateral plasma membrane of small intestine cells, delivery of copper to the brain and recovery of copper from proximal tubules of the kidney [125]. Normally, ATP7A is located in the trans-Golgi network, which enables it to supply cuproenzymes [126,127]. When cells are exposed to high copper concentrations, it traffics to the plasma membrane to export the excess copper [128,129]. ATP7A can also deliver copper to intracellular exocytic vesicles and specialised cell compartments such as secretory granules or melanosomes [130]. ATP7B is closely related to ATP7A in both structure and function, and transports copper across membranes in an ATP-dependent fashion [131]. We could not detect *Atp7b* mRNA expression in primary mouse macrophages (S. L. Stafford, unpublished work), but various lines of evidence support a role for *Atp7a* in these cells. Atp7a mRNA and protein expression was robustly up-regulated by LPS and IFNγ in RAW264.7 mouse macrophages [110], and *Salmonella* infection also increased *Atp7a* mRNA levels in primary mouse macrophages [111]. Furthermore, in IFNγ-stimulated RAW264.7 cells, Atp7a partially co-localized to phagosomes during engulfment of latex beads, and knock-down of *Atp7a* impaired the ability of macrophages to kill *E. coli* [110]. Collectively, these data strongly support the idea that macrophages primed by activating agents such as IFNγ can utilize Atp7a for phagosomal delivery of copper to target pathogens for destruction.

CP (ceruloplasmin)

CP is a multicopper oxidase that is widely distributed in verte-
rates. It is mainly produced by the liver and is predominantly
found in the plasma [132]. It is a 132 kDa α-2 glycoprotein con-
taining six copper ions, and functions as a ferroxidase [133]. It has
roles in iron homoeostasis [134] and antioxidant defence [135],
as well as metabolism of copper [136], biogenic amines [137]
and NO (nitric oxide) [138]. CP is an acute phase protein [139],
and several pro-inflammatory stimuli including IFNγ [140], IL-
1, IL-6 [141], TNFα and LPS [142] induce CP synthesis. It is
also expressed as a GPI (glycosylphosphatidylinositol)-anchored
form [143], and both soluble and GPI-anchored Cp mRNA, are
robustly induced by LPS in mouse macrophages [111]. More
than 95% of serum copper is bound by CP, with the remain-
ing copper being bound primarily with albumin and transcuprein
[144]. Surprisingly, aceruloplasminemic patients suffer no cop-
per imbalance; rather, they have impaired iron efflux from cells

resulting in complications with iron homoeostasis, which leads
to neurodegeneration [145]. CP is thus a key protein that provides
a functional link between copper and iron metabolism [146]. It
primarily does so through the FPN1 (ferroportin 1) iron exporter;
CP ferroxidase activity is required for cell surface FPN1 expres-
sion and iron export [147]. Consequently, inducible copper traf-
ficking in macrophages probably enables copper loading into CP,
which is required for iron export from macrophages. Iron export
from macrophages provides an additional defence mechanism by
restricting bacterial growth, as has been clearly demonstrated in
the case of *Salmonella* [148]. Interestingly, knock-down of Atp7a
in mouse macrophages did not affect CP expression, but reduced
CP enzymatic activity [110]. This is consistent with Atp7a con-
tributing to iron export from macrophages, in addition to direct
delivery of copper to the phagosome.

Interplay between zinc and copper in
macrophage-mediated host defence

As reviewed above, there are remarkable parallels between zinc
and copper with respect to macrophage antimicrobial pathways.
Both these metal ions promote macrophage antimicrobial re-
sponses, and their intracellular localizations are dynamically reg-
ulated in macrophages responding to pathogen challenge. Fur-
thermore, both zinc and copper can be delivered to the mac-
rophage phagosome, raising the question of whether there is
interplay between the two ions in macrophage-mediated host de-
fence. Both zinc and copper are soft metal ions that can inactivate
the exposed Fe-S cluster of key bacterial dehydratase enzymes,
including 6-phosphogluconate dehydratase (Entner-Douderoff
Pathway), fumarase A [TCA (tricarboxylic acid) cycle] and iso-
propylmalate isomerase (leucine biosynthesis) [149]. This prop-
erty may be harnessed within the intramacrophage environment,
perhaps in a synergistic fashion. The pro-oxidant properties of
copper ions have already been noted, while zinc, although not
a redox active metal ion, has the ability to bind to protein thiol
groups in the cell. It is established that zinc inhibits thioredoxin
reductase, for example [150], and this could perturb antioxid-
ant defences linked to thioredoxins. Thus, copper and zinc could
independently enhance oxidative stress during macrophage an-
timicrobial responses, even though zinc is more generally asso-
ciated with antioxidant effects. The importance of intracellular
copper and zinc buffering by low molecular mass thiols in the
protection against metal-ion toxicity was recently demonstrated
for pneumococcus, where it was shown that mutants lacking the
ability to import glutathione were hypersensitive to copper and
zinc [151].

CONCLUSIONS AND FUTURE
DIRECTIONS

The interplay between zinc and macrophages is complex. Zinc
appears to enhance the microbicidal activity of macrophages,
while at the same time limiting excessive inflammatory responses
that may be deleterious to the host. The redistribution of the
similarly, a cell impermeable copper chelator inhibited the form-
ation of LPS- and *Salmonella*-inducible copper-containing ves-
icles in macrophages [111]. These stimuli also up-regulated
*Ctrl*1 and *Ctrl*2 expression in primary mouse macrophages and
RAW264.7 cells [110,111], implicating one or both of these
transporters in copper uptake by macrophages.

ATP7A and ATP7B

ATP7A is a 178 kDa, Cu(I)-transporting P<sub>1B</sub>-type ATPase [120-122], and defects in *ATP7A* are responsible for Menkes disease. ATP7A is expressed in the majority of tissues except for the liver, and has roles in biosynthesis and homoeostasis [123,124]. It is essential for dietary uptake of copper by releasing copper at the basolateral plasma membrane of small intestine cells, delivery of copper to the brain and recovery of copper from proximal tubules of the kidney [125]. Normally, ATP7A is located in the trans-Golgi network, which enables it to supply cuproenzymes [126,127]. When cells are exposed to high copper concentrations, it traffics to the plasma membrane to export the excess copper [128,129]. ATP7A can also deliver copper to intracellular exocytic vesicles and specialised cell compartments such as secretory granules or melanosomes [130]. ATP7B is closely related to ATP7A in both structure and function, and transports copper across membranes in an ATP-dependent fashion [131]. We could not detect *Atp7b* mRNA expression in primary mouse macrophages (S. L. Stafford, unpublished work), but various lines of evidence support a role for *Atp7a* in these cells. Atp7a mRNA and protein expression was robustly up-regulated by LPS and IFNγ in RAW264.7 mouse macrophages [110], and *Salmonella* infection also increased *Atp7a* mRNA levels in primary mouse macrophages [111]. Furthermore, in IFNγ-stimulated RAW264.7 cells, Atp7a partially co-localized to phagosomes during engulfment of latex beads, and knock-down of *Atp7a* impaired the ability of macrophages to kill *E. coli* [110]. Collectively, these data strongly support the idea that macrophages primed by activating agents such as IFNγ can utilize Atp7a for phagosomal delivery of copper to target pathogens for destruction.

CP (ceruloplasmin)

CP is a multicopper oxidase that is widely distributed in verte-
rates. It is mainly produced by the liver and is predominantly
found in the plasma [132]. It is a 132 kDa α-2 glycoprotein con-
taining six copper ions, and functions as a ferroxidase [133]. It has
roles in iron homoeostasis [134] and antioxidant defence [135],
as well as metabolism of copper [136], biogenic amines [137]
and NO (nitric oxide) [138]. CP is an acute phase protein [139],
and several pro-inflammatory stimuli including IFNγ [140], IL-
1, IL-6 [141], TNFα and LPS [142] induce CP synthesis. It is
also expressed as a GPI (glycosylphosphatidylinositol)-anchored
form [143], and both soluble and GPI-anchored Cp mRNA, are
robustly induced by LPS in mouse macrophages [111]. More
than 95% of serum copper is bound by CP, with the remain-
ing copper being bound primarily with albumin and transcuprein
[144]. Surprisingly, aceruloplasminemic patients suffer no cop-
per imbalance; rather, they have impaired iron efflux from cells

resulting in complications with iron homoeostasis, which leads
to neurodegeneration [145]. CP is thus a key protein that provides
a functional link between copper and iron metabolism [146]. It
primarily does so through the FPN1 (ferroportin 1) iron exporter;
CP ferroxidase activity is required for cell surface FPN1 expres-
sion and iron export [147]. Consequently, inducible copper traf-
ficking in macrophages probably enables copper loading into CP,
which is required for iron export from macrophages. Iron export
from macrophages provides an additional defence mechanism by
restricting bacterial growth, as has been clearly demonstrated in
the case of *Salmonella* [148]. Interestingly, knock-down of Atp7a
in mouse macrophages did not affect CP expression, but reduced
CP enzymatic activity [110]. This is consistent with Atp7a con-
tributing to iron export from macrophages, in addition to direct
delivery of copper to the phagosome.

