Sian L Stafford

Nilesh J Bokil

Maud E S Achard

Ronan Kapetanovic

Mark A Schembri

Alastair G Mcewan

Matthew J Sweet
email: m.sweet@imb.uq.edu.au

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

Keywords: copper transporter (CTR), host defence, innate immunity, monocyte, Toll-like receptor, zinc transporter ceruloplasmin, CTR, copper transporter, DC, dendritic cells, FPN1, ferroportin 1, GPI, glycosylphosphatidylinositol, IFNγ , interferon γ, IKKβ, IκB

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.

INTRODUCTION

The immune system requires essential micronutrients and trace elements such as iron, zinc, copper and selenium for optimal function [START_REF] Failla | Trace elements and host defense: recent advances and continuing challenges[END_REF]. 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 [START_REF] Failla | Trace elements and host defense: recent advances and continuing challenges[END_REF]. 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 animal models, have conclusively demonstrated that zinc is essential 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 approximately 50 years ago [START_REF] Prasad | Discovery of human zinc deficiency: 50 years later[END_REF]. The first case was reported in Iran in a male patient who presented with severe anaemia and growth retardation [START_REF] Prasad | Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia[END_REF]. This condition was associated with hepatosplenomegaly, 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 [START_REF] Prasad | Zinc metabolism in patients with the syndrome of iron deficiency anemia, hepatosplenomegaly, dwarfism, and hypognadism[END_REF], where a regimen of zinc supplementation reversed hypogonadism and growth retardation [START_REF] Sandstead | Human zinc deficiency, endocrine manifestations and response to treatment[END_REF]. In addition to developmental anomalies and anaemia, the patients also suffered from severe immune deficiency leading to intercurrent infection and death before the age of 25 [START_REF] Prasad | Zinc in human health: effect of zinc on immune cells[END_REF]. Furthermore, experimentally induced mild zinc deficiency in human subjects [START_REF] Prasad | Experimental zinc deficiency in humans[END_REF] resulted in a decrease in the ratio of CD4 + to CD8 + T-cells, and a decreased 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 individuals with chronic alcoholism. The 2002 WHO world health report attributed about 800 000 deaths (1.4 % of global mortalities) to zinc deficiency, and estimated that it contributed to 16 % of lower respiratory tract infections, 18 % of malaria and 10 % of diarrhoeal disease [START_REF] Guilbert | The world health report 2002-reducing risks, promoting healthy life[END_REF]. 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,[START_REF] Fraker | Effect of zinc deficiency on the immune response of the young adult A/J mouse[END_REF]. 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,[START_REF] Knoell | Zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis[END_REF]. The enhanced inflammatory response correlated with increased activity of NF-κB (nuclear factor κB) and expression of NF-κBdependent 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 [START_REF] Sobocinski | Involvement of hepatic metallothioneins in hypozincemia associated with bacterial infection[END_REF], 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. [START_REF] Yasuno | The disposition and intestinal absorption of zinc in rats[END_REF] 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 [START_REF] Beker Aydemir | Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia)[END_REF]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 [START_REF] Goode | Evidence of cellular zinc depletion in hospitalized but not in healthy elderly subjects[END_REF]. A more recent study demonstrated that plasma zinc concentrations were decreased in critically ill patients, and were dramatically lower in patients with septic shock [START_REF] Besecker | A comparison of zinc metabolism, inflammation, and disease severity in critically ill infected and noninfected adults early after intensive care unit admission[END_REF]. 