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Multiple impacts of zinc on immune function Cite this: Metallomics, 2014, 6, 1175
Hajo Haase and Lothar Rink* Even though zinc is essential for virtually all processes in the human body, observations during zinc deficiency indicate that the absence of this trace element most severely affects the immune response. Numerous investigations of the cellular and molecular requirements for zinc in the immune system have
Received 27th November 2013, Accepted 5th February 2014
indicated that there is not just one single function of zinc underlying this essentiality. In fact, there is a
DOI: 10.1039/c3mt00353a
three of the major fields: the role of zinc as a second messenger in signal transduction, the importance
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of zinc for immune cell function, and the competition for zinc between the host and the pathogen, a concept known as nutritional immunity.
wide range of different roles of zinc in immunity. This review summarizes the recent developments in
Introduction The trace element zinc is essential for a vast number of organ systems, and virtually every aspect of human biology involves zinc in some way.1 In particular, it has been known for over 50 years that zinc is an important factor for the immune system.2 Consequently, alleviating zinc deficiency by supplementation reduces mortality from infectious diseases, such as diarrhea and pneumonia,3 and zinc also affects additional functions of the immune system, such as tumor immunology.4 Over the years, several of the immunological and biochemical mechanisms Institute of Immunology, Medical Faculty, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail: [email protected]; Fax: +49 (0)241 8082613; Tel: +49 (0)241 8080208
Hajo Haase received his diploma in chemistry in 1998 and his PhD in natural sciences in 2001, both from the University of Bremen, Germany. From 2001 to 2003 he was a postdoc at the Center for Biochemical and Biophysical Sciences and Medicine at Harvard Medical School in Boston. Since 2003, he has been an assistant professor at the Institute of Immunology of the RWTH Aachen University Hajo Haase Hospital in Germany. His work focuses mainly on the role of zinc ions as an intracellular second messenger in cells of the immune system and the interference of immunotoxic heavy metal ions with zinc signaling.
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underlying this essentiality have been identified.5 First of all, zinc is a constituent of an extraordinarily high number of proteins. According to in silico studies, anywhere from 2400 to up to 4000 enzymes and a comparable number of transcription factors in the human proteome contain known zinc binding motifs.6,7 In addition, recent years have witnessed a rapid increase in the knowledge about further immunological roles of zinc. A comprehensive overview of zinc’s role in immunity will not be the subject of this minireview, because it has been summarized in many previous reviews.8–11 Instead, an update will be provided on recent developments in three major aspects of the immunobiology of zinc: (1) its role in the signal transduction of immune cells, (2) the impact of zinc on immune cell function, and (3) the competition for zinc between the host and the pathogen, which has become known as ‘nutritional immunity’.
Lothar Rink received his diploma in biology in 1990 and his PhD in natural sciences in 1995, both from the University of Hamburg, Germany. From 1995 to 1999 he worked at the Institute of Immunology and Transfusion ¨beck Medicine at University of Lu until he became an assistant professor at this institution. In 2001 he became full professor of immunology and head of the Institute of Immunology at RWTH Lothar Rink Aachen University. Key aspects of his work are the immunobiology of zinc especially in regard to homeostasis of the immune system and during ageing.
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Fig. 1 Activation of the different T cell subpopulations. Naı¨ve T cells are activated through antigen presentation by DCs and macrophages. Subsequently, they are armed T-helper (TH) cells (green) and cytotoxic T cells (red), which can participate in the immune response. For TH cells, the activation also involves the differentiation into several functionally diverse subpopulations.
The immune defense relies on two major groups of cells, which both depend on zinc on multiple levels:12 innate and adaptive immune cells. Innate immune cells mediate the immediate part of the immune response.13 It is mainly sustained by macrophages and neutrophil granulocytes, which directly attack different pathogens such as bacteria and fungi. Adaptive immunity is mediated by B and T cells. Through somatic recombination, each newly formed B and T cell has an individual receptor with the potential to recognize and bind a random biological structure, the so-called antigen. During an infection, pathogens are phagocytosed by macrophages and dendritic cells (DCs) and their antigens presented to T cells (Fig. 1). Out of the myriad of diverse T cells with different receptors, the ones recognizing antigens of the infecting pathogen are activated and the population of these few cells is expanded by proliferation. The main function of B cells is the production of antibodies, which act by neutralizing pathogens, or marking them for an attack by other immune cells or soluble factors. T cells can be further divided into T helper (TH) cells, which support other immune cells’ functions, and cytotoxic T cells, which directly eliminate virus-infected cells and tumor cells.