Interplay between zinc and copper in
macrophage-mediated host defence

As reviewed above, there are remarkable parallels between zinc
and copper with respect to macrophage antimicrobial pathways.
Both these metal ions promote macrophage antimicrobial re-
sponses, and their intracellular localizations are dynamically reg-
ulated in macrophages responding to pathogen challenge. Fur-
thermore, both zinc and copper can be delivered to the mac-
rophage phagosome, raising the question of whether there is
interplay between the two ions in macrophage-mediated host de-
fence. Both zinc and copper are soft metal ions that can inactivate
the exposed Fe-S cluster of key bacterial dehydratase enzymes,
including 6-phosphogluconate dehydratase (Entner-Douderoff
Pathway), fumarase A [TCA (tricarboxylic acid) cycle] and iso-
propylmalate isomerase (leucine biosynthesis) [149]. This prop-
erty may be harnessed within the intramacrophage environment,
perhaps in a synergistic fashion. The pro-oxidant properties of
copper ions have already been noted, while zinc, although not
a redox active metal ion, has the ability to bind to protein thiol
groups in the cell. It is established that zinc inhibits thioredoxin
reductase, for example [150], and this could perturb antioxid-
ant defences linked to thioredoxins. Thus, copper and zinc could
independently enhance oxidative stress during macrophage an-
timicrobial responses, even though zinc is more generally asso-
ciated with antioxidant effects. The importance of intracellular
copper and zinc buffering by low molecular mass thiols in the
protection against metal-ion toxicity was recently demonstrated
for pneumococcus, where it was shown that mutants lacking the
ability to import glutathione were hypersensitive to copper and
zinc [151].

CONCLUSIONS AND FUTURE
DIRECTIONS

The interplay between zinc and macrophages is complex. Zinc
appears to enhance the microbicidal activity of macrophages,
while at the same time limiting excessive inflammatory responses
that may be deleterious to the host. The redistribution of the
<figure><img src="image_11.png" /></figure>

intracellular zinc pool to phagosomes and other vesicular com-
partments may enable macrophages to harness the activity of this
metal ion for microbial destruction. Conversely, this sequestration
away from the cytoplasm, as well as inducible export of cytoplasm-
mic zinc from macrophages, may also enable these cells to starve
certain pathogens of zinc to limit growth. The specific transport-
ers involved in trafficking zinc within and out of macrophages
are not well understood, but there are several obvious candid-
ates within the SLC30A (e.g. SLC30A1, SLC30A4, SLC30A6)
and SLC39A (e.g. SLC39A6, SLC39A8, SLC39A14) families.
Functional analysis of these genes should reveal new insights into
macrophage responses to pathogen challenge. Macrophages may
utilize copper in host defence strategies through several mech-
anisms including acute and delayed generation of ROS, as well
as iron export as a means of limiting bacterial growth. Further
experimental evidence is still required to support each of these
potential mechanisms. Many of the copper transport genes have
also been implicated in macrophage-mediated host defence (e.g.
CTR1, CTR2, ATP7A, CP). Analysis of macrophage-specific
knock outs for the above zinc and copper transport genes is now
required to determine their *in vivo* roles in macrophage antimi-
crobial pathways. An understanding of zinc and copper traffick-
ing in macrophages in response to distinct classes of pathogens
(e.g. Gram-positive against Gram-negative bacteria, cytoplasmic
against vesicular intramacrophage pathogens) may also yield in-
sights into the requirements for these pathways in different in-
fectious disease settings.

AUTHOR CONTRIBUTION

All authors contributed to the development of the scope of the
review and to critical analysis of relevant literature. Sian Stafford,
Nilesh Bokil, Ronan Kapetanovic and Matthew Sweet wrote the ma-
nuscript, and Maud Achard, Mark Schembri and Alastair McEwan
critiqued and edited the manuscript.

FUNDING

This work was supported by project grants from the National
Health and Medical Research Council of Australia [grant numbers
ID631531 (to M.J.S.), ID519722 (to A.G.M. and M.A.S.)]. M.A.S.
and M.J.S. are the recipients of Australian Research Council Future
Fellowships [grant numbers FT100100662 and FT100100657]
and M.J.S. holds an honorary NHMRC Senior Research Fellowship
[grant number APP1003470]. R.K. is supported by an ARC DECRA
Fellowship [grant number DE130100470]. S.L.S. is the recipient of
a University of Queensland Postgraduate Scholarship, and N.J.B. is
the recipient of an Endeavour international postgraduate research
scholarship and a University of Queensland research scholarship.

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6 Haase, H. and Rink, L. (2009) Functional significance of zinc-related signaling pathways in immune cells. *Annu. Rev. Nutr.* **29**, 133–152

7 Rink, L. and Haase, H. (2007) Zinc homeostasis and immunity. *Trends Immunol.* **28**, 1–4

8 Prasad, A. S. (2008) Zinc in human health: effect of zinc on immune cells. *Mol. Med.* **14**, 353–357

9 Andreini, C., Banci, L., Bertini, I. and Rosato, A. (2006) Zinc through the three domains of life. *J. Proteome Res.* **5**, 3173–3178

10 Prasad, A. S. (2012) Discovery of human zinc deficiency: 50 years later. *J. Trace Elem. Med. Biol.* **26**, 66–69

11 Prasad, A. S., Halsted, J. A. and Nadimi, M. (1961) Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia. *Am. J. Med.* **31**, 532–546

12 Prasad, A. S., Miale, Jr. A., Farid, Z., Sandstead, H. H. and Schulert, A. R. (1963) Zinc metabolism in patients with the syndrome of iron deficiency anemia, hepatosplenomegaly, dwarfism, and hypogonadism. *J. Lab. Clin. Med.* **61**, 537–549

13 Sandstead, H. H., Prasad, A. S., Schulert, A. R., Farid, Z., Miale, Jr, A., Bassilly, S. and Darby, W. J. (1967) Human zinc deficiency, endocrine manifestations and response to treatment. *Am. J. Clin. Nutr.* **20**, 422–442

14 Prasad, A. S., Rabbani, P, Abbasii, A., Bowersox, E. and Fox, M. R. (1978) Experimental zinc deficiency in humans. *Ann. Intern. Med.* **89**, 483–490

15 Beck, F. W., Prasad, A. S., Kaplan, J., Fitzgerald, J. T. and Brewer, G. J. (1997) Changes in cytokine production and T cell subpopulations in experimentally induced zinc-deficient humans. *Am. J. Physiol.* **272**, E1002–1007

16 Guilbert, J. J. (2003) The world health report 2002–reducing risks, promoting healthy life. *Educ. Health (Abingdon)* **16**, 230

17 Fraker, P J., DePasquale-Jardieu, P., Zwickl, C. M. and Luecke, R. W. (1978) Regeneration of T-cell helper function in zinc-deficient adult mice. *Proc. Natl. Acad. Sci. U.S.A.* **75**, 5660–5664

18 Fraker, P J., Haas, S. M. and Luecke, R. W. (1977) Effect of zinc deficiency on the immune response of the young adult A/J mouse. *J. Nutr.* **107**, 1889–1895

19 Bao, S., Liu, M.-J., Lee, B., Besecker, B., Lai, J.-P., Guttridge, D. C. and Knoell, D. L. (2010) Zinc modulates the innate immune response *in vivo* to polymicrobial sepsis through regulation of NF-κB. *Am. J. Physiol. Lung Cell Mol. Physiol.* **298**, L744–L754

20 Knoell, D. L., Julian, M. W., Bao, S., Besecker, B., Macre, J. E., Leikauf, G. D., DiSilvestro, R. A. and Crouser, E. D. (2009) Zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis. *Crit. Care Med.* **37**, 1380–1388

21 Sobocinski, P. Z., Canterbury, Jr, W. J., Mapes, C. A. and Dinterman, R. E. (1978) Involvement of hepatic metallothioneins in hypozincemia associated with bacterial infection. *Am. J. Physiol.* **234**, E399–E406

22 Kushner, I. (1982) The phenomenon of the acute phase response. *Ann. NY Acad. Sci.* **389**, 39–48

23 Gaetke, L. M., McClain, C. J., Talwalkar, R. T. and Shedlofsky, S. I. (1997) Effects of endotoxin on zinc metabolism in human volunteers. *Am. J. Physiol.* **272**, E952–956
<figure><img src="image_11.png" /></figure>

intracellular zinc pool to phagosomes and other vesicular compartments may enable macrophages to harness the activity of this metal ion for microbial destruction. Conversely, this sequestration away from the cytoplasm, as well as inducible export of cytoplasmic zinc from macrophages, may also enable these cells to starve certain pathogens of zinc to limit growth. The specific transporters involved in trafficking zinc within and out of macrophages are not well understood, but there are several obvious candidates within the SLC30A (e.g. SLC30A1, SLC30A4, SLC30A6) and SLC39A (e.g. SLC39A6, SLC39A8, SLC39A14) families. Functional analysis of these genes should reveal new insights into macrophage responses to pathogen challenge. Macrophages may utilize copper in host defence strategies through several mechanisms including acute and delayed generation of ROS, as well as iron export as a means of limiting bacterial growth. Further experimental evidence is still required to support each of these potential mechanisms. Many of the copper transport genes have also been implicated in macrophage-mediated host defence (e.g. CTR1, CTR2, ATP7A, CP). Analysis of macrophage-specific knock outs for the above zinc and copper transport genes is now required to determine their *in vivo* roles in macrophage antimicrobial pathways. An understanding of zinc and copper trafficking in macrophages in response to distinct classes of pathogens (e.g. Gram-positive against Gram-negative bacteria, cytoplasmic against vesicular intramacrophage pathogens) may also yield insights into the requirements for these pathways in different infectious disease settings.

## AUTHOR CONTRIBUTION

All authors contributed to the development of the scope of the review and to critical analysis of relevant literature. Sian Stafford, Nilesh Bokil, Ronan Kapetanovic and Matthew Sweet wrote the manuscript, and Maud Achard, Mark Schembri and Alastair McEwan critiqued and edited the manuscript.

## FUNDING

This work was supported by project grants from the National Health and Medical Research Council of Australia [grant numbers ID631531 (to M.J.S.), ID519722 (to A.G.M. and M.A.S.)]. M.A.S. and M.J.S. are the recipients of Australian Research Council Future Fellowships [grant numbers FT100100662 and FT100100657] and M.J.S. holds an honorary NHMRC Senior Research Fellowship [grant number APP1003470]. R.K. is supported by an ARC DECRA Fellowship [grant number DE130100470]. S.L.S. is the recipient of a University of Queensland Postgraduate Scholarship, and N.J.B. is the recipient of an Endeavour international postgraduate research scholarship and a University of Queensland research scholarship.