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 infectious disease, both clinically and in animal models. More than three decades ago, Snyder and Walker [START_REF] Snyder | Inhibition of lethality in endotoxin-challenged mice treated with zinc chloride[END_REF] showed that ZnCl 2 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 polymicrobial sepsis model when C57Bl/6 mice received prophylactic zinc gluconate treatment [START_REF] Nowak | Prophylactic zinc supplementation reduces bacterial load and improves survival in a murine model of sepsis[END_REF]. Similarly, zinc supplementation decreased the parasite load in the blood in Wistar rats infected with Trypanosoma cruzi [START_REF] Brazao | Zinc supplementation increases resistance to experimental infection by Trypanosoma cruzi[END_REF], and restored the ability of alcoholfed 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 [START_REF] Singh | Effect of zinc on immune functions and host resistance against infection and tumor challenge[END_REF], a rat model of Escherichia coli-mediated prostatitis [START_REF] Cho | Antibacterial effect of intraprostatic zinc injection in a rat model of chronic bacterial prostatitis[END_REF], and a Rhesus monkey model of severe diarrhoeal disease caused by enteropathogenic E. coli [START_REF] Kelleher | Supplementation of infant formula with the probiotic Lactobacillus reuteri and zinc: impact on enteric infection and nutrition in infant rhesus monkeys[END_REF]. More importantly, many studies have demonstrated that zinc supplementation has beneficial effects in clinical settings, particularly in severe diarrhoeal diseases and respiratory tract infections. Pooled analysis of randomized controlled trials of zinc supplementation in children suffering from severe diarrhoea and pneumonia showed that this treatment regime reduced the incidence 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 supplementation reduced the frequency and duration of acute and persistent diarrhoea in infants by up to 18 % [START_REF] Lukacik | A meta-analysis of the effects of oral zinc in the treatment of acute and persistent diarrhea[END_REF]. Indeed, a WHO and UNICEF report recommended the inclusion of zinc in oral rehydration solution to treat gastroenteritis in infants and children [START_REF] Wardlaw | Diarrhoea: why children are still dying and what can be done[END_REF]. 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 infection [START_REF] Valavi | The efficacy of zinc supplementation on outcome of children with severe pneumonia. A randomized double-blind placebo-controlled clinical trial[END_REF]. Zinc supplementation has also been reported to show beneficial effects for a range of other infectious diseases including shigellosis, leprosy, tuberculosis and leishmaniasis [START_REF] Prasad | Zinc: role in immunity, oxidative stress and chronic inflammation[END_REF]. Finally, Mocchegiani et al. [START_REF] Mocchegiani | Benefit of oral zinc supplementation as an adjunct to zidovudine (AZT) therapy against opportunistic infections in AIDS[END_REF] reported that zinc supplementation (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 [START_REF] Patro | Oral zinc for the treatment of acute gastroenteritis in Polish children: a randomized, double-blind, placebo-controlled trial[END_REF], and zinc and vitamin A supplementation in pulmonary tuberculosis showed no therapeutic benefit [START_REF] Lawson | Randomized controlled trial of zinc and vitamin A as co-adjuvants for the treatment of pulmonary tuberculosis[END_REF]. Thus, zinc supplementation may be effective in controlling specific infections only in individuals with notable zinc deficiency, or alternatively, other environmental and/or genetic factors may impact on efficacy of this treatment. Furthermore, zinc supplementation may actually exacerbate disease severity for some pathogens. Vitamin A and zinc supplementation was assessed in gastrointestinal parasitic infections among Mexican children [START_REF] Long | Effect of vitamin A and zinc supplementation on gastrointestinal parasitic infections among Mexican children[END_REF]. Zinc reduced the incidence of Giardia lamblia and Entamoeba histolytica infections, but increased Ascaris lumbricoides-associated diarrhoea. Animal model studies also suggest that high-dose zinc supplementation may exert undesirable effects that are independent of immune function; for example, leading to hippocampus-dependent memory impairment [START_REF] Yang | High dose zinc supplementation induces hippocampal zinc deficiency and memory impairment with inhibition of BDNF signaling[END_REF]. Taken together, the above literature suggests that zinc has an essential role in immune function and protecting against infectious 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 [START_REF] Haase | Functional significance of zinc-related signaling pathways in immune cells[END_REF]. 