Zinc in immune cell signaling Among the most exciting developments in zinc biochemistry is certainly its molecular role as a second messenger in immune cells.14,15 A growing number of signaling pathways were discovered to involve zinc signals, including activation of T cells by their T cell receptor and by the cytokine interleukin (IL)-2,16–18 the major stimulus for T cell proliferation once they have been activated. Furthermore, zinc signals occur in response to activation of immune cells via one type of antibody-binding receptors, socalled Fce receptors, on their surface,19,20 and after triggering of pattern recognition receptors (PRRs).21–23 The latter are predominantly found on cells of the innate immune system, enabling them to recognize conserved pathogen associated molecular patterns (PAMPs). One of these PAMPs is lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, and it activates its
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corresponding PRR Toll-like receptor (TLR)4. The investigation of zinc signaling in response to TLR4 had previously given contradictory results. Activation of DCs via TLR4 led to reduced expression of the import protein ZIP6, thereby also reducing free intracellular zinc, an event that is required for maturation of these cells.23 In contrast, LPS causes elevated zinc in macrophages by upregulating ZIP8.24 A rapid increase of free zinc in macrophages has been shown to be a zinc signal required for directly inhibiting protein tyrosine phosphatases (PTP), thereby conserving TLR4-induced phosphorylation signals.22 Some of these differences may very well be based on the different cell types in which TLR4 signaling was investigated. However, a recent paper from our group indicates that the two major intracellular signaling pathways of TLR4, originating from the adaptor proteins MyD88 or TRIF, are differentially regulated by zinc. An early zinc signal is required for phosphatase inhibition and production of inflammatory cytokines through the MyD88 signaling pathway, whereas basal zinc levels at a later time point negatively regulate TRIF-dependent production of the cytokine interferon (IFN)-b, the pathogenicidal reactive nitrogen species nitric monoxide, and mRNA expression of the surface molecules CD80 and CD86,21 which contribute to T cell activation during antigen presentation. Notably, the latter has previously been shown to be negatively regulated by zinc in DCs, as well.23 Therefore, it seems that zinc has multiple targets in TLR4 signaling and might act in balancing the two branches of TLR signaling. This is supported by the observation that several other TLRs (1, 2, 5, 6, 7, 8, and 9) also trigger zinc signals, with the exception of TLR3. Notably, TLR3 is also the only TLR which signals exclusively via TRIF, and not via the zinc-dependent MyD88 pathway.21 The function of zinc in signal transduction is not limited to a role as a general PTP inhibitor, which unselectively blocks dephosphorylation of all cellular tyrosine residues alike. Firstly, the sensitivity of PTPs to inhibition by zinc differs over a wide concentration range. Some concentrations might even be too high to occur in the living cells at all, whereas PTPb has a Ki value of 21 pM for zinc, indicating a predominantly zinc-inhibited form of this enzyme at physiological levels of free intracellular zinc, which may only be activated by withdrawal of the ion.25 Secondly, subcellular localization of zinc signals is putting intracellular zinc homeostasis into the limelight. It is controlled by zinc transport proteins from the ZIP (SLC39A) and ZnT (SLC30A) families,26 and several of these zinc transporters have been shown to be involved in regulating the activities in immune cells (Table 1). For example, ZIP6 mediates the influx of extracellular zinc in response to T cell receptor (TCR) stimulation. This leads to a localized zinc signal in the immediate vicinity of the receptor’s intracellular region, which inhibits recruitment of SHP-1, a phosphatase that negatively affects TCR signaling. Thereby the zinc signal augments downstream signaling by the kinase ZAP70 and calcium ions in a defined region of the cytoplasm.18
Cellular immunological functions of zinc Despite the fact that the immune system has been investigated for decades by literally thousands of research groups, novel cellular
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Table 1
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Zinc transporters that regulate immune cell function
Transporter
Cell type
Function
Ref.
ZIP6
T cells
18
ZIP6
DCs
ZIP8
T cells
ZIP8
Monocytes/macrophages
ZIP14 ZnT4/7 (ZIP2)
Macrophages Macrophages
ZnT5
Mast cells
Zinc influx into the subsynaptic area of the cytoplasm promotes T cell receptor signaling, lowering its activation threshold Reduced expression after stimulation with LPS leads to less free intracellular zinc, which is required for DC maturation Release of lysosomal zinc inhibits the phosphatase calcineurin, augmenting phosphorylation of the transcription factor CREB and the production of IFNg Upregulation and concomitant zinc influx are involved in a negative feedback loop of NFkB signaling during an inflammatory response LPS-stimulated upregulation attenuates the production of IL-6 and TNF-a Zinc deprivation of phagocytosed Histoplasma capsulatum by sequestration into the Golgi apparatus (ZnT4/7) and uptake of extracellular zinc (ZIP2) Necessary for the mast cell-mediated delayed-type allergic response through activation of PKCb and NFkB
immune functions are still being uncovered and, subsequently, roles of zinc in these events are described. One example is neutrophil extracellular traps (NETs), a defense mechanism of neutrophil granulocytes against bacteria and fungi. During NET formation, neutrophil granulocytes release a matrix of DNA, serving as a scaffold for histones and antimicrobial proteins in which extracellular pathogens are captured.27 A protein kinase C (PKC)-dependent oxidative intracellular release of zinc is an essential component of the ROS-dependent signal transduction leading to NET formation.28 Another aspect is the polarization of TH cells into their different subpopulations (Fig. 1). TH cells regulate the functions of other immune cells. This includes the activation of macrophages to kill phagocytosed pathogens by TH1 cells, a support of antibody production by B cells from TH2 cells, augmentation of inflammation by TH17 cells, or the downregulation of the immune response by regulatory T cells. Together with the growing knowledge of the various TH subsets and the processes that regulate their differentiation, the role of zinc in these events has been elucidated. The initial observation showed that zinc shifts the balance between cell-mediated (TH1) and humoral (TH2) immunity in favor of TH1. This involves upregulating the expression of two crucial factors for the differentiation into TH1 cells: the cytokine IFNg and the transcription factor T-bet.29 In addition, zinc has been shown to suppress several T cellmediated immune reactions. Supplementation of human subjects with zinc for one week inhibited the mixed lymphocyte reaction, an ex vivo model for the allogeneic immune response after transplantations.30 In mice, zinc treatment suppressed TH17 development and related autoimmune disease,31 and it reduced the severity score of experimental autoimmune encephalomyelitis, which is an animal model of multiple sclerosis.32 Unpublished data from our group show that zinc supplementation promotes the formation of another TH subpopulation, regulatory T cells, which decrease T cell allogenic (TH1) and allergic (TH2) reactions as well as TH17-mediated autoimmune encephalomyelitis.