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6. Haase, H. and Rink, L. (2009) Functional significance of zinc-related signaling pathways in immune cells. Annu. Rev. Nutr. **29**, 133-152

7. Rink, L. and Haase, H. (2007) Zinc homeostasis and immunity. Trends Immunol. **28**, 1-4

8. Prasad, A. S. (2008) Zinc in human health: effect of zinc on immune cells. Mol. Med. **14**, 353-357

9. Andreini, C., Banci, L., Bertini, I. and Rosato, A. (2006) Zinc through the three domains of life. J. Proteome Res. **5**, 3173-3178

10. Prasad, A. S. (2012) Discovery of human zinc deficiency: 50 years later. J. Trace Elem. Med. Biol. **26**, 66-69

11. Prasad, A. S., Halsted, J. A. and Nadimi, M. (1961) Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia. Am. J. Med. **31**, 532-546

12. Prasad, A. S., Miale, Jr. A., Farid, Z., Sandstead, H. H. and Schulert, A. R. (1963) Zinc metabolism in patients with the syndrome of iron deficiency anemia, hepatosplenomegaly, dwarfism, and hypogonadism. J. Lab. Clin. Med. **61**, 537-549

13. Sandstead, H. H., Prasad, A. S., Schulert, A. R., Farid, Z., Miale, Jr., A., Bassilly, S. and Darby, W. J. (1967) Human zinc deficiency, endocrine manifestations and response to treatment. Am. J. Clin. Nutr. **20**, 422-442

14. Prasad, A. S., Rabbani, P, Abbasii, A., Bowersox, E. and Fox, M. R. (1978) Experimental zinc deficiency in humans. Ann. Intern. Med. **89**, 483-490

15. Beck, F. W., Prasad, A. S., Kaplan, J., Fitzgerald, J. T. and Brewer, G. J. (1997) Changes in cytokine production and T cell subpopulations in experimentally induced zinc-deficient humans. Am. J. Physiol. **272**, E1002-1007

16. Guilbert, J. J. (2003) The world health report 2002-reducing risks, promoting healthy life. Educ. Health (Abingdon) **16**, 230

17. Fraker, P J., DePasquale-Jardieu, P., Zwickl, C. M. and Luecke, R. W. (1978) Regeneration of T-cell helper function in zinc-deficient adult mice. Proc. Natl. Acad. Sci. U.S.A. **75**, 5660-5664

18. Fraker, P J., Haas, S. M. and Luecke, R. W. (1977) Effect of zinc deficiency on the immune response of the young adult A/J mouse. J. Nutr. **107**, 1889-1895

19. Bao, S., Liu, M.-J., Lee, B., Besecker, B., Lai, J.-P., Guttridge, D. C. and Knoell, D. L. (2010) Zinc modulates the innate immune response *in vivo* to polymicrobial sepsis through regulation of NF-κB. Am. J. Physiol. Lung Cell Mol. Physiol. **298**, L744-L754

20. Knoell, D. L., Julian, M. W., Bao, S., Besecker, B., Macre, J. E., Leikauf, G. D., DiSilvestro, R. A. and Crouser, E. D. (2009) Zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis. Crit. Care Med. **37**, 1380-1388

21. Sobocinski, P. Z., Canterbury, Jr, W. J., Mapes, C. A. and Dinterman, R. E. (1978) Involvement of hepatic metallothioneins in hypozincemia associated with bacterial infection. Am. J. Physiol. **234**, E399-E406

22. Kushner, I. (1982) The phenomenon of the acute phase response. Ann. NY Acad. Sci. **389**, 39-48

23. Gaetke, L. M., McClain, C. J., Talwalkar, R. T. and Shedlofsky, S. I. (1997) Effects of endotoxin on zinc metabolism in human volunteers. Am. J. Physiol. **272**, E952-E956
24 Yasuno, T., Okamoto, H., Nagai, M., Kimura, S., Yamamoto, T., Nagano, K., Furubayashi, T., Yoshikawa, Y., Yasui, H., Katsumi, H. et al. (2011) The disposition and intestinal absorption of zinc in rats. Eur. J. Pharm. Sci. **44**, 410–415

25 Beker Aydemir, T., Chang, S. M., Guthrie, G. J., Maki, A. B., Ryu, M. S., Karabiyik, A. and Cousins, R. J. (2012) Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia). PLoS ONE **7**, 24

26 Liuzzi, J. P., Lichten, L. A., Rivera, S., Blanchard, R. K., Aydemir, T. B., Knutson, M. D., Ganz, T. and Cousins, R. J. (2005) Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response. Proc. Natl. Acad. Sci. U.S.A. **102**, 6843–6848

27 Goode, H. F., Penn, N. D., Kelleher, J. and Walker, B. E. (1991) Evidence of cellular zinc depletion in hospitalized but not in healthy elderly subjects. Age Ageing **20**, 345–348

28 Besecker, B. Y., Exline, M. C., Hollyfield, J., Phillips, G., DiSilvestro, R. A., Wewers, M. D. and Knoell, D. L. (2011) A comparison of zinc metabolism, inflammation, and disease severity in critically ill infected and noninfected adults early after intensive care unit admission. Am. J. Clin. Nutr. **93**, 1356–1364

29 Snyder, S. L. and Walker, R. I. (1976) Inhibition of lethality in endotoxin-challenged mice treated with zinc chloride. Infect. Immun. **13**, 998–1000

30 Nowak, J. E., Harmon, K., Caldwell, C. C. and Wong, H. R. (2012) Prophylactic zinc supplementation reduces bacterial load and improves survival in a murine model of sepsis. Pediatr. Crit. Care Med. **13**, e323–329

31 Brazao, V., Caetano, L. C., Del Vecchio Filipin, M., Paula Alonso Toldo, M., Caetano, L. N. and do Prado, J. C., Jr. (2008) Zinc supplementation increases resistance to experimental infection by *Trypanosoma cruzi*. Vet. Parasitol. **154**, 32–37

32 Mehta, A. J., Joshi, P. C., Fan, X., Brown, L. A., Ritzenthaler, J. D., Roman, J. and Guidot, D. M. (2011) Zinc supplementation restores PU.1 and Nrf2 nuclear binding in alveolar macrophages and improves redox balance and bacterial clearance in the lungs of alcohol-fed rats. Alcohol. Clin. Exp. Res. **35**, 1519–1528

33 Singh, K. P., Zaidi, S. I., Raisuddin, S., Saxena, A. K., Murthy, R. C. and Ray, P. K. (1992) Effect of zinc on immune functions and host resistance against infection and tumor challenge. Immunopharmacol. Immunotoxicol. **14**, 813–840

34 Cho, Y. H., Lee, S. J., Lee, J. Y., Kim, S. W., Lee, C. B., Lee, W. Y. and Yoon, M. S. (2002) Antibacterial effect of intraprostatic zinc injection in a rat model of chronic bacterial prostatitis. Int. J. Antimicrob. Agents **19**, 576–582

35 Kelleher, S. L., Casas, I., Carbajal, N. and Lonnerdal, B. (2002) Supplementation of infant formula with the probiotic *Lactobacillus reuteri* and zinc: impact on enteric infection and nutrition in infant rhesus monkeys. J. Pediatr. Gastroenterol. Nutr. **35**, 162–168

36 Bhutta, Z. A., Black, R. E., Brown, K. H., Gardner, J. M., Gore, S., Hidayat, A., Khatun, F., Martorell, R., Ninh, N. X., Penny, M. E. et al. (1999) Prevention of diarrhea and pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized controlled trials. Zinc Investigators’ Collaborative Group. J. Pediatr. **135**, 689–697

37 Lukacik, M., Thomas, R. L. and Aranda, J. V. (2008) A meta-analysis of the effects of oral zinc in the treatment of acute and persistent diarrhea. Pediatrics **121**, 326–336

38 Wardlaw, T., Salama, P., Brocklehurst, C., Chopra, M. and Mason, E. (2010) Diarrhoea: why children are still dying and what can be done. Lancet **375**, 870–872

39 Valavi, E., Hakimzadeh, M., Shamsizadeh, A., Aminzadeh, M. and Alghasi, A. (2011) The efficacy of zinc supplementation on outcome of children with severe pneumonia. A randomized double-blind placebo-controlled clinical trial. Indian J. Pediatr. **78**, 1079–1084

40 Prasad, A. S. (2009) Zinc: role in immunity, oxidative stress and chronic inflammation. Curr. Opin. Clin. Nutr. Metab. Care **12**, 646–652

41 Mocchegiani, E., Vecchia, S., Ancarani, F., Scalise, G. and Fabris, N. (1995) Benefit of oral zinc supplementation as an adjunct to zidovudine (AZT) therapy against opportunistic infections in AIDS. Int. J. Immunopharmacol. **17**, 719–727

42 Patro, B., Szymanski, H. and Szajewska, H. (2010) Oral zinc for the treatment of acute gastroenteritis in Polish children: a randomized, double-blind, placebo-controlled trial. J. Pediatr. **157**, 984–988.e1

43 Lawson, L., Thacher, T. D., Yassin, M. A., Onuoha, N. A., Usman, A., Emenyonu, N. E., Shenkin, A., Davies, P. D. and Cuevas, L. E. (2010) Randomized controlled trial of zinc and vitamin A as co-adjuants for the treatment of pulmonary tuberculosis. Trop. Med. Int. Health **15**, 1481–1490

44 Long, K. Z., Rosado, J. L., Montoya, Y., de Lourdes Solano, M., Hertzmark, E., DuPont, H. L. and Santos, J. I. (2007) Effect of vitamin A and zinc supplementation on gastrointestinal parasitic infections among Mexican children. Pediatrics **120**, e846–e855