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 Staphylococcus aureus by peritoneal macrophages [START_REF] Nowak | Prophylactic zinc supplementation reduces bacterial load and improves survival in a murine model of sepsis[END_REF]. 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 [START_REF] Brazao | Zinc supplementation increases resistance to experimental infection by Trypanosoma cruzi[END_REF]46]. Several other studies have also reported that zinc promotes macrophage phagocytic capacity and/or pathogen clearance by these cells [47][START_REF] Lastra | Zinc intervention on macrophages and lymphocytes response[END_REF][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 promoted the phagosomal accumulation of zinc in Mycobacterium avium-infected mouse macrophages, and phagosomal zinc also accumulated over time in response to infection with Mycobacterium tuberculosis [START_REF] Wagner | Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system[END_REF]. 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 macrophages, M. tuberculosis expressed ctpC, which encodes a zinc efflux pump [START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF]. 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 [START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF]. Such evidence 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 essential 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 [START_REF] Mcdevitt | A molecular mechanism for bacterial susceptibility to zinc[END_REF]. 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 macrophage 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 [START_REF] Houghton | Macrophage elastase kills bacteria within murine macrophages[END_REF]. MMP7 is involved in the activation of defensins by cleaving the pro form of αand 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 employed as part of the macrophage response to Histoplasma capsulatum [START_REF] Winters | Metallomic analysis of macrophages infected with Histoplasma capsulatum reveals a fundamental role for zinc in host defenses[END_REF], 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 (granulocyte/macrophage colony stimulating factor)-derived murine peritoneal and bone marrow macrophages with H. capsulatum decreased the intracellular zinc concentration. The TLR (Toll-like receptor) 4 agonist LPS from Gram-negative bacteria also reduced the intracellular zinc concentration within mouse DC (dendritic cells) [START_REF] Kitamura | Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function[END_REF], 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 extracellular space [START_REF] Achouiti | Myeloid-related protein-14 contributes to protective immunity in Gram-negative pneumonia derived sepsis[END_REF][60][START_REF] Sohnle | Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14)[END_REF]. Not surprisingly, some pathogens have evolved mechanisms to thwart zinc starvation by the host. For example, Salmonella enterica serovar Typhimurium (S. Typhimurium) thrives in the inflamed gut by expressing ZnuABC, a high-affinity zinc transporter that overcomes calprotectinmediated zinc chelation [62]. The utilization of zinc in macrophage antimicrobial pathways versus zinc sequestration as a nutrient starvation strategy to limit microbial growth highlights the complexity of zinc involvement in macrophage functions. Zinc trafficking may have distinct functions in these cells, depending on the specific infectious agent encountered and/or the zinc concentrations that are present. Indeed, the effects of zinc on macrophage inflammatory responses vary in a concentrationdependent manner [START_REF] Haase | Signal transduction in monocytes: the role of zinc ions[END_REF].