Zinc in nutritional immunity The concept of ‘nutritional immunity’ was originally described for iron, but has now been extended to other essential trace elements,
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23 16 24 59 55 20
including zinc.33 It describes the competition between the host and the invading pathogen for an important resource, during which both make great efforts to control its availability. For the pathogen, zinc is an essential nutrient required for survival and spreading, and approximately 5 percent of a bacterial proteome consists of zinc-containing proteins.6 Only 0.1% of total body zinc is located in the plasma. This means that 99.9% is within cells, and, therefore not easily accessible for extracellular pathogens. Moreover, most of plasma zinc is bound to proteins, further reducing its availability. Hence, sequestering the relatively small remaining pool of unbound plasma zinc seems to be a facile way for depriving pathogens of an essential nutrient and it is utilized during the immune response through several different mechanisms (Fig. 2). In addition to barriers and a direct attack by the immune system, this is an efficient further strategy for innate immune defense of the host to protect the body against infections. On the systemic level, redistribution of zinc during inflammation has been extensively investigated.26 Central events include inflammatory cytokines, such as IL-6, causing upregulation of the ZIP14 transporter on hepatocytes. Subsequently, metallothionein (MT)-bound zinc accumulates in the liver, whereby plasma zinc levels are diminished.34 Low serum zinc is associated with poor prognosis in pediatric septic shock35 and animal sepsis models.36,37 These data suggest that zinc supplementation could be a valuable therapeutic tool against sepsis. However, as shown by the group of Wong, only prophylactic zinc supplementation (most likely improving the immune system’s zinc status) was beneficial in a murine model of intraperitoneal sepsis, whereas multiple attempts to achieve similar results by supplementing zinc after the induction of sepsis were ineffective.38 Immune cells compromised by zinc deficiency could lead to impaired clearance of bacteria, allowing them to spread more effectively. Alternatively, zinc deficiency augments inflammation and might increase the initial inflammatory response, including the accompanying collateral damage to host tissues.39 Restoring immune function by zinc supplementation prior to the onset of sepsis seems to be beneficial, but treatment of acute infections by zinc holds the risk that the beneficial effects for the immune system could be foiled by annulling nutritional immunity. In addition to changes on the systemic scale, some antimicrobial peptides from the S100 family act by chelating zinc locally. These proteins are upregulated in keratinocytes exposed
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Fig. 2 Zinc metabolism in nutritional immunity. The immune system can modulate zinc at three different levels, either systemically (A), or in a limited extracellular environment (B), or in specialized intracellular compartments (C). (A) Zinc accumulation in the liver is caused by upregulation of ZIP14 and MT in hepatocytes in response to proinflammatory cytokines. (B) Antimicrobial proteins, especially calprotectin (a dimer of S100 A8 and A9), can be secreted by neutrophils as free proteins or associated with NETs. They exert their antimicrobial activity by chelation of essential metal ions, such as zinc. (C) Intracellular pathogens can be killed within macrophages by two opposed mechanisms either by zinc deprivation (as shown for H. capsulatum), or intoxication by excess zinc (as shown for M. tuberculosis).
to TH17 cytokines.40 The S100A7 protein (psoriasin) is secreted by keratinocytes and kills Escherichia coli by sequestration of zinc.41 Secretion of calprotectin (a heterodimer of the S100 proteins A8 and A9, also known as calgranulin A/B or MRP 8/14) by neutrophil granulocytes inhibits the growth of Staphylococcus aureus in abscesses by sequestration of zinc and manganese.42 Calprotectin accounts for up to 50% of the cytoplasmic protein content in neutrophil granulocytes and can reach concentrations of up to 1 mg ml 1 in abscesses.33 An in vivo relevance of zinc chelation by calprotectin as a pathogenicidal mechanism has been shown in several infection models.43–46 During NETosis, between 50% and 60% of total cellular calprotectin is externalized, half of it into the surrounding liquid and half incorporated into NETs.46 Here, calprotectin causes a zinc-limited microenvironment. As a constituent of NETs, calprotectin contributes significantly to their antifungal activity against Candida albicans and Cryptococcus neoformans, a process which was reversible by immunodepletion of calprotectin or addition
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of Zn2+ and Mn2+.46 A mouse model of pneumonia-derived sepsis caused by Klebsiella pneumoniae showed a higher bacterial burden in animals deficient in S100A9, and in vitro studies demonstrated that NET-mediated growth inhibition of K. pneumoniae involves calprotectin and is reversed by addition of zinc. Taken together, these results indicate zinc chelation as a likely mechanism for the pathogenicidal effects of calprotectin.43 Gram-positive S. aureus and gram-negative Pseudomonas aeruginosa were also susceptible to NET-associated calprotectin in vitro,43 and the calprotectin-mediated chelation of Zn2+ contributes to growth inhibition of Aspergillus fumigatus hyphae by NETs.47 Uptake systems for metal ions are significant virulence factors for numerous pathogens.48 For several bacteria, the ZnuABC system has been shown to be an important factor during infection, suggesting that counteracting zinc deprivation may be a general requirement for successful infection.33 Not only can Salmonella typhimurium overcome calprotectin-mediated zinc starvation during gut inflammation by ZnuABC transporter expression. Moreover, this mechanism is so effective that it is even giving S. typhimurium a competitive advantage over other microbes in colonizing the inflamed gut mucosa.44 Still, the response of pathogens to zinc deprivation goes beyond the elevated expression of import proteins, e.g., Candida albicans scavenges endothelial cell zinc by secreting the protein Pra1. Pra1 then re-associates with the fungus via the zinc transporter Zrt1 to deliver its zinc.49 Notably, pathogens are not limited to a single defense strategy. Two recent papers illustrated that Neisseria meningitidis can utilize at least two different mechanisms to overcome zinc restriction. On the one hand, the high-affinity zinc uptake receptor ZnuD prevents NET-mediated retardation of N. meningitidis proliferation.50 On the other hand, N. meningitidis responds to zinc limitation with another particularly elegant defense mechanism, which involves elevated expression of the outer membrane protein CbpA. It acts as a receptor for calprotectin, enabling N. meningitidis to acquire calprotectin-bound zinc as a nutrient.51 Some recent papers report that the competition for zinc even continues after pathogens have been phagocytosed by a macrophage. Notably, these immune cells use two opposed strategies to kill microorganisms inside their phagosomes; either starvation by zinc sequestration or intoxication by zinc excess. In addition to iron and manganese deprivation by the natural resistance-associated macrophage protein (NRAMP)-1, macrophages damage pathogens in their phagosomes, such as Mycobacterium tuberculosis, by accumulation of toxic amounts of zinc and copper.52 M. tuberculosis in the phagosomes of macrophages is exposed to high amounts of zinc and shows signs of heavy metal poisoning. A P1-type ATPase confers resistance to zinc intoxication, and a similar strategy has also been confirmed for E. coli and the transporter ZntA.53 Zinc accumulation requires NADPH oxidase activity, leading to the hypothesis that zinc is oxidatively released from cytosolic MT and then pumped into the endosomal compartment.53 In a single report, zinc intoxication has also been suggested to be a systemic antimicrobial mechanism against Streptococcus pneumoniae, acting via inhibition of manganese uptake.54
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However, this does not only contradict abovementioned studies reporting a systemic deprivation of zinc as a general response to inflammation. It also describes an increase in serum zinc to levels above 600 mM, exceeding the concentrations present in many tissues. Even if these results were to be confirmed, two open questions remain: from where could such a high amount of zinc originate, and how does the host tolerate a concentration that should be toxic for it as well? Intraphagosomal deprivation of zinc has been reported for Histoplasma capsulatum.55 Here, zinc is removed from the phagosome, and either stored in the cytoplasm bound to MT or transported into the Golgi by ZnTs 4 and 7. Strangely, even more zinc is taken up from the extracellular space via ZIP2. At first glance, it seems illogical for the macrophage to take up more zinc while trying to remove it from the pathogen, but zinc deficiency renders H. capsulatum more susceptible to ROS.56 Hence, zinc deficiency could act in two ways: directly impairing the pathogens’ growth or survival, while indirectly contributing to ROS-mediated defense mechanisms of the host. For the macrophage, on the other hand, zinc and MT exhibit a considerable antioxidant capacity.57 Hence, the immune cell might use accumulation of zinc-saturated MT to elevate its tolerance against reactive oxygen species, while depriving the pathogen of zinc undermines the latter’s antioxidant capacity. Killing of phagocytosed pathogens involves an oxidative burst, i.e., the massive production of reactive oxygen and nitrogen species and changes in the intracellular zinc distribution may contribute to effective intracellular pathogen clearance.58 So far, it remains puzzling how a macrophage decides when to harm the pathogen by zinc deprivation and when to intoxicate it by excess zinc.58 Bacteria have evolved transporters to overcome zinc deprivation, including ABC family transporters and TonB-dependent transport systems such as ZnuD, as well as mechanisms against intoxication, such as P-type ATPases, cation diffusion facilitators (CDFs), and RND family transporters.48 Maybe the differential response of macrophages reflects an adapted reaction toward specific susceptibilities of the individual infectious agent, even though we do not yet know how such susceptibility would be identified by the macrophage.