45 Yang, Y., Jing, X. P., Zhang, S. P., Gu, R. X., Tang, F. X., Wang, X. L., Xiong, Y., Qiu, M., Sun, X. Y., Ke, D. et al. (2013) High dose zinc supplementation induces hippocampal zinc deficiency and memory impairment with inhibition of BDNF signaling. PLoS ONE **8**, e55384

46 Wirth, J. J., Fraker, P. J. and Kierszenbaum, F. (1989) Zinc requirement for macrophage function: effect of zinc deficiency on uptake and killing of a protozoan parasite. Immunology **68**, 114–119

47 Humphrey, P. A., Ashraf, M. and Lee, C. M. (1997) Hepatic cells’ mitotic and peritoneal macrophage phagocytic activities during *Trypanosoma musculi* infection in zinc-deficient mice. J. Natl. Med. Assoc. **89**, 259–267

48 Lastra, M. D., Pastelin, R., Camacho, A., Monroy, B. and Aguilar, A. E. (2001) Zinc intervention on macrophages and lymphocytes response. J. Trace Elem. Med. Biol. **15**, 5–10

49 Salvin, S. B., Horecker, B. L., Pan, L. X. and Rabin, B. S. (1987) The effect of dietary zinc and prothymosin alpha on cellular immune responses of RF/J mice. Clin. Immunol. Immunopathol. **43**, 281–288

50 Wagner, D., Maser, J., Lai, B., Cai, Z. H., Barry, C. E., Bentrup, K. H. Z., Russell, D. G. and Bermudez, L. E. (2005) Elemental analysis of *Mycobacterium avium*, *Mycobacterium tuberculosis*-, and *Mycobacterium smegmatis*-containing phagosomes indicates pathogen-induced microenvironments within the host cell’s endosomal system. J. Immunol. **174**, 1491–1500

51 Botella, H., Peyron, P., Levillain, F., Poincloux, R., Poquet, Y., Brandli, I., Wang, C., Tailleux, L., Tilleul, S., Charriere, G. M. et al. (2011) *Mycobacterial p(1)-type ATPases* mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe **10**, 248–259

52 Lemire, J. A., Harrison, J. J. and Turner, R. J. (2013) Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat. Rev. Microbiol. **11**, 371–384

53 McDevitt, C. A., Ogunniyi, A. D., Valkov, E., Lawrence, M. C., Kobe, B., McEwan, A. G. and Paton, J. C. (2011) A molecular mechanism for bacterial susceptibility to zinc. PLoS Pathog. **7**, e1002357

54 Visse, R. and Nagase, H. (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ. Res. **92**, 827–839

55 Houghton, A. M., Hartzell, W. O., Robbins, C. S., Gomis-Ruth, F. X. and Shapiro, S. D. (2009) Macrophage elastase kills bacteria within murine macrophages. Nature **460**, 637–641

56 Wilson, C. L., Schmidt, A. P., Pirila, E., Valore, E. V., Ferri, N., Sorsa, T., Ganz, T. and Parks, W. C. (2009) Differential processing of {alpha}- and {beta}-defensin precursors by matrix metalloproteinase-7 (MMP-7). J. Biol. Chem. **284**, 8301–8311
24 Yasuno, T., Okamoto, H., Nagai, M., Kimura, S., Yamamoto, T., Nagano, K., Furubayashi, T., Yoshikawa, Y., Yasui, H., Katsumi, H. et al. (2011) The disposition and intestinal absorption of zinc in rats. Eur. J. Pharm. Sci. **44**, 410–415

25 Beker Aydemir, T., Chang, S. M., Guthrie, G. J., Maki, A. B., Ryu, M. S., Karabiyik, A. and Cousins, R. J. (2012) Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia). PLoS ONE **7**, 24

26 Liuzzi, J. P., Lichten, L. A., Rivera, S., Blanchard, R. K., Aydemir, T. B., Knutson, M. D., Ganz, T. and Cousins, R. J. (2005) Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response. Proc. Natl. Acad. Sci. U.S.A. **102**, 6843–6848

27 Goode, H. F., Penn, N. D., Kelleher, J. and Walker, B. E. (1991) Evidence of cellular zinc depletion in hospitalized but not in healthy elderly subjects. Age Ageing **20**, 345–348

28 Besecker, B. Y., Exline, M. C., Hollyfield, J., Phillips, G., DiSilvestro, R. A., Wewers, M. D. and Knoell, D. L. (2011) A comparison of zinc metabolism, inflammation, and disease severity in critically ill infected and noninfected adults early after intensive care unit admission. Am. J. Clin. Nutr. **93**, 1356–1364

29 Snyder, S. L. and Walker, R. I. (1976) Inhibition of lethality in endotoxin-challenged mice treated with zinc chloride. Infect. Immun. **13**, 998–1000

30 Nowak, J. E., Harmon, K., Caldwell, C. C. and Wong, H. R. (2012) Prophylactic zinc supplementation reduces bacterial load and improves survival in a murine model of sepsis. Pediatr. Crit. Care Med. **13**, e323–329

31 Brazao, V., Caetano, L. C., Del Vecchio Filipin, M., Paula Alonso Toldo, M., Caetano, L. N. and do Prado, J. C., Jr. (2008) Zinc supplementation increases resistance to experimental infection by *Trypanosoma cruzi*. Vet. Parasitol. **154**, 32–37

32 Mehta, A. J., Joshi, P. C., Fan, X., Brown, L. A., Ritzenthaler, J. D., Roman, J. and Guidot, D. M. (2011) Zinc supplementation restores PU.1 and Nrf2 nuclear binding in alveolar macrophages and improves redox balance and bacterial clearance in the lungs of alcohol-fed rats. Alcohol. Clin. Exp. Res. **35**, 1519–1528

33 Singh, K. P., Zaidi, S. I., Raisuddin, S., Saxena, A. K., Murthy, R. C. and Ray, P. K. (1992) Effect of zinc on immune functions and host resistance against infection and tumor challenge. Immunopharmacol. Immunotoxicol. **14**, 813–840

34 Cho, Y. H., Lee, S. J., Lee, J. Y., Kim, S. W., Lee, C. B., Lee, W. Y. and Yoon, M. S. (2002) Antibacterial effect of intraprostatic zinc injection in a rat model of chronic bacterial prostatitis. Int. J. Antimicrob. Agents **19**, 576–582

35 Kelleher, S. L., Casas, I., Carbajal, N. and Lonnerdal, B. (2002) Supplementation of infant formula with the probiotic *Lactobacillus reuteri* and zinc: impact on enteric infection and nutrition in infant rhesus monkeys. J. Pediatr. Gastroenterol. Nutr. **35**, 162–168

36 Bhutta, Z. A., Black, R. E., Brown, K. H., Gardner, J. M., Gore, S., Hidayat, A., Khatun, F., Martorell, R., Ninh, N. X., Penny, M. E. et al. (1999) Prevention of diarrhea and pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized controlled trials. Zinc Investigators’ Collaborative Group. J. Pediatr. **135**, 689–697

37 Lukacik, M., Thomas, R. L. and Aranda, J. V. (2008) A meta-analysis of the effects of oral zinc in the treatment of acute and persistent diarrhea. Pediatrics **121**, 326–336

38 Wardlaw, T., Salama, P., Brocklehurst, C., Chopra, M. and Mason, E. (2010) Diarrhoea: why children are still dying and what can be done. Lancet **375**, 870–872

39 Valavi, E., Hakimzadeh, M., Shamsizadeh, A., Aminzadeh, M. and Alghasi, A. (2011) The efficacy of zinc supplementation on outcome of children with severe pneumonia. A randomized double-blind placebo-controlled clinical trial. Indian J. Pediatr. **78**, 1079–1084

40 Prasad, A. S. (2009) Zinc: role in immunity, oxidative stress and chronic inflammation. Curr. Opin. Clin. Nutr. Metab. Care **12**, 646–652

41 Mocchegiani, E., Veccia, S., Ancarani, F., Scalise, G. and Fabris, N. (1995) Benefit of oral zinc supplementation as an adjunct to zidovudine (AZT) therapy against opportunistic infections in AIDS. Int. J. Immunopharmacol. **17**, 719–727

42 Patro, B., Szymanski, H. and Szajewska, H. (2010) Oral zinc for the treatment of acute gastroenteritis in Polish children: a randomized, double-blind, placebo-controlled trial. J. Pediatr. **157**, 984–988.e1

43 Lawson, L., Thacher, T. D., Yassin, M. A., Onuoha, N. A., Usman, A., Emenyonu, N. E., Shenkin, A., Davies, P. D. and Cuevas, L. E. (2010) Randomized controlled trial of zinc and vitamin A as co-adjuvants for the treatment of pulmonary tuberculosis. Trop. Med. Int. Health **15**, 1481–1490

44 Long, K. Z., Rosado, J. L., Montoya, Y., de Lourdes Solano, M., Hertzmark, E., DuPont, H. L. and Santos, J. I. (2007) Effect of vitamin A and zinc supplementation on gastrointestinal parasitic infections among Mexican children. Pediatrics **120**, e846–e855

45 Yang, Y., Jing, X. P., Zhang, S. P., Gu, R. X., Tang, F. X., Wang, X. L., Xiong, Y., Qiu, M., Sun, X. Y., Ke, D. et al. (2013) High dose zinc supplementation induces hippocampal zinc deficiency and memory impairment with inhibition of BDNF signaling. PLoS ONE **8**, e55384

46 Wirth, J. J., Fraker, P. J. and Kierszenbaum, F. (1989) Zinc requirement for macrophage function: effect of zinc deficiency on uptake and killing of a protozoan parasite. Immunology **68**, 114–119