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 [START_REF] Haase | Signal transduction in monocytes: the role of zinc ions[END_REF][START_REF] Haase | Functional significance of zinc-related signaling pathways in immune cells[END_REF]. Indeed, approximately 5 % of genes were reported to be zinc-responsive in a human monocytic cell line [START_REF] Cousins | A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells[END_REF]. Such effects are likely to be an important component of zincmediated 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 [START_REF] Haase | Signal transduction in monocytes: the role of zinc ions[END_REF][START_REF] Haase | Functional significance of zinc-related signaling pathways in immune cells[END_REF], 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-1dependent cytokines such as IL-1β [START_REF] Schroder | The inflammasomes[END_REF]. However, one study has reported that IL-1β release downstream of the NLRP3 inflammasome was dependent on zinc [START_REF] Brough | Pannexin-1-dependent caspase-1 activation and secretion of IL-1beta is regulated by zinc[END_REF]. 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 [START_REF] Haase | Signal transduction in monocytes: the role of zinc ions[END_REF]. 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 [START_REF] Haase | Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes[END_REF]. 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 [START_REF] Kitamura | Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function[END_REF]. 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 [START_REF] Aydemir | Zinc supplementation of young men alters metallothionein, zinc transporter, and cytokine gene expression in leukocyte populations[END_REF], whereas dietary zinc depletion suppressed LPS-inducible TNF production from whole blood [START_REF] Ryu | Genomic analysis, cytokine expression, and microRNA profiling reveal biomarkers of human dietary zinc depletion and homeostasis[END_REF]. 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 [START_REF] Prasad | Zinc enhances the expression of interleukin-2 and interleukin-2 receptors in HUT-78 cells by way of NF-kappaB activation[END_REF], and in response to LPS in human and mouse monocytes/macrophages [START_REF] Haase | Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes[END_REF]. 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 [START_REF] Liu | ZIP8 regulates host defense through zinc-mediated inhibition of NF-kappaB[END_REF], while others have also shown that zinc indirectly inhibits IKKβ via cGMPdependent activation of PKA (protein kinase A) [START_REF] Von Bulow | 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[END_REF]. Zinc also up-regulates the expression of A20, a negative regulator of inflammatory responses [START_REF] Prasad | Zinc-suppressed inflammatory cytokines by induction of A20-mediated inhibition of nuclear factor-kappaB[END_REF]. 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 [START_REF] Haase | Signal transduction in monocytes: the role of zinc ions[END_REF][START_REF] Haase | Functional significance of zinc-related signaling pathways in immune cells[END_REF]. Various other primary TLR-activated signalling pathways including MAPK p38 and ERK (extracellular-signal-regulated kinase) are also zinc-dependent [START_REF] Haase | Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes[END_REF]. 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 Fe 2 + [START_REF] Powell | The antioxidant properties of zinc[END_REF]. Zn 2 + 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 homoeostasis is maintained through MT, a family of zincbinding proteins controlling cellular zinc distribution in a redoxdependent manner [START_REF] Maret | Thiolate ligands in metallothionein confer redox activity on zinc clusters[END_REF]. LPS rapidly up-regulates MT expression in human macrophages [START_REF] Leibbrandt | Activation of human monocytes with lipopolysaccharide induces metallothionein expression and is diminished by zinc[END_REF]; presumably this may act to sequester Interplay between zinc trafficking, inflammatory signalling and antimicrobial responses in macrophages 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 [START_REF] Hood | Nutritional immunity: transition metals at the pathogen-host interface[END_REF]. 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 [START_REF] Haase | Signal transduction in monocytes: the role of zinc ions[END_REF] and mobilizes the intracellular zinc pool. TLR-induced SLC30A family members (e.g. SLC30A1, 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 [START_REF] Rink | Zinc homeostasis and immunity[END_REF] 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 [START_REF] Andreini | Zinc through the three domains of life[END_REF].