Conclusion Altogether, recent discoveries provide an intriguing new look into the multifaceted immunobiology of zinc. They finally bring us closer to understanding the immunological changes resulting from zinc deficiency and supplementation, which were described many years ago. Zinc supplementation is certainly important to address zinc deficiency-induced immune dysfunction. However, therapeutic application of zinc as a pharmacological agent during infection seems to be more complex. All the different impacts of zinc on the immune system will have to be considered, in order to achieve a desired therapeutic effect without interfering with other zinc-dependent processes in the immune response.
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43 A. Achouiti, T. Vogl, C. F. Urban, M. Rohm, T. J. Hommes, M. A. van Zoelen, S. Florquin, J. Roth, C. van’t Veer, A. F. de Vos and T. van der Poll, PLoS Pathog., 2012, 8, e1002987. 44 J. Z. Liu, S. Jellbauer, A. J. Poe, V. Ton, M. Pesciaroli, T. E. KehlFie, N. A. Restrepo, M. P. Hosking, R. A. Edwards, A. Battistoni, P. Pasquali, T. E. Lane, W. J. Chazin, T. Vogl, J. Roth, E. P. Skaar and M. Raffatellu, Cell Host Microbe, 2012, 11, 227–239. 45 M. I. Hood, B. L. Mortensen, J. L. Moore, Y. Zhang, T. E. Kehl-Fie, N. Sugitani, W. J. Chazin, R. M. Caprioli and E. P. Skaar, PLoS Pathog., 2012, 8, e1003068. 46 C. F. Urban, D. Ermert, M. Schmid, U. Abu-Abed, C. Goosmann, W. Nacken, V. Brinkmann, P. R. Jungblut and A. Zychlinsky, PLoS Pathog., 2009, 5, e1000639. 47 A. McCormick, L. Heesemann, J. Wagener, V. Marcos, D. Hartl, J. Loeffler, J. Heesemann and F. Ebel, Microbes Infect., 2010, 12, 928–936. 48 M. I. Hood and E. P. Skaar, Nat. Rev. Microbiol., 2012, 10, 525–537. 49 F. Citiulo, I. D. Jacobsen, P. Miramon, L. Schild, S. Brunke, P. Zipfel, M. Brock, B. Hube and D. Wilson, PLoS Pathog., 2012, 8, e1002777. 50 M. Lappann, S. Danhof, F. Guenther, S. Olivares-Florez, I. L. Mordhorst and U. Vogel, Mol. Microbiol., 2013, 89, 433–449. 51 M. Stork, J. Grijpstra, M. P. Bos, T. C. Manas, N. Devos, J. T. Poolman, W. J. Chazin and J. Tommassen, PLoS Pathog., 2013, 9, e1003733. 52 H. Botella, G. Stadthagen, G. Lugo-Villarino, C. de Chastellier and O. Neyrolles, Trends Microbiol., 2012, 20, 106–112. 53 H. Botella, P. Peyron, F. Levillain, R. Poincloux, Y. Poquet, I. Brandli, C. Wang, L. Tailleux, S. Tilleul, G. M. Charriere, S. J. Waddell, M. Foti, G. Lugo-Villarino, Q. Gao, I. Maridonneau-Parini, P. D. Butcher, P. R. Castagnoli, B. Gicquel, C. de Chastellier and O. Neyrolles, Cell Host Microbe, 2011, 10, 248–259. 54 C. A. McDevitt, A. D. Ogunniyi, E. Valkov, M. C. Lawrence, B. Kobe, A. G. McEwan and J. C. Paton, PLoS Pathog., 2011, 7, e1002357. 55 K. Subramanian Vignesh, J. A. Landero Figueroa, A. Porollo, J. A. Caruso and G. S. Deepe, Jr., Immunity, 2013, 39, 697–710. 56 V. K. Subramanian, J. A. Landero Figueroa, A. Porollo, J. A. Caruso and G. S. Deepe, Jr., Immunity, 2013, 39, 697–710. 57 W. Maret, Antioxid. Redox Signaling, 2006, 8, 1419–1441. 58 H. Haase, Immunity, 2013, 39, 623–624. 59 A. Sayadi, A. T. Nguyen, F. A. Bard and E. A. Bard-Chapeau, Inflammation Res., 2013, 62, 133–143.