47 Humphrey, P. A., Ashraf, M. and Lee, C. M. (1997) Hepatic cells’ mitotic and peritoneal macrophage phagocytic activities during *Trypanosoma musculi* infection in zinc-deficient mice. J. Natl. Med. Assoc. **89**, 259–267

48 Lastra, M. D., Pastelin, R., Camacho, A., Monroy, B. and Aguilar, A. E. (2001) Zinc intervention on macrophages and lymphocytes response. J. Trace Elem. Med. Biol. **15**, 5–10

49 Salvin, S. B., Horecker, B. L., Pan, L. X. and Rabin, B. S. (1987) The effect of dietary zinc and prothymosin alpha on cellular immune responses of RF/J mice. Clin. Immunol. Immunopathol. **43**, 281–288

50 Wagner, D., Maser, J., Lai, B., Cai, Z. H., Barry, C. E., Bentrup, K. H. Z., Russell, D. G. and Bermudez, L. E. (2005) Elemental analysis of *Mycobacterium avium*, *Mycobacterium tuberculosis*-, and *Mycobacterium smegmatis*-containing phagosomes indicates pathogen-induced microenvironments within the host cell’s endosomal system. J. Immunol. **174**, 1491–1500

51 Botella, H., Peyron, P., Levillain, F., Poincloux, R., Poquet, Y., Brandli, I., Wang, C., Tailleux, L., Tilleul, S., Charriere, G. M. et al. (2011) Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe **10**, 248–259

52 Lemire, J. A., Harrison, J. J. and Turner, R. J. (2013) Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat. Rev. Microbiol. **11**, 371–384

53 McDevitt, C. A., Ogunniyi, A. D., Valkov, E., Lawrence, M. C., Kobe, B., McEwan, A. G. and Paton, J. C. (2011) A molecular mechanism for bacterial susceptibility to zinc. PLoS Pathog. **7**, e1002357

54 Visse, R. and Nagase, H. (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ. Res. **92**, 827–839

55 Houghton, A. M., Hartzell, W. O., Robbins, C. S., Gomis-Ruth, F. X. and Shapiro, S. D. (2009) Macrophage elastase kills bacteria within murine macrophages. Nature **460**, 637–641

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<figure><img src="image_12.png" /></figure>

57 Winters, M. S., Chan, Q., Caruso, J. A. and Deepe, Jr, G. S. (2010) Metallomic analysis of macrophages infected with *Histoplasma capsulatum* reveals a fundamental role for zinc in host defenses. J. Infect. Dis. **202**, 1136–1145

58 Kitamura, H., Morikawa, H., Kamon, H., Iguchi, M., Hojyo, S., Fukada, T., Yamashita, S., Kaisho, T., Akira, S., Murakami, M. et al. (2006) Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function. Nat. Immunol. **7**, 971–977

59 Achouiti, A., Vogl, T., Urban, C. F., Rohm, M., Hommes, T. J., van Zoelen, M. A., Florquin, S., Roth, J., van 't Veer, C., de Vos, A. F. et al. (2012) Myeloid-related protein-14 contributes to protective immunity in Gram-negative pneumonia derived sepsis. PLoS Pathog. **8**, e1002987

60 Corbin, B. D., Seeley, E. H., Raab, A., Feldmann, J., Miller, M. R., Torres, V. J., Anderson, K. L., Dattilo, B. M., Dunman, P. M., Gerads, R. et al. (2008) Metal chelation and inhibition of bacterial growth in tissue abscesses. Science **319**, 962–965

61 Sohnle, P. G., Hunter, M. J., Hahn, B. and Chazin, W. J. (2000) Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14). J. Infect. Dis. **182**, 1272–1275

62 Seing, I., Stahl, C., Nordenfelt, L., Bulow, P and Ekberg, K. (2012) Policy and practice of work ability: a negotiation of responsibility in organizing return to work. J. Occup. Rehabil. **22**, 553–564

63 Cousins, R. J., Blanchard, R. K., Popp, M. P., Liu, L., Cao, J., Moore, J. B. and Green, C. L. (2003) A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells. Proc. Natl. Acad. Sci. U.S.A. **100**, 6952–6957

64 Schroder, K. and Tschopp, J. (2010) The inflammasomes. Cell **140**, 821–832

65 Brough, D., Pelegrin, P. and Rothwell, N. J. (2009) Pannexin-1-dependent caspase-1 activation and secretion of IL-1beta is regulated by zinc. Eur. J. Immunol. **39**, 352–358

66 Haase, H., Ober-Blobaum, J. L., Engelhardt, G., Hebel, S., Heit, A., Heine, H. and Rink, L. (2008) Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes. J. Immunol. **181**, 6491–6502

67 Aydemir, T. B., Blanchard, R. K. and Cousins, R. J. (2006) Zinc supplementation of young men alters metallothionein, zinc transporter, and cytokine gene expression in leukocyte populations. Proc. Natl. Acad. Sci. U.S.A. **103**, 1699–1704

68 Ryu, M. S., Langkamp-Henken, B., Chang, S. M., Shankar, M. N. and Cousins, R. J. (2011) Genomic analysis, cytokine expression, and microRNA profiling reveal biomarkers of human dietary zinc depletion and homeostasis. Proc. Natl. Acad. Sci. U.S.A. **108**, 20970–20975

69 Prasad, A. S., Bao, B., Beck, F. W. and Sarkar, F. H. (2002) Zinc enhances the expression of interleukin-2 and interleukin-2 receptors in HUT-78 cells by way of NF-kappaB activation. J. Lab. Clin. Med. **140**, 272–289

70 Uzzo, R. G., Crispen, P. L., Golovine, K., Makhov, P., Horwitz, E. M. and Kolenko, V. M. (2006) Diverse effects of zinc on NF-kappaB and AP-1 transcription factors: implications for prostate cancer progression. Carcinogenesis **27**, 1980–1990

71 Zhou, Z., Wang, L., Song, Z., Saari, J. T., McClain, C. J. and Kang, Y. J. (2004) Abrogation of nuclear factor-kappaB activation is involved in zinc inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production and liver injury. Am. J. Pathol. **164**, 1547–1556

72 Liu, M. J., Bao, S., Galvez-Peralta, M., Pyle, C. J., Rudawsky, A. C., Pavlovicz, R. E., Killilea, D. W., Li, C., Nebert, D. W., Wewers, M. D. et al. (2013) ZIP8 regulates host defense through zinc-mediated inhibition of NF-kappaB. Cell Rep. **3**, 386–400

73 von Bulow, V., Dubben, S., Engelhardt, G., Hebel, S., Plumakers, B., Heine, H., Rink, L. and Haase, H. (2007) Zinc-dependent suppression of TNF-alpha production is mediated by protein kinase A-induced inhibition of Raf-1, I kappa B kinase beta, and NF-kappa B. J. Immunol. **179**, 4180–4186

74 Prasad, A. S., Bao, B., Beck, F. W. and Sarkar, F. H. (2011) Zinc-suppressed inflammatory cytokines by induction of A20-mediated inhibition of nuclear factor-kappaB. Nutrition **27**, 816–823

75 Powell, S. R. (2000) The antioxidant properties of zinc. J. Nutr. **130**, 1447S–1454S

76 Klotz, L. O., Kroncke, K. D., Buchczyk, D. P. and Sies, H. (2003) Role of copper, zinc, selenium and tellurium in the cellular defense against oxidative and nitrosative stress. J. Nutr. **133**, 1448S–1451S

77 Kong, X., Thimmulappa, R., Kombairaju, P and Biswal, S. (2010) NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signalling and sepsis-induced mortality in Nrf2-deficient mice. J. Immunol. **185**, 569–577

78 Maret, W. and Vallee, B. L. (1998) Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc. Natl. Acad. Sci. U.S.A. **95**, 3478–3482

79 Leibbrandt, M. E. and Koropatnick, J. (1994) Activation of human monocytes with lipopolysaccharide induces metallothionein expression and is diminished by zinc. Toxicol. Appl. Pharmacol. **124**, 72–81

80 Inoue, K., Takano, H., Shimada, A. and Atho, M. (2009) Metallothionein as an anti-inflammatory mediator. *Mediators Inflamm.* **2009**

81 Inoue, K., Takano, H., Shimada, A., Wada, E., Yanagisawa, R., Sakurai, M., Satoh, M. and Yoshikawa, T. (2006) Role of metallothionein in coagulatory disturbance and systemic inflammation induced by lipopolysaccharide in mice. FASEB J. **20**, 533–535

82 Takano, H., Inoue, K., Yanagisawa, R., Sato, M., Shimada, A., Morita, T., Sawada, M., Nakamura, K., Sanbongi, C. and Yoshikawa, T. (2004) Protective role of metallothionein in acute lung injury induced by bacterial endotoxin. Thorax **59**, 1057–1062

83 Lichten, L. A. and Cousins, R. J. (2009) Mammalian zinc transporters: nutritional and physiologic regulation. Annu. Rev. Nutr. **29**, 153–176

84 Overbeck, S., Uciechowski, P., Ackland, M. L., Ford, D. and Rink, L. (2008) Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT-1 to ZnT-9. J. Leukoc. Biol. **83**, 368–380

85 Palmiter, R. D., Cole, T. B. and Findley, S. D. (1996) ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J. **15**, 1784–1791

86 Liuzzi, J. P., Bobo, J. A., Lichten, L. A., Samuelson, D. A. and Cousins, R. J. (2004) Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis. Proc. Natl. Acad. Sci. U.S.A. **101**, 14355–14360

87 Huang, L., Kirschke, C. P and Gitschier, J. (2002) Functional characterization of a novel mammalian zinc transporter, ZnT6. J. Biol. Chem. **277**, 26389–26395

88 Kirschke, C. P and Huang, L. (2003) ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J. Biol. Chem. **278**, 4096–4102