the free intracellular zinc that is generated immediately after TLR activation [START_REF] Haase | Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes[END_REF], and would be broadly consistent with the antiinflammatory roles of MT, as identified by gene knock-out studies [80][START_REF] Inoue | Role of metallothionein in coagulatory disturbance and systemic inflammation induced by lipopolysaccharide in mice[END_REF][START_REF] Takano | Protective role of metallothionein in acute lung injury induced by bacterial endotoxin[END_REF]. Zinc homoeostasis 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 [START_REF] Lichten | Mammalian zinc transporters: nutritional and physiologic regulation[END_REF]. 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 [START_REF] Lichten | Mammalian zinc transporters: nutritional and physiologic regulation[END_REF]. 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 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 [START_REF] Winters | Metallomic analysis of macrophages infected with Histoplasma capsulatum reveals a fundamental role for zinc in host defenses[END_REF] and LPS [START_REF] Kitamura | Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function[END_REF] 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 [START_REF] Overbeck | Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT-1 to ZnT-9[END_REF], 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 [START_REF] Kitamura | Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function[END_REF], 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 [START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF]. 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 [START_REF] Wagner | Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system[END_REF][START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF] and perhaps other infectious agents. Indeed, several members of this family including SLC30A2 [START_REF] Palmiter | ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration[END_REF][START_REF] Liuzzi | Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis[END_REF], SLC30A6 [START_REF] Huang | Functional characterization of a novel mammalian zinc transporter, ZnT6[END_REF], SLC30A7 [START_REF] Kirschke | ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus[END_REF] 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 downregulated mRNA levels of Slc39a6 and Slc39a10 in these cells [START_REF] Kitamura | Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function[END_REF]. Furthermore, Slc39a6 overexpression blocked the LPStriggered 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 Tcells 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 [START_REF] Yu | Regulation of T cell receptor signaling by activation-induced zinc influx[END_REF]. 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 [START_REF] Besecker | The human zinc transporter SLC39A8 (Zip8) is critical in zinc-mediated cytoprotection in lung epithelia[END_REF]. 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 [START_REF] Aydemir | Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-gamma expression in activated human T cells[END_REF]. 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 [START_REF] Besecker | A comparison of zinc metabolism, inflammation, and disease severity in critically ill infected and noninfected adults early after intensive care unit admission[END_REF], 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 difficult 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 inflammatory responses [START_REF] Liu | ZIP8 regulates host defense through zinc-mediated inhibition of NF-kappaB[END_REF]. The authors confirmed that LPS and TNF up-regulated SLC39A8 expression, and that the mechanism involved direct regulation by the transcription factor NF-κB. The authors also showed that this up-regulation of SLC39A8 enabled 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 inflammatory responses [START_REF] Sayadi | Zip14 expression induced by lipopolysaccharides in macrophages attenuates inflammatory response[END_REF]. Slc39a14 mRNA and protein was also LPS-inducible in mice, and Slc39a14 -/ -mice have impaired zinc uptake, altered plasma zinc and IL-6 levels after LPS administration, as well as dysregulated metabolism [START_REF] Beker Aydemir | Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia)[END_REF]. Furthermore, the up-regulation of Slc39a14 was previously reported to be IL-6 dependent [26], suggesting that there may be some level of interdependence 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 importers 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 microbicidal 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 enzymes 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 H 2 O 2 from superoxide in neutrophils and monocytes [START_REF] Maggini | Selected vitamins and trace elements support immune function by strengthening epithelial barriers and cellular and humoral immune responses[END_REF]. Several lines of evidence indicate that copper deficiency perturbs immune function. Firstly, copper deficiency, in addition to causing neurological dysfunctions, also results in haematological abnormalities, most commonly neutropenia and anaemia [97]. Copper-deficient patients also displayed decreased numbers of myeloid precursors 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 reported in Menkes disease [START_REF] Prohaska | Copper deficiency suppresses the immune response of mice[END_REF][99][100][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 [START_REF] Prohaska | Copper deficiency suppresses the immune response of mice[END_REF]. Subsequent studies have reported increased 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 copperdeficient 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 isolated 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 responses, suggesting that copper is likely to regulate the functions of multiple immune cell types [109]. As with the zinc literature, several studies have demonstrated that copper regulates macrophage antimicrobial functions. Macrophages from copperdeficient rats were unimpaired in their ability to phagocytose erythrocytes, but had a defective respiratory burst and were compromised in their ability to kill C. albicans [105]. In vitro studies 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 macrophages [START_REF] Wagner | Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system[END_REF], suggesting that this metal ion may have some role in macrophage antimicrobial responses. Consistent with this, exogenous 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 macrophages, in keeping with the well-known capacity of Cu(I) to catalyse the generation of hydroxyl radical from H 2 O 2 . This study also showed that both IFNγ and LPS regulated copper-trafficking pathways in macrophages. Studies on the intramacrophage pathogen S. Typhimurium also provide functional evidence for a role in macrophage antimicrobial pathways. Infection of mouse macrophages with S. Typhimurium, as well as treatment with LPS, promoted the accumulation of copper within intracellular vesicles. This response peaked at about 14 h post-stimulation. Furthermore, a cell impermeable copper chelator (bathocuproinedisulfonic acid) reduced vesicular copper accumulation in macrophages, and this impaired the ability of primary mouse macrophages to kill S. Typhimurium [111]. The use of a Salmonella copper-responsive promoter reporter strain also provided evidence 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 promotes a slower accumulation of ROS, which is derived from the mitochondria [113]. Hence, it is conceivable that the delayed vesicular 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 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 [START_REF] Wagner | Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system[END_REF]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] . This is consistent with Atp7a contributing 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 responses, and their intracellular localizations are dynamically regulated in macrophages responding to pathogen challenge. Furthermore, both zinc and copper can be delivered to the macrophage phagosome, raising the question of whether there is interplay between the two ions in macrophage-mediated host defence. 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 isopropylmalate isomerase (leucine biosynthesis) [149]. This property 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 antioxidant defences linked to thioredoxins. Thus, copper and zinc could independently enhance oxidative stress during macrophage antimicrobial responses, even though zinc is more generally associated 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 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.