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MINIREVIEW
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Multiple impacts of zinc on immune function Cite this: Metallomics, 2014, 6, 1175
Hajo Haase and Lothar Rink* Even though zinc is essential for virtually all processes in the human body, observations during zinc deficiency indicate that the absence of this trace element most severely affects the immune response. Numerous investigations of the cellular and molecular requirements for zinc in the immune system have
Received 27th November 2013, Accepted 5th February 2014
indicated that there is not just one single function of zinc underlying this essentiality. In fact, there is a
DOI: 10.1039/c3mt00353a
three of the major fields: the role of zinc as a second messenger in signal transduction, the importance
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of zinc for immune cell function, and the competition for zinc between the host and the pathogen, a concept known as nutritional immunity.
wide range of different roles of zinc in immunity. This review summarizes the recent developments in
Introduction The trace element zinc is essential for a vast number of organ systems, and virtually every aspect of human biology involves zinc in some way.1 In particular, it has been known for over 50 years that zinc is an important factor for the immune system.2 Consequently, alleviating zinc deficiency by supplementation reduces mortality from infectious diseases, such as diarrhea and pneumonia,3 and zinc also affects additional functions of the immune system, such as tumor immunology.4 Over the years, several of the immunological and biochemical mechanisms Institute of Immunology, Medical Faculty, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail: [email protected]; Fax: +49 (0)241 8082613; Tel: +49 (0)241 8080208
Hajo Haase received his diploma in chemistry in 1998 and his PhD in natural sciences in 2001, both from the University of Bremen, Germany. From 2001 to 2003 he was a postdoc at the Center for Biochemical and Biophysical Sciences and Medicine at Harvard Medical School in Boston. Since 2003, he has been an assistant professor at the Institute of Immunology of the RWTH Aachen University Hajo Haase Hospital in Germany. His work focuses mainly on the role of zinc ions as an intracellular second messenger in cells of the immune system and the interference of immunotoxic heavy metal ions with zinc signaling.
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underlying this essentiality have been identified.5 First of all, zinc is a constituent of an extraordinarily high number of proteins. According to in silico studies, anywhere from 2400 to up to 4000 enzymes and a comparable number of transcription factors in the human proteome contain known zinc binding motifs.6,7 In addition, recent years have witnessed a rapid increase in the knowledge about further immunological roles of zinc. A comprehensive overview of zinc’s role in immunity will not be the subject of this minireview, because it has been summarized in many previous reviews.8–11 Instead, an update will be provided on recent developments in three major aspects of the immunobiology of zinc: (1) its role in the signal transduction of immune cells, (2) the impact of zinc on immune cell function, and (3) the competition for zinc between the host and the pathogen, which has become known as ‘nutritional immunity’.
Lothar Rink received his diploma in biology in 1990 and his PhD in natural sciences in 1995, both from the University of Hamburg, Germany. From 1995 to 1999 he worked at the Institute of Immunology and Transfusion ¨beck Medicine at University of Lu until he became an assistant professor at this institution. In 2001 he became full professor of immunology and head of the Institute of Immunology at RWTH Lothar Rink Aachen University. Key aspects of his work are the immunobiology of zinc especially in regard to homeostasis of the immune system and during ageing.
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Fig. 1 Activation of the different T cell subpopulations. Naı¨ve T cells are activated through antigen presentation by DCs and macrophages. Subsequently, they are armed T-helper (TH) cells (green) and cytotoxic T cells (red), which can participate in the immune response. For TH cells, the activation also involves the differentiation into several functionally diverse subpopulations.
The immune defense relies on two major groups of cells, which both depend on zinc on multiple levels:12 innate and adaptive immune cells. Innate immune cells mediate the immediate part of the immune response.13 It is mainly sustained by macrophages and neutrophil granulocytes, which directly attack different pathogens such as bacteria and fungi. Adaptive immunity is mediated by B and T cells. Through somatic recombination, each newly formed B and T cell has an individual receptor with the potential to recognize and bind a random biological structure, the so-called antigen. During an infection, pathogens are phagocytosed by macrophages and dendritic cells (DCs) and their antigens presented to T cells (Fig. 1). Out of the myriad of diverse T cells with different receptors, the ones recognizing antigens of the infecting pathogen are activated and the population of these few cells is expanded by proliferation. The main function of B cells is the production of antibodies, which act by neutralizing pathogens, or marking them for an attack by other immune cells or soluble factors. T cells can be further divided into T helper (TH) cells, which support other immune cells’ functions, and cytotoxic T cells, which directly eliminate virus-infected cells and tumor cells.
Zinc in immune cell signaling Among the most exciting developments in zinc biochemistry is certainly its molecular role as a second messenger in immune cells.14,15 A growing number of signaling pathways were discovered to involve zinc signals, including activation of T cells by their T cell receptor and by the cytokine interleukin (IL)-2,16–18 the major stimulus for T cell proliferation once they have been activated. Furthermore, zinc signals occur in response to activation of immune cells via one type of antibody-binding receptors, socalled Fce receptors, on their surface,19,20 and after triggering of pattern recognition receptors (PRRs).21–23 The latter are predominantly found on cells of the innate immune system, enabling them to recognize conserved pathogen associated molecular patterns (PAMPs). One of these PAMPs is lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, and it activates its
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corresponding PRR Toll-like receptor (TLR)4. The investigation of zinc signaling in response to TLR4 had previously given contradictory results. Activation of DCs via TLR4 led to reduced expression of the import protein ZIP6, thereby also reducing free intracellular zinc, an event that is required for maturation of these cells.23 In contrast, LPS causes elevated zinc in macrophages by upregulating ZIP8.24 A rapid increase of free zinc in macrophages has been shown to be a zinc signal required for directly inhibiting protein tyrosine phosphatases (PTP), thereby conserving TLR4-induced phosphorylation signals.22 Some of these differences may very well be based on the different cell types in which TLR4 signaling was investigated. However, a recent paper from our group indicates that the two major intracellular signaling pathways of TLR4, originating from the adaptor proteins MyD88 or TRIF, are differentially regulated by zinc. An early zinc signal is required for phosphatase inhibition and production of inflammatory cytokines through the MyD88 signaling pathway, whereas basal zinc levels at a later time point negatively regulate TRIF-dependent production of the cytokine interferon (IFN)-b, the pathogenicidal reactive nitrogen species nitric monoxide, and mRNA expression of the surface molecules CD80 and CD86,21 which contribute to T cell activation during antigen presentation. Notably, the latter has previously been shown to be negatively regulated by zinc in DCs, as well.23 Therefore, it seems that zinc has multiple targets in TLR4 signaling and might act in balancing the two branches of TLR signaling. This is supported by the observation that several other TLRs (1, 2, 5, 6, 7, 8, and 9) also trigger zinc signals, with the exception of TLR3. Notably, TLR3 is also the only TLR which signals exclusively via TRIF, and not via the zinc-dependent MyD88 pathway.21 The function of zinc in signal transduction is not limited to a role as a general PTP inhibitor, which unselectively blocks dephosphorylation of all cellular tyrosine residues alike. Firstly, the sensitivity of PTPs to inhibition by zinc differs over a wide concentration range. Some concentrations might even be too high to occur in the living cells at all, whereas PTPb has a Ki value of 21 pM for zinc, indicating a predominantly zinc-inhibited form of this enzyme at physiological levels of free intracellular zinc, which may only be activated by withdrawal of the ion.25 Secondly, subcellular localization of zinc signals is putting intracellular zinc homeostasis into the limelight. It is controlled by zinc transport proteins from the ZIP (SLC39A) and ZnT (SLC30A) families,26 and several of these zinc transporters have been shown to be involved in regulating the activities in immune cells (Table 1). For example, ZIP6 mediates the influx of extracellular zinc in response to T cell receptor (TCR) stimulation. This leads to a localized zinc signal in the immediate vicinity of the receptor’s intracellular region, which inhibits recruitment of SHP-1, a phosphatase that negatively affects TCR signaling. Thereby the zinc signal augments downstream signaling by the kinase ZAP70 and calcium ions in a defined region of the cytoplasm.18
Cellular immunological functions of zinc Despite the fact that the immune system has been investigated for decades by literally thousands of research groups, novel cellular
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Table 1
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Zinc transporters that regulate immune cell function
Transporter
Cell type
Function
Ref.