89 Yu, M., Lee, W. W., Tomar, D., Pryshchep, S., Czesnikiewicz-Guzik, M., Lamar, D. L., Li, G., Singh, K., Tian, L., Weyand, C. M. et al. (2011) Regulation of T cell receptor signaling by activation-induced zinc influx. J. Exp. Med. **208**, 775–785

90 Besecker, B., Bao, S., Bohacova, B., Papp, A., Sadee, W. and Knoell, D. L. (2008) The human zinc transporter SLC39A8 (Zip8) is critical in zinc-mediated cytoprotection in lung epithelia. Am. J. Physiol. Lung Cell. Mol. Physiol. **294**, 4
<figure><img src="image_12.png" /></figure>

57 Winters, M. S., Chan, Q., Caruso, J. A. and Deepe, Jr, G. S. (2010) Metallomic analysis of macrophages infected with *Histoplasma capsulatum* reveals a fundamental role for zinc in host defenses. J. Infect. Dis. **202**, 1136–1145

58 Kitamura, H., Morikawa, H., Kamon, H., Iguchi, M., Hojyo, S., Fukada, T., Yamashita, S., Kaisho, T., Akira, S., Murakami, M. et al. (2006) Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function. Nat. Immunol. **7**, 971–977

59 Achouiti, A., Vogl, T., Urban, C. F., Rohm, M., Hommes, T. J., van Zoelen, M. A., Florquin, S., Roth, J., van 't Veer, C., de Vos, A. F. et al. (2012) Myeloid-related protein-14 contributes to protective immunity in Gram-negative pneumonia derived sepsis. PLoS Pathog. **8**, e1002987

60 Corbin, B. D., Seeley, E. H., Raab, A., Feldmann, J., Miller, M. R., Torres, V. J., Anderson, K. L., Dattilo, B. M., Dunman, P. M., Gerads, R. et al. (2008) Metal chelation and inhibition of bacterial growth in tissue abscesses. Science **319**, 962–965

61 Sohnle, P. G., Hunter, M. J., Hahn, B. and Chazin, W. J. (2000) Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14). J. Infect. Dis. **182**, 1272–1275

62 Seing, I., Stahl, C., Nordenfelt, L., Bulow, P and Ekberg, K. (2012) Policy and practice of work ability: a negotiation of responsibility in organizing return to work. J. Occup. Rehabil. **22**, 553–564

63 Cousins, R. J., Blanchard, R. K., Popp, M. P., Liu, L., Cao, J., Moore, J. B. and Green, C. L. (2003) A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells. Proc. Natl. Acad. Sci. U.S.A. **100**, 6952–6957

64 Schroder, K. and Tschopp, J. (2010) The inflammasomes. Cell **140**, 821–832

65 Brough, D., Pelegrin, P. and Rothwell, N. J. (2009) Pannexin-1-dependent caspase-1 activation and secretion of IL-1beta is regulated by zinc. Eur. J. Immunol. **39**, 352–358

66 Haase, H., Ober-Blobaum, J. L., Engelhardt, G., Hebel, S., Heit, A., Heine, H. and Rink, L. (2008) Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes. J. Immunol. **181**, 6491–6502

67 Aydemir, T. B., Blanchard, R. K. and Cousins, R. J. (2006) Zinc supplementation of young men alters metallothionein, zinc transporter, and cytokine gene expression in leukocyte populations. Proc. Natl. Acad. Sci. U.S.A. **103**, 1699–1704

68 Ryu, M. S., Langkamp-Henken, B., Chang, S. M., Shankar, M. N. and Cousins, R. J. (2011) Genomic analysis, cytokine expression, and microRNA profiling reveal biomarkers of human dietary zinc depletion and homeostasis. Proc. Natl. Acad. Sci. U.S.A. **108**, 20970–20975

69 Prasad, A. S., Bao, B., Beck, F. W. and Sarkar, F. H. (2002) Zinc enhances the expression of interleukin-2 and interleukin-2 receptors in HUT-78 cells by way of NF-kappaB activation. J. Lab. Clin. Med. **140**, 272–289

70 Uzzo, R. G., Crispen, P. L., Golovine, K., Makhov, P., Horwitz, E. M. and Kolenko, V. M. (2006) Diverse effects of zinc on NF-kappaB and AP-1 transcription factors: implications for prostate cancer progression. Carcinogenesis **27**, 1980–1990

71 Zhou, Z., Wang, L., Song, Z., Saari, J. T., McClain, C. J. and Kang, Y. J. (2004) Abrogation of nuclear factor-kappaB activation is involved in zinc inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production and liver injury. Am. J. Pathol. **164**, 1547–1556

72 Liu, M. J., Bao, S., Galvez-Peralta, M., Pyle, C. J., Rudawsky, A. C., Pavlovicz, R. E., Killilea, D. W., Li, C., Nebert, D. W., Wewers, M. D. et al. (2013) ZIP8 regulates host defense through zinc-mediated inhibition of NF-kappaB. Cell Rep. **3**, 386–400

73 von Bulow, V., Dubben, S., Engelhardt, G., Hebel, S., Plumakers, B., Heine, H., Rink, L. and Haase, H. (2007) Zinc-dependent suppression of TNF-alpha production is mediated by protein kinase A-induced inhibition of Raf-1, I kappa B kinase beta, and NF-kappa B. J. Immunol. **179**, 4180–4186

74 Prasad, A. S., Bao, B., Beck, F. W. and Sarkar, F. H. (2011) Zinc-suppressed inflammatory cytokines by induction of A20-mediated inhibition of nuclear factor-kappaB. Nutrition **27**, 816–823

75 Powell, S. R. (2000) The antioxidant properties of zinc. J. Nutr. **130**, 1447S–1454S

76 Klotz, L. O., Kroncke, K. D., Buchczyk, D. P. and Sies, H. (2003) Role of copper, zinc, selenium and tellurium in the cellular defense against oxidative and nitrosative stress. J. Nutr. **133**, 1448S–1451S

77 Kong, X., Thimmulappa, R., Kombairaju, P and Biswal, S. (2010) NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signalling and sepsis-induced mortality in Nrf2-deficient mice. J. Immunol. **185**, 569–577

78 Maret, W. and Vallee, B. L. (1998) Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc. Natl. Acad. Sci. U.S.A. **95**, 3478–3482

79 Leibbrandt, M. E. and Koropatnick, J. (1994) Activation of human monocytes with lipopolysaccharide induces metallothionein expression and is diminished by zinc. Toxicol. Appl. Pharmacol. **124**, 72–81

80 Inoue, K., Takano, H., Shimada, A. and Atho, M. (2009) Metallothionein as an anti-inflammatory mediator. *Mediators Inflamm.* **2009**

81 Inoue, K., Takano, H., Shimada, A., Wada, E., Yanagisawa, R., Sakurai, M., Satoh, M. and Yoshikawa, T. (2006) Role of metallothionein in coagulatory disturbance and systemic inflammation induced by lipopolysaccharide in mice. FASEB J. **20**, 533–535

82 Takano, H., Inoue, K., Yanagisawa, R., Sato, M., Shimada, A., Morita, T., Sawada, M., Nakamura, K., Sanbongi, C. and Yoshikawa, T. (2004) Protective role of metallothionein in acute lung injury induced by bacterial endotoxin. Thorax **59**, 1057–1062

83 Lichten, L. A. and Cousins, R. J. (2009) Mammalian zinc transporters: nutritional and physiologic regulation. Annu. Rev. Nutr. **29**, 153–176

84 Overbeck, S., Uciechowski, P., Ackland, M. L., Ford, D. and Rink, L. (2008) Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT-1 to ZnT-9. J. Leukoc. Biol. **83**, 368–380

85 Palmiter, R. D., Cole, T. B. and Findley, S. D. (1996) ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J. **15**, 1784–1791

86 Liuzzi, J. P., Bobo, J. A., Lichten, L. A., Samuelson, D. A. and Cousins, R. J. (2004) Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis. Proc. Natl. Acad. Sci. U.S.A. **101**, 14355–14360

87 Huang, L., Kirschke, C. P and Gitschier, J. (2002) Functional characterization of a novel mammalian zinc transporter, ZnT6. J. Biol. Chem. **277**, 26389–26395

88 Kirschke, C. P and Huang, L. (2003) ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J. Biol. Chem. **278**, 4096–4102

89 Yu, M., Lee, W. W., Tomar, D., Pryshchep, S., Czesnikiewicz-Guzik, M., Lamar, D. L., Li, G., Singh, K., Tian, L., Weyand, C. M. et al. (2011) Regulation of T cell receptor signaling by activation-induced zinc influx. J. Exp. Med. **208**, 775–785

90 Besecker, B., Bao, S., Bohacova, B., Papp, A., Sadee, W. and Knoell, D. L. (2008) The human zinc transporter SLC39A8 (Zip8) is critical in zinc-mediated cytoprotection in lung epithelia. Am. J. Physiol. Lung Cell. Mol. Physiol. **294**, 4
91 Aydemir, T. B., Liuzzi, J. P., McClellan, S. and Cousins, R. J. (2009) Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-gamma expression in activated human T cells. J. Leukoc. Biol. **86**, 337–348

92 Idaghdour, Y., Quinlan, J., Goulet, J. P., Berghout, J., Gbeha, E., Bruat, V., de Malliard, T., Grenier, J. C., Gomez, S., Gros, P et al. (2012) Evidence for additive and interaction effects of host genotype and infection in malaria. Proc. Natl. Acad. Sci. U.S.A. **109**, 16786–16793

93 Sayadi, A., Nguyen, A. T., Bard, F. A. and Bard-Chapeau, E. A. (2013) Zip14 expression induced by lipopolysaccharides in macrophages attenuates inflammatory response. Inflamm. Res. **62**, 133–143