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Figure 1

 1 Figure 1Interplay between zinc trafficking, inflammatory signalling and antimicrobial responses in macrophages 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[START_REF] Hood | Nutritional immunity: transition metals at the pathogen-host interface[END_REF]. 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[START_REF] Haase | Signal transduction in monocytes: the role of zinc ions[END_REF] and mobilizes the intracellular zinc pool. TLR-induced SLC30A family members (e.g. SLC30A1, 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[START_REF] Rink | Zinc homeostasis and immunity[END_REF] 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).

Figure 2

 2 Figure2Nearest-neighbour-joining generated phylogenetic tree of human and mouse SLC30A and SLC39A family members 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.

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Figure 3

 3 Figure 3 Possible mechanisms by which copper may contribute to macrophage antimicrobial responses 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.

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  . 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 , a cell impermeable copper chelator inhibited the formation of LPS-and Salmonella-inducible copper-containing vesicles in macrophages[111]. These stimuli also up-regulated Ctr1 and Ctr2 expression in primary mouse macrophages and RAW264.7 cells[110,111], implicating one or both of these transporters in copper uptake by macrophages. 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 expression and iron export[147]. Consequently, inducible copper trafficking 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]

	ATP7A and ATP7B
	ATP7A is a 178 kDa, Cu(I)-transporting P 1B -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 exo-
	cytic vesicles and specialised cell compartments such as secret-
	ory 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 macro-
	phages (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 infec-
	tion also increased Atp7a mRNA levels in primary mouse macro-
	phages [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 verteb-
	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

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

similarlyform

[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 remaining copper being bound primarily with albumin and transcuprein

[144]

. Surprisingly, aceruloplasminemic patients suffer no copper imbalance; rather, they have impaired iron efflux from cells resulting in complications

c 2013 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/ by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

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.

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.


Filip Dámek

Bastien Fremaux

Dominique Aubert

Sandra Thoumire

Delphine Le Roux

Sandra Vuillermet

Maxime Delsart

Isabelle Villena

Radu Blaga

Detailed anatomical distribution of Toxoplasma gondii in tissues of infected pigs

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.

DETAILED ANATOMICAL DISTRIBUTION OF TOXOPLASMA GONDII IN TISSUES OF INFECTED PIGS

• Unexpectedly low T. gondii DNA prevalence in brains by qPCR.

• Uneven concentration of tissue cysts within the tissues of the tested animals.

• Significant individual differences observed between the pigs.

• Tissue cyst-infected pigs' tissues found to be positive more frequently but without a significantly higher parasite burden estimate. • Longer incubation period may support parasite distribution into more tissues.

RESULTS

36 muscles (pelvic limb-14, thoracic limb-7, head-5, core-10) + 14 organs

  co-funded from the TOXOSOURCES and ToxSauQMRA projects funded by the European Union's Horizon 2020 Research and Innovation programme under grant agreement No. 773830: One Health European Joint Programme, France-Agri-Mer: contract number 0917003490 and Med-Vet-Net European network of Excellence for Zoonoses research.

Figure 1 .

 1 Figure 1. Anatomical drawing of a selection of muscles for sample collection.

Filip Dámek 1 , Bastien Fremaux 2 , Dominique Aubert 3 , Sandra Thoumire 1 , Delphine Le Roux 1 , Sandra Vuillermet 1 , Maxime Delsart 4 , Isabelle Villena 3 , Radu Blaga 1 1

  Anses, INRAE, Ecole Nationale Vétérinaire d'Alfort, Laboratoire de Santé Animale, BIPAR, Maisons-Alfort, France, 2 IFIP -Institut du Porc, Maisons-Alfort, France, 3 National Reference Center on Toxoplasmosis, Toxoplasma Biological Resources Center, CHU Reims and EA7510, SFR CAP-Santé, University of Reims Champagne-Ardenne, USC EpiToxo ANSES, France, 4 EPIMAI, USC Anses, Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France

Table 1

 1

. Selection of tested muscles, positive tissues are highlighted in red, negative tissues in white, head muscles missing in carcasses of the naturally infected pigs in black.