ZIP6
T cells
18
ZIP6
DCs
ZIP8
T cells
ZIP8
Monocytes/macrophages
ZIP14 ZnT4/7 (ZIP2)
Macrophages Macrophages
ZnT5
Mast cells
Zinc influx into the subsynaptic area of the cytoplasm promotes T cell receptor signaling, lowering its activation threshold Reduced expression after stimulation with LPS leads to less free intracellular zinc, which is required for DC maturation Release of lysosomal zinc inhibits the phosphatase calcineurin, augmenting phosphorylation of the transcription factor CREB and the production of IFNg Upregulation and concomitant zinc influx are involved in a negative feedback loop of NFkB signaling during an inflammatory response LPS-stimulated upregulation attenuates the production of IL-6 and TNF-a Zinc deprivation of phagocytosed Histoplasma capsulatum by sequestration into the Golgi apparatus (ZnT4/7) and uptake of extracellular zinc (ZIP2) Necessary for the mast cell-mediated delayed-type allergic response through activation of PKCb and NFkB
immune functions are still being uncovered and, subsequently, roles of zinc in these events are described. One example is neutrophil extracellular traps (NETs), a defense mechanism of neutrophil granulocytes against bacteria and fungi. During NET formation, neutrophil granulocytes release a matrix of DNA, serving as a scaffold for histones and antimicrobial proteins in which extracellular pathogens are captured.27 A protein kinase C (PKC)-dependent oxidative intracellular release of zinc is an essential component of the ROS-dependent signal transduction leading to NET formation.28 Another aspect is the polarization of TH cells into their different subpopulations (Fig. 1). TH cells regulate the functions of other immune cells. This includes the activation of macrophages to kill phagocytosed pathogens by TH1 cells, a support of antibody production by B cells from TH2 cells, augmentation of inflammation by TH17 cells, or the downregulation of the immune response by regulatory T cells. Together with the growing knowledge of the various TH subsets and the processes that regulate their differentiation, the role of zinc in these events has been elucidated. The initial observation showed that zinc shifts the balance between cell-mediated (TH1) and humoral (TH2) immunity in favor of TH1. This involves upregulating the expression of two crucial factors for the differentiation into TH1 cells: the cytokine IFNg and the transcription factor T-bet.29 In addition, zinc has been shown to suppress several T cellmediated immune reactions. Supplementation of human subjects with zinc for one week inhibited the mixed lymphocyte reaction, an ex vivo model for the allogeneic immune response after transplantations.30 In mice, zinc treatment suppressed TH17 development and related autoimmune disease,31 and it reduced the severity score of experimental autoimmune encephalomyelitis, which is an animal model of multiple sclerosis.32 Unpublished data from our group show that zinc supplementation promotes the formation of another TH subpopulation, regulatory T cells, which decrease T cell allogenic (TH1) and allergic (TH2) reactions as well as TH17-mediated autoimmune encephalomyelitis.
Zinc in nutritional immunity The concept of ‘nutritional immunity’ was originally described for iron, but has now been extended to other essential trace elements,
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23 16 24 59 55 20
including zinc.33 It describes the competition between the host and the invading pathogen for an important resource, during which both make great efforts to control its availability. For the pathogen, zinc is an essential nutrient required for survival and spreading, and approximately 5 percent of a bacterial proteome consists of zinc-containing proteins.6 Only 0.1% of total body zinc is located in the plasma. This means that 99.9% is within cells, and, therefore not easily accessible for extracellular pathogens. Moreover, most of plasma zinc is bound to proteins, further reducing its availability. Hence, sequestering the relatively small remaining pool of unbound plasma zinc seems to be a facile way for depriving pathogens of an essential nutrient and it is utilized during the immune response through several different mechanisms (Fig. 2). In addition to barriers and a direct attack by the immune system, this is an efficient further strategy for innate immune defense of the host to protect the body against infections. On the systemic level, redistribution of zinc during inflammation has been extensively investigated.26 Central events include inflammatory cytokines, such as IL-6, causing upregulation of the ZIP14 transporter on hepatocytes. Subsequently, metallothionein (MT)-bound zinc accumulates in the liver, whereby plasma zinc levels are diminished.34 Low serum zinc is associated with poor prognosis in pediatric septic shock35 and animal sepsis models.36,37 These data suggest that zinc supplementation could be a valuable therapeutic tool against sepsis. However, as shown by the group of Wong, only prophylactic zinc supplementation (most likely improving the immune system’s zinc status) was beneficial in a murine model of intraperitoneal sepsis, whereas multiple attempts to achieve similar results by supplementing zinc after the induction of sepsis were ineffective.38 Immune cells compromised by zinc deficiency could lead to impaired clearance of bacteria, allowing them to spread more effectively. Alternatively, zinc deficiency augments inflammation and might increase the initial inflammatory response, including the accompanying collateral damage to host tissues.39 Restoring immune function by zinc supplementation prior to the onset of sepsis seems to be beneficial, but treatment of acute infections by zinc holds the risk that the beneficial effects for the immune system could be foiled by annulling nutritional immunity. In addition to changes on the systemic scale, some antimicrobial peptides from the S100 family act by chelating zinc locally. These proteins are upregulated in keratinocytes exposed
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Fig. 2 Zinc metabolism in nutritional immunity. The immune system can modulate zinc at three different levels, either systemically (A), or in a limited extracellular environment (B), or in specialized intracellular compartments (C). (A) Zinc accumulation in the liver is caused by upregulation of ZIP14 and MT in hepatocytes in response to proinflammatory cytokines. (B) Antimicrobial proteins, especially calprotectin (a dimer of S100 A8 and A9), can be secreted by neutrophils as free proteins or associated with NETs. They exert their antimicrobial activity by chelation of essential metal ions, such as zinc. (C) Intracellular pathogens can be killed within macrophages by two opposed mechanisms either by zinc deprivation (as shown for H. capsulatum), or intoxication by excess zinc (as shown for M. tuberculosis).