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92 Idaghdour, Y., Quinlan, J., Goulet, J. P., Berghout, J., Gbeha, E., Bruat, V., de Malliard, T., Grenier, J. C., Gomez, S., Gros, P et al. (2012) Evidence for additive and interaction effects of host genotype and infection in malaria. Proc. Natl. Acad. Sci. U.S.A. **109**, 16786–16793

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94 Veldhuis, N. A., Valova, V. A., Gaeth, A. P., Palstra, N., Hannan, K. M., Michell, B. J., Kelly, L. E., Jennings, I., Kemp, B. E., Pearson, R. B. et al. (2009) Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase. Int. J. Biochem. Cell Biol. **41**, 2403–2412

95 Steiger, D., Fetchko, M., Vardanyan, A., Atanesyan, L., Steiner, K., Turski, M. L., Thiele, D. J., Georgiev, O. and Schaffner, W. (2010) The *Drosophila* copper transporter Ctr1C functions in male fertility. J. Biol. Chem. **285**, 17089–17097

96 Maggini, S., Wintergerst, E. S., Beveridge, S. and Hornig, D. H. (2007) Selected vitamins and trace elements support immune function by strengthening epithelial barriers and cellular and humoral immune responses. Br. J. Nutr. **98** (Suppl 1), S29–S35

97 Halfdanarson, T. R., Kumar, N., Li, C.-Y., Phylisky, R. L. and Hogan, W. J. (2008) Hematological manifestations of copper deficiency: a retrospective review. Eur. J. Haematol. **80**, 523–531

98 Prohaska, J. R. and Lukasewycz, O. A. (1981) Copper deficiency suppresses the immune response of mice. Science **213**, 559–561

99 Kreuder, J., Otten, A., Fuder, H., Tumer, Z., Tonnesen, T., Horn, N. and Dralle, D. (1993) Clinical and biochemical consequences of copper-histidine therapy in Menkes disease. Eur. J. Pediatr. **152**, 828–832

100 Gunn, T. R., Macfarlane, S. and Phillips, L. I. (1984) Difficulties in the neonatal diagnosis of Menkes' kinky hair syndrome–trichopoliodystrophy. Clin. Pediatr. **23**, 514–516

101 Yuen, P., Lin, H. J. and Hutchinson, J. H. (1979) Copper deficiency in a low birthweight infant. Arch. Dis. Child. **54**, 553–555

102 Danks, D. M., Campbell, P. E., Stevens, B. J., Mayne, V. and Cartwright, E. (1972) Menkes's kinky hair syndrome. An inherited defect in copper absorption with widespread effects. Pediatrics **50**, 188–201

103 Jones, D. G. and Suttle, N. F. (1983) The effect of copper deficiency on the resistance of mice to infection with *Pasteurella haemolytica*. J. Comp. Pathol. **93**, 143–149

104 Newberne, P. M., Hunt, C. E. and Young, V. R. (1968) The role of diet and the reticuloendothelial system in the response of rats to *Salmonella* Typhimurium infection. Br. J. Exp. Pathol. **49**, 448–457

105 Babu, U. and Failla, M. L. (1990) Respiratory burst and candidacidal activity of peritoneal macrophages are impaired in copper-deficient rats. J. Nutr. **120**, 1692–1699

106 Crocker, A., Lee, C., Aboko-Cole, G. and Durham, C. (1992) Interaction of nutrition and infection: effect of copper deficiency on resistance to *Trypanosoma lewisi*. J. Natl. Med. Assoc. **84**, 697–706

107 Jones, D. G. and Suttle, N. F. (1981) Some effects of copper deficiency on leukocyte function in sheep and cattle. Res. Vet. Sci. **31**, 151–156

108 Boyne, R. and Arthur, J. R. (1981) Effects if selenium and copper deficiency on neutrophil function in cattle. J. Comp. Pathol. **91**, 271–276

109 Percival, S. S. (1998) Copper and immunity. Am. J. Clin. Nutr. **67**, 1064S–1068S

110 White, C., Lee, J., Kambe, T., Fritsche, K. and Petris, M. J. (2009) A role for the ATP7A copper-transporting ATPase in macrophage bactericidal activity. J. Biol. Chem. **284**, 33949–33956

111 Achard, M. E., Stafford, S. L., Bokil, N. J., Chartres, J., Bernhardt, P. V., Schembri, M. A., Sweet, M. J. and McEwan, A. G. (2012) Copper redistribution in murine macrophages in response to *Salmonella* infection. Biochem. J. **444**, 51–57

112 Osman, D., Waldron, K. J., Denton, H., Taylor, C. M., Grant, A. J., Mastroeni, P., Robinson, N. J. and Cavet, J. S. (2010) Copper homeostasis in Salmonella is atypical and copper–CueP is a major periplasmic metal complex. J. Biol. Chem. **285**, 25259–25268

113 West, A. P., Brodsky, I. E., Rahner, C., Woo, D. K., Erdjument-Bromage, H., Tempst, P., Walsh, M. C., Choi, Y., Shadel, G. S. and Ghosh, S. (2011) TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature **472**, 476–480

114 Zhou, B. and Gitschier, J. (1997) hCTR1: a human gene for copper uptake identified by complementation in yeast. Proc. Natl. Acad. Sci. U.S.A. **94**, 7481–7486

115 Aller, S. G. and Unger, V. M. (2006) Projection structure of the human copper transporter CTR1 at 6-A resolution reveals a compact trimer with a novel channel-like architecture. Proc. Natl. Acad. Sci. U.S.A. **103**, 3627–3632

116 Petris, M. J., Smith, K., Lee, J. and Thiele, D. J. (2003) Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1. J. Biol. Chem. **278**, 9639–9646

117 Klomp, A. E., Tops, B. B., Van Denberg, I. E., Berger, R. and Klomp, L. W. (2002) Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1). Biochem. J. **364**, 497–505

118 Bertinato, J., Swist, E., Plouffe, L. J., Brooks, S. P and L'Abbé, M. R. (2008) Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells. Biochem. J. **409**, 731–740

119 van den Berghe, P. V. E., Folmer, D. E., Malingre, H. E. M., van Beurden, E., Klomp, A. E. M., van de Sluis, B., Merkx, M., Berger, R. and Klomp, L. W. J. (2007) Human copper transporter 2 is localized in late endosomes and facilitates cellular copper uptake. Biochem. J. **407**, 49–59

120 Lutsenko, S., LeShane, E. S. and Shinde, U. (2007) Biochemical basis of regulation of human copper-transporting ATPases. Arch. Biochem. Biophys. **463**, 134–148

121 Camakaris, J., Petris, M. J., Bailey, L., Shen, P., Lockhart, P., Glover, T. W., Barcroft, C., Patton, J. and Mercer, J. F. (1995) Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux. Hum. Mol. Genet. **4**, 2117–2123

122 Voskoboinik, I., Brooks, H., Smith, S., Shen, P and Camakaris, J. (1998) ATP-dependent copper transport by the Menkes protein in membrane vesicles isolated from cultured Chinese hamster ovary cells. FEBS Lett. **435**, 178–182

123 Vulpe, C., Levinson, B., Whitney, S., Packman, S. and Gitschier, J. (1993) Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat. Genet. **3**, 7–13

124 Paynter, J. A., Grimes, A., Lockhart, P and Mercer, J. F. (1994) Expression of the Menkes gene homologue in mouse tissues lack of effect of copper on the mRNA levels. FEBS Lett. **351**, 186–190

125 Ke, B. X., Llanos, R. M., Wright, M., Deal, Y. and Mercer, J. F. (2006) Alteration of copper physiology in mice overexpressing the human Menkes protein ATP7A. Am. J. Physiol. Regul. Integr. Comp. Physiol. **290**, R1460–R1467

126 Pase, L., Voskoboinik, I., Greenough, M. and Camakaris, J. (2004) Copper stimulates trafficking of a distinct pool of the Menkes copper ATPase (ATP7A) to the plasma membrane and diverts it into a rapid recycling pool. Biochem. J. **378**, 1031–1037
<figure><img src="image_13.png" /></figure>

127 Petris, M. J., Camakaris, J., Greenough, M., La Fontaine, S. and Mercer, J. F. B. (1998) A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network. Hum. Mol. Genet. **7**, 2063–2071

128 Petris, M. J., Mercer, J. F., Culvenor, J. G., Lockhart, P., Gleeson, P. A. and Camakaris, J. (1996) Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J. **15**, 6084–6095

129 Cobbold, C., Coventry, J., Ponnambalam, S. and Monaco, A. P. (2003) The Menkes disease ATPase (ATP7A) is internalized via a Rac-1 regulated, clathrin- and caveolae-independent pathway. Hum. Mol. Genet. **12**, 1523–1533

130 Lutsenko, S., Gupta, A., Burkhead, J. L. and Zuzel, V. (2008) Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance. Arch. Biochem. Biophys. **476**, 22–32

131 Linz, R. and Lutsenko, S. (2007) Copper-transporting ATPases ATP7A and ATP7B: cousins, not twins. J. Bioenerg. Biomembr. **39**, 403–407

132 Yang, F., Naylor, S. L., Lum, J. B., Cutshaw, S., McCombs, J. L., Naberhaus, K. H., McGill, J. R., Adrian, G. S., Moore, C. M., Barnett, D. R. et al. (1986) Characterization, mapping, and expression of the human ceruloplasmin gene. Proc. Natl. Acad. Sci. U.S.A. **83**, 3257–3261

133 Osaki, S. and Johnson, D. A. (1969) Mobilization of liver iron by ferroxidase (ceruloplasmin). J. Biol. Chem. **244**, 5757–5758

134 Osaki, S., Johnson, D. A. and Frieden, E. (1966) The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum. J. Biol. Chem. **241**, 2746–2751

135 Gutteridge, J. M. (1983) Antioxidant properties of ceruloplasmin towards iron- and copper-dependent oxygen radical formation. FEBS Lett. **157**, 37–40