R Soliva

ConocoPhilips R A Schultz

A Taboada

C05 Strain Localisation in Porous Sandstone as a Function of Tectonic Setting, Burial and Material Properties

The analysis of three cataclastic band sets from Provence (France) reveals that the band density, their conjugate angles, their ratio of shear displacement to compaction, and the amount of cataclasis are different and can be expressed as a function of tectonic setting and petrophysical properties. We identify (1) a dense and closely spaced network of shear enhanced (reverse) compaction bands, (2) a regularly spaced less dense network of reverse compactional shear bands, and (3) a localized network of normal shear bands. The field data show that the localization of strain is favored in an extensional setting and is characterized by shear bands with a large shear to compaction ratio and a low conjugate band angle. In contrast, distributed strain is favored in a contractional setting and is characterized by compactional bands with a low ratio of shear to compaction and a large conjugate band angle. To explain the mechanical origin of this strain localization, we quantify the yield strength and the stress evolution in extensional and contractional regimes. We propose a model of strain localisation in porous sandstone as a function of tectonic stresses, burial depth, material properties, strain hardening and fluid pressure.

Introduction

Deformation bands are brittle-ductile deformation structures in porous sandstones in which the petrophysical properties can be roughly different than the surrounding host rock. The origin of the band network geometry and the mechanical reasons for which they can be more or less cataclastic (i.e. efficient or transient membrane seal barrier) remains poorly analysed in the light of tectonic conditions, burial and material properties.

In this study, we present an analysis of three different band systems at two localities in Provence (France), which clarify the dependence of strain localisation to tectonic stresses and material properties. We examine (1) within the same lithology and petrophysical properties the dependence of strain distribution as a function of the tectonic regime, and (2) in two different lithologies, the dependence of strain distribution on sandstone material properties for the same tectonic conditions. Based on these observations and the theory of plastic yielding in porous granular sandstones, we propose a model of band system formation as a function tectonic stresses, burial, material properties, strain hardening and fluid pressures.

Field study

We have identified (1) a dense and closely spaced network of shear enhanced (reverse) compaction bands, (2) a regularly spaced less dense network of reverse compactional shear bands, and (3) a localized network of normal shear bands (red, green and bleu, respectively in Fihures 1).

The first set is composed of conjugate shear-enhanced compaction bands (SECB), very closely spaced and showing wide variations in band thickness distribution. In thin section, force chains are recognized oblique to the bands, and bisecting their conjugate geometry at 45•. The orientation of the force chains (parallel to beds) suggest that shear, if any, must be of reverse movement and would then be in the opposite sense to the apparent offset due to compaction.

The second set is composed of conjugate E-W trending compactional shear bands of reverse sense. They show a regular spacing and a positive correlation between shear displacement and compaction. In thin section, a moderate cataclasis is observed and chains of fragmented particles are bisecting their conjugate geometry at ~38° and therefore nearly parallel to the bedding. The mean ratio of shear displacement to compaction is 7.6 (with 0.8 < D s /D c < 20 and a standard deviation of 4.1), then close to 8 times higher than in the SECB band system.

The third set consists of conjugate N-S oriented shear bands of normal sense, interacting with strikeslip fault zones by increasing their dip and their density. They show no clear regular spacing (clustered around the strike-slip faults) and a positive correlation between shear displacement and compaction. The degree of cataclasis is clearly the highest of the three studied band sets and force chains are bisecting their conjugate geometry at ~20° and are therefore nearly vertical. The ratio of shear displacement to compaction is more variable than the other band sets with an average value of 23.7 (with 0.4 < D s /D c < 90 and a standard deviation of 19.7), and then also clearly the highest of the three studied band systems.