to TH17 cytokines.40 The S100A7 protein (psoriasin) is secreted by keratinocytes and kills Escherichia coli by sequestration of zinc.41 Secretion of calprotectin (a heterodimer of the S100 proteins A8 and A9, also known as calgranulin A/B or MRP 8/14) by neutrophil granulocytes inhibits the growth of Staphylococcus aureus in abscesses by sequestration of zinc and manganese.42 Calprotectin accounts for up to 50% of the cytoplasmic protein content in neutrophil granulocytes and can reach concentrations of up to 1 mg ml 1 in abscesses.33 An in vivo relevance of zinc chelation by calprotectin as a pathogenicidal mechanism has been shown in several infection models.43–46 During NETosis, between 50% and 60% of total cellular calprotectin is externalized, half of it into the surrounding liquid and half incorporated into NETs.46 Here, calprotectin causes a zinc-limited microenvironment. As a constituent of NETs, calprotectin contributes significantly to their antifungal activity against Candida albicans and Cryptococcus neoformans, a process which was reversible by immunodepletion of calprotectin or addition
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of Zn2+ and Mn2+.46 A mouse model of pneumonia-derived sepsis caused by Klebsiella pneumoniae showed a higher bacterial burden in animals deficient in S100A9, and in vitro studies demonstrated that NET-mediated growth inhibition of K. pneumoniae involves calprotectin and is reversed by addition of zinc. Taken together, these results indicate zinc chelation as a likely mechanism for the pathogenicidal effects of calprotectin.43 Gram-positive S. aureus and gram-negative Pseudomonas aeruginosa were also susceptible to NET-associated calprotectin in vitro,43 and the calprotectin-mediated chelation of Zn2+ contributes to growth inhibition of Aspergillus fumigatus hyphae by NETs.47 Uptake systems for metal ions are significant virulence factors for numerous pathogens.48 For several bacteria, the ZnuABC system has been shown to be an important factor during infection, suggesting that counteracting zinc deprivation may be a general requirement for successful infection.33 Not only can Salmonella typhimurium overcome calprotectin-mediated zinc starvation during gut inflammation by ZnuABC transporter expression. Moreover, this mechanism is so effective that it is even giving S. typhimurium a competitive advantage over other microbes in colonizing the inflamed gut mucosa.44 Still, the response of pathogens to zinc deprivation goes beyond the elevated expression of import proteins, e.g., Candida albicans scavenges endothelial cell zinc by secreting the protein Pra1. Pra1 then re-associates with the fungus via the zinc transporter Zrt1 to deliver its zinc.49 Notably, pathogens are not limited to a single defense strategy. Two recent papers illustrated that Neisseria meningitidis can utilize at least two different mechanisms to overcome zinc restriction. On the one hand, the high-affinity zinc uptake receptor ZnuD prevents NET-mediated retardation of N. meningitidis proliferation.50 On the other hand, N. meningitidis responds to zinc limitation with another particularly elegant defense mechanism, which involves elevated expression of the outer membrane protein CbpA. It acts as a receptor for calprotectin, enabling N. meningitidis to acquire calprotectin-bound zinc as a nutrient.51 Some recent papers report that the competition for zinc even continues after pathogens have been phagocytosed by a macrophage. Notably, these immune cells use two opposed strategies to kill microorganisms inside their phagosomes; either starvation by zinc sequestration or intoxication by zinc excess. In addition to iron and manganese deprivation by the natural resistance-associated macrophage protein (NRAMP)-1, macrophages damage pathogens in their phagosomes, such as Mycobacterium tuberculosis, by accumulation of toxic amounts of zinc and copper.52 M. tuberculosis in the phagosomes of macrophages is exposed to high amounts of zinc and shows signs of heavy metal poisoning. A P1-type ATPase confers resistance to zinc intoxication, and a similar strategy has also been confirmed for E. coli and the transporter ZntA.53 Zinc accumulation requires NADPH oxidase activity, leading to the hypothesis that zinc is oxidatively released from cytosolic MT and then pumped into the endosomal compartment.53 In a single report, zinc intoxication has also been suggested to be a systemic antimicrobial mechanism against Streptococcus pneumoniae, acting via inhibition of manganese uptake.54
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However, this does not only contradict abovementioned studies reporting a systemic deprivation of zinc as a general response to inflammation. It also describes an increase in serum zinc to levels above 600 mM, exceeding the concentrations present in many tissues. Even if these results were to be confirmed, two open questions remain: from where could such a high amount of zinc originate, and how does the host tolerate a concentration that should be toxic for it as well? Intraphagosomal deprivation of zinc has been reported for Histoplasma capsulatum.55 Here, zinc is removed from the phagosome, and either stored in the cytoplasm bound to MT or transported into the Golgi by ZnTs 4 and 7. Strangely, even more zinc is taken up from the extracellular space via ZIP2. At first glance, it seems illogical for the macrophage to take up more zinc while trying to remove it from the pathogen, but zinc deficiency renders H. capsulatum more susceptible to ROS.56 Hence, zinc deficiency could act in two ways: directly impairing the pathogens’ growth or survival, while indirectly contributing to ROS-mediated defense mechanisms of the host. For the macrophage, on the other hand, zinc and MT exhibit a considerable antioxidant capacity.57 Hence, the immune cell might use accumulation of zinc-saturated MT to elevate its tolerance against reactive oxygen species, while depriving the pathogen of zinc undermines the latter’s antioxidant capacity. Killing of phagocytosed pathogens involves an oxidative burst, i.e., the massive production of reactive oxygen and nitrogen species and changes in the intracellular zinc distribution may contribute to effective intracellular pathogen clearance.58 So far, it remains puzzling how a macrophage decides when to harm the pathogen by zinc deprivation and when to intoxicate it by excess zinc.58 Bacteria have evolved transporters to overcome zinc deprivation, including ABC family transporters and TonB-dependent transport systems such as ZnuD, as well as mechanisms against intoxication, such as P-type ATPases, cation diffusion facilitators (CDFs), and RND family transporters.48 Maybe the differential response of macrophages reflects an adapted reaction toward specific susceptibilities of the individual infectious agent, even though we do not yet know how such susceptibility would be identified by the macrophage.