136 Harris, E. D. (1993) The transport of copper. Prog. Clin. Biol. Res. **380**, 163–179

137 Zaitsev, V. N., Zaitseva, I., Papiz, M. and Lindley, P. F. (1999) An X-ray crystallographic study of the binding sites of the azide inhibitor and organic substrates to ceruloplasmin, a multi-copper oxidase in the plasma. J. Biol. Inorg. Chem. **4**, 579–587

138 Bianchini, A., Musci, G. and Calabrese, L. (1999) Inhibition of endothelial nitric-oxide synthase by ceruloplasmin. J. Biol. Chem. **274**, 20265–20270

139 Lee, K. H., Yun, S. J., Nam, K. N., Gho, Y. S. and Lee, E. H. (2007) Activation of microglial cells by ceruloplasmin. Brain Res. **1171**, 1–8

140 Mazumder, B., Mukhopadhyay, C. K., Prok, A., Cathcart, M. K. and Fox, P. L. (1997) Induction of ceruloplasmin synthesis by IFN-γ in human monocytic cells. J. Immunol. **159**, 1938–1944

141 Ramadori, G., Van Damme, J., Rieder, H. and Meyer zum Buschenfelde, K. H. (1988) Interleukin 6, the third mediator of acute-phase reaction, modulates hepatic protein synthesis in human and mouse. Comparison with interleukin 1 beta and tumor necrosis factor-alpha. Eur. J. Immunol. **18**, 1259–1264

142 Fleming, R. E., Whitman, I. P. and Gitlin, J. D. (1991) Induction of ceruloplasmin gene expression in rat lung during inflammation and hyperoxia. Am. J. Physiol. **260**, L68–74

143 Patel, B. N. and David, S. (1997) A novel glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed by mammalian astrocytes. J. Biol. Chem. **272**, 20185–20190

144 Gray, L. W., Kidane, T. Z., Nguyen, A., Akagi, S., Petrasek, K., Chu, Y. L., Cabrera, A., Kantardjieff, K., Mason, A. Z. and Linder, M. C. (2009) Copper proteins and ferroxidases in human plasma and that of wild-type and ceruloplasmin knockout mice. Biochem. J. **419**, 237–245

145 Meyer, L. A., Durley, A. P., Prohaska, J. R. and Harris, Z. L. (2001) Copper transport and metabolism are normal in ceruloplasminemic mice. J. Biol. Chem. **276**, 36857–36861

146 Das, D., Tapryal, N., Goswami, S. K., Fox, P. L. and Mukhopadhyay, C. K. (2007) Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene. Biochem. J. **402**, 135–141

147 De Domenico, I., McVey Ward, D., Bonaccorsi di Patti, M. C., Jeong, S. Y., David, S., Musci, G. and Kaplan, J. (2007) Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin. EMBO J. **26**, 2823–2831

148 Nairz, M., Theurl, I., Ludwiczek, S., Theurl, M., Mair, S. M., Fritsche, G. and Weiss, G. (2007) The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular *Salmonella* typhimurium. Cell. Microbiol. **9**, 2126–2140

149 Xu, F. F. and Imlay, J. A. (2012) Silver(I), mercury(II), cadmium(II), and zinc(II) target exposed enzymic iron-sulfur clusters when they toxify *Escherichia coli*. Appl. Environ. Microbiol. **78**, 3614–3621

150 Turanov, A. A., Su, D. and Gladyshev, V. N. (2006) Characterization of alternative cytosolic forms and cellular targets of mouse mitochondrial thioredoxin reductase. J. Biol. Chem. **281**, 22953–22963

151 Potter, A. J., Trappetti, C. and Paton, J. C. (2012) *Streptococcus pneumoniae* uses glutathione to defend against oxidative stress and metal ion toxicity. J. Bacteriol. **194**, 6248–6254

152 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. **28**, 2731–2739

Received 28 January 2013/4 June 2013; accepted 5 June 2013

Published as Immediate Publication 5 June 2013, doi 10.1042/BSR20130014
<figure><img src="image_13.png" /></figure>

127 Petris, M. J., Camakaris, J., Greenough, M., La Fontaine, S. and Mercer, J. F. B. (1998) A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network. Hum. Mol. Genet. **7**, 2063–2071

128 Petris, M. J., Mercer, J. F., Culvenor, J. G., Lockhart, P., Gleeson, P. A. and Camakaris, J. (1996) Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J. **15**, 6084–6095

129 Cobbold, C., Coventry, J., Ponnambalam, S. and Monaco, A. P. (2003) The Menkes disease ATPase (ATP7A) is internalized via a Rac-1 regulated, clathrin- and caveolae-independent pathway. Hum. Mol. Genet. **12**, 1523–1533

130 Lutsenko, S., Gupta, A., Burkhead, J. L. and Zuzel, V. (2008) Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance. Arch. Biochem. Biophys. **476**, 22–32

131 Linz, R. and Lutsenko, S. (2007) Copper-transporting ATPases ATP7A and ATP7B: cousins, not twins. J. Bioenerg. Biomembr. **39**, 403–407

132 Yang, F., Naylor, S. L., Lum, J. B., Cutshaw, S., McCombs, J. L., Naberhaus, K. H., McGill, J. R., Adrian, G. S., Moore, C. M., Barnett, D. R. et al. (1986) Characterization, mapping, and expression of the human ceruloplasmin gene. Proc. Natl. Acad. Sci. U.S.A. **83**, 3257–3261

133 Osaki, S. and Johnson, D. A. (1969) Mobilization of liver iron by ferroxidase (ceruloplasmin). J. Biol. Chem. **244**, 5757–5758

134 Osaki, S., Johnson, D. A. and Frieden, E. (1966) The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum. J. Biol. Chem. **241**, 2746–2751

135 Gutteridge, J. M. (1983) Antioxidant properties of ceruloplasmin towards iron- and copper-dependent oxygen radical formation. FEBS Lett. **157**, 37–40

136 Harris, E. D. (1993) The transport of copper. Prog. Clin. Biol. Res. **380**, 163–179

137 Zaitsev, V. N., Zaitseva, I., Papiz, M. and Lindley, P. F. (1999) An X-ray crystallographic study of the binding sites of the azide inhibitor and organic substrates to ceruloplasmin, a multi-copper oxidase in the plasma. J. Biol. Inorg. Chem. **4**, 579–587

138 Bianchini, A., Musci, G. and Calabrese, L. (1999) Inhibition of endothelial nitric-oxide synthase by ceruloplasmin. J. Biol. Chem. **274**, 20265–20270

139 Lee, K. H., Yun, S. J., Nam, K. N., Gho, Y. S. and Lee, E. H. (2007) Activation of microglial cells by ceruloplasmin. Brain Res. **1171**, 1–8

140 Mazumder, B., Mukhopadhyay, C. K., Prok, A., Cathcart, M. K. and Fox, P. L. (1997) Induction of ceruloplasmin synthesis by IFN-γ in human monocytic cells. J. Immunol. **159**, 1938–1944

141 Ramadori, G., Van Damme, J., Rieder, H. and Meyer zum Buschenfelde, K. H. (1988) Interleukin 6, the third mediator of acute-phase reaction, modulates hepatic protein synthesis in human and mouse. Comparison with interleukin 1 beta and tumor necrosis factor-alpha. Eur. J. Immunol. **18**, 1259–1264

142 Fleming, R. E., Whitman, I. P. and Gitlin, J. D. (1991) Induction of ceruloplasmin gene expression in rat lung during inflammation and hyperoxia. Am. J. Physiol. **260**, L68–74

143 Patel, B. N. and David, S. (1997) A novel glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed by mammalian astrocytes. J. Biol. Chem. **272**, 20185–20190

144 Gray, L. W., Kidane, T. Z., Nguyen, A., Akagi, S., Petrasek, K., Chu, Y. L., Cabrera, A., Kantardjieff, K., Mason, A. Z. and Linder, M. C. (2009) Copper proteins and ferroxidases in human plasma and that of wild-type and ceruloplasmin knockout mice. Biochem. J. **419**, 237–245

145 Meyer, L. A., Durley, A. P., Prohaska, J. R. and Harris, Z. L. (2001) Copper transport and metabolism are normal in ceruloplasminemic mice. J. Biol. Chem. **276**, 36857–36861

146 Das, D., Tapryal, N., Goswami, S. K., Fox, P. L. and Mukhopadhyay, C. K. (2007) Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene. Biochem. J. **402**, 135–141

147 De Domenico, I., McVey Ward, D., Bonaccorsi di Patti, M. C., Jeong, S. Y., David, S., Musci, G. and Kaplan, J. (2007) Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin. EMBO J. **26**, 2823–2831

148 Nairz, M., Theurl, I., Ludwiczek, S., Theurl, M., Mair, S. M., Fritsche, G. and Weiss, G. (2007) The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular *Salmonella* typhimurium. Cell. Microbiol. **9**, 2126–2140

149 Xu, F. F. and Imlay, J. A. (2012) Silver(I), mercury(II), cadmium(II), and zinc(II) target exposed enzymic iron-sulfur clusters when they toxify *Escherichia coli*. Appl. Environ. Microbiol. **78**, 3614–3621

150 Turanov, A. A., Su, D. and Gladyshev, V. N. (2006) Characterization of alternative cytosolic forms and cellular targets of mouse mitochondrial thioredoxin reductase. J. Biol. Chem. **281**, 22953–22963

151 Potter, A. J., Trappetti, C. and Paton, J. C. (2012) *Streptococcus pneumoniae* uses glutathione to defend against oxidative stress and metal ion toxicity. J. Bacteriol. **194**, 6248–6254

152 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. **28**, 2731–2739

Received 28 January 2013/4 June 2013; accepted 5 June 2013

Published as Immediate Publication 5 June 2013, doi 10.1042/BSR20130014