Using thicknesses of sediments still present and potentially eroded above, we estimate that all the band sets have been buried between 300 and 700 m (400 m at Uchaux and 600 m at Orange). Both the orientation and kinematic analysis of the first (compaction) and the second (reverse movement) band sets relate them to the Pyrenean compression. The tectonic origin of the third band set is probably related to the Oligocene rifting. Localization of strain appears to be favored in extensional tectonic settings and is characterized by normal sense shear bands with a large shear to compaction ratio, a large amount of cataclasis and a low conjugate band angle. In contrast, spatially more distributed strain is favored in contractional tectonic settings and is characterized by reverse sense compactional bands with moderate cataclasis, a low ratio of shear to compaction and a large conjugate band angle.

3 rd International Conference on Fault and Top Seals -From Characterization to Modelling Montpellier, France, 1-3 October 2012

Figure 2 Graph of q vs p stresses including Druker-Prager -cap envelopes, burial and tectonic stress paths. The cap sides of the envelopes have been using porosity and grain size values for the Uchaux and Orange porous sandstones. Burial and tectonic stress paths have been calculated using a K 0 = 0.7. Colored stars represent the expected stress conditions to create the three deformation bands sets studied. Two different depths, 400 and 600 m are used for Uchaux and Orange, respectively. These graphs show that shear strain localization is favored in extensional stress conditions whereas strain distribution is favored for contractional conditions.

Mechanics

To explain the mechanical origin of this strain localization, we propose a model of stress evolution and plastic yielding in porous granular sandstones (Figure 2). The stress path model suggests that strain localization with a large component of shear is favored in extensional settings since the deviation of stresses from a burial path must evolve by an increase of deviatoric stresses and a reduction of the mean stress. This stress reduction, inherent to extensional settings implies a high probability of the stress path to cross the critical state line or the cap side of the envelope in its upper part. In contrast, distributed strain on multiple bands having a large component of compaction is likely to occur in contractional settings because of the high rate of increase of the mean stress and the larger probability to cross the middle or lower part of the yield cap. A stress permutation scenario related to the horizontal stress increase from a lithostatic state is proposed for contractional settings to explain the occurrence of reverse sense compactional bands and the low probability to obtain strike slip sense bands.

3 rd International Conference on Fault and Top Seals -From Characterization to Modelling Montpellier, France, 1-3 October 2012

Figure 3 Model of strain localization or distribution as shear or compaction dominated bands respectively, as a function of several parameters including mean and differential stress, sandstone grain size, porosity, pore pressure and strain hardening. Both the strain distribution, the sense of shear, the amount of cataclasis and the conjugate angles are controlled by the relative interplay between the parameters cited above.

Conclusions

The main results are summarized in the following points:

(1) In the same lithology (Uchaux porous sandstones) it is possible to obtain significant differences in the band network geometry and degree of cataclasis as a function of the tectonic regime applied (Pyrenean compression and Oligocene extension).

(2) In two different lithologies (Uchaux and Orange sandstones), it is possible to obtain two different band network geometry and degree of cataclasis in the same contractional tectonic regime (Pyrenean compression).

(3) The average ratio of shear displacement to compaction has been estimated to be around 1 for the shear enhanced compaction bands from Uchaux, around 8 for the compactional shear bands from Orange and much more variable but around 24 for the shear bands from Uchaux (Figure 1B). (4) The band kinematics, i.e. shear displacement to compaction, has been related to the mean and the differential stresses, grain size, porosity, fluid pressure and strain hardening (Figure 3). (5) Band initiation as strike-slip seems least probable in compressive context because of the large coefficient of Earth pressure at rest (ratio between the lateral to the axial stresses) inherent to such a compliant, frictional and incohesive granular material

3Figure 1

 1 Figure 1 Strain localisation in porous sandstones as shown by the scaling properties of the bands and the three different degrees of cataclasis observed in each type of band. A Cumulative frequency diagram of band spacing. B Graph of shear displacement vs. compaction. C, D and E are photomicrographs of the three band sets. C. Thin section photomicrograph of the normal shear bands from Uchaux showing a high degree of cataclasis. D Thin section photomicrograph and fracture mapping of the reverse compactional shear bands from Orange, showing moderate cataclasis. E. Thin section photomicrograph and fracture mapping of the reverse SECB from Uchaux, showing a low degree of cataclasis.