Conclusion Altogether, recent discoveries provide an intriguing new look into the multifaceted immunobiology of zinc. They finally bring us closer to understanding the immunological changes resulting from zinc deficiency and supplementation, which were described many years ago. Zinc supplementation is certainly important to address zinc deficiency-induced immune dysfunction. However, therapeutic application of zinc as a pharmacological agent during infection seems to be more complex. All the different impacts of zinc on the immune system will have to be considered, in order to achieve a desired therapeutic effect without interfering with other zinc-dependent processes in the immune response.
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References 1 L. Rink, Zinc in Human Health, IOS Press, Amsterdam, 2011. 2 A. S. Prasad, A. Miale, Jr., Z. Farid, H. H. Sandstead and A. R. Schulert, J. Lab. Clin. Med., 1963, 61, 537–549. 3 M. Y. Yakoob, E. Theodoratou, A. Jabeen, A. Imdad, T. P. Eisele, J. Ferguson, A. Jhass, I. Rudan, H. Campbell, R. E. Black and Z. A. Bhutta, BMC Public Health, 2011, 11(suppl 3), S23. 4 E. John, T. C. Laskow, W. J. Buchser, B. R. Pitt, P. H. Basse, L. H. Butterfield, P. Kalinski and M. T. Lotze, J. Transl. Med., 2010, 8, 118. 5 H. Haase and L. Rink, BioFactors, 2013, DOI: 10.1002/biof.1114. 6 C. Andreini, I. Bertini and A. Rosato, Acc. Chem. Res., 2009, 42, 1471–1479. 7 M. Brylinski and J. Skolnick, Proteins, 2011, 79, 735–751. 8 P. J. Fraker and L. E. King, Annu. Rev. Nutr., 2004, 24, 277–298. 9 N. Wellinghausen and L. Rink, J. Leukocyte Biol., 1998, 64, 571–577. 10 A. S. Prasad, J. Trace Elem. Med. Biol., 2012, 26, 66–69. 11 E. Rosenkranz, A. S. Prasad and L. Rink, Immunobiology and Hematology of Zinc, in Zinc in Human Health, ed. L. Rink, IOS Press, Amsterdam, 2011, pp. 195–233. 12 N. Wellinghausen, H. Kirchner and L. Rink, Immunol. Today, 1997, 18, 519–521. 13 S. E. Turvey and D. H. Broide, J. Allergy Clin. Immunol., 2010, 125, S24–S32. 14 T. Fukada, S. Yamasaki, K. Nishida, M. Murakami and T. Hirano, J. Biol. Inorg. Chem., 2011, 16, 1123–1134. 15 H. Haase and L. Rink, Annu. Rev. Nutr., 2009, 29, 133–152. 16 T. B. Aydemir, J. P. Liuzzi, S. McClellan and R. J. Cousins, J. Leukocyte Biol., 2009, 86, 337–348. 17 J. Kaltenberg, L. M. Plum, J. L. Ober-Blobaum, A. Honscheid, L. Rink and H. Haase, Eur. J. Immunol., 2010, 40, 1496–1503. 18 M. Yu, W. W. Lee, D. Tomar, S. Pryshchep, M. CzesnikiewiczGuzik, D. L. Lamar, G. Li, K. Singh, L. Tian, C. M. Weyand and J. J. Goronzy, J. Exp. Med., 2011, 208, 775–785. 19 K. Kabu, S. Yamasaki, D. Kamimura, Y. Ito, A. Hasegawa, E. Sato, H. Kitamura, K. Nishida and T. Hirano, J. Immunol., 2006, 177, 1296–1305. 20 K. Nishida, A. Hasegawa, S. Nakae, K. Oboki, H. Saito, S. Yamasaki and T. Hirano, J. Exp. Med., 2009, 206, 1351–1364. 21 A. Brieger, L. Rink and H. Haase, J. Immunol., 2013, 191, 1808–1817. 22 H. Haase, J. L. Ober-Blobaum, G. Engelhardt, S. Hebel, A. Heit, H. Heine and L. Rink, J. Immunol., 2008, 181, 6491–6502. 23 H. Kitamura, H. Morikawa, H. Kamon, M. Iguchi, S. Hojyo, T. Fukada, S. Yamashita, T. Kaisho, S. Akira, M. Murakami and T. Hirano, Nat. Immunol., 2006, 7, 971–977. 24 M. J. Liu, S. Bao, M. Galvez-Peralta, C. J. Pyle, A. C. Rudawsky, R. E. Pavlovicz, D. W. Killilea, C. Li, D. W. Nebert, M. D. Wewers and D. L. Knoell, Cell Rep., 2013, 3, 386–400. 25 M. Wilson, C. Hogstrand and W. Maret, J. Biol. Chem., 2012, 287, 9322–9326. 26 L. A. Lichten and R. J. Cousins, Annu. Rev. Nutr., 2009, 29, 153–176.
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