Essential Role of Zinc and Zinc Transporters in Myeloid Cell Function and Host Defense against Infection.

Zinc is an essential micronutrient known to play a vital role in host defense against pathogens. Diets that are deficient in zinc lead to impaired immunity and delayed recovery from and worse outcomes following infection. Sustained insufficient zinc intake leads to dysregulation of the innate immune response and increases susceptibility to infection whereas zinc supplementation in at-risk populations has been shown to restore host defense and reduce pathogen-related morbidity and mortality. Upon infection, zinc deficiency leads to increased pathology due to imbalance in key signaling networks that result in excessive inflammation and collateral tissue damage. In particular, zinc impacts macrophage function, a critical front-line cell in host defense, in addition to other immune cells. Deficits in zinc adversely impact macrophage function resulting in dysregulation of phagocytosis, intracellular killing, and cytokine production. An additional work in this field has revealed a vital role for several zinc transporter proteins that are required for proper bioredistribution of zinc within mononuclear cells to achieve an optimal immune response against invading microorganisms. In this review, we will discuss the most recent developments regarding zinc's role in innate immunity and protection against pathogen invasion.

Zinc Modulates Endotoxin-Induced Human Macrophage Inflammation through ZIP8 Induction and C/EBPß Inhibition.

Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-κB pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNFα, IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBPß, a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.

Zinc modulates the innate immune response in vivo to polymicrobial sepsis through regulation of NF-kappaB.

Zinc is an essential element that facilitates coordination of immune activation during the host response to infection. We recently reported that zinc deficiency increases systemic inflammation, vital organ damage, and mortality in a small animal model of sepsis. To investigate potential mechanisms that cause these phenomena, we used the same animal model and observed that zinc deficiency increases bacterial burden and enhances NF-kappaB activity in vital organs including the lung. We conducted further studies in the lung to determine the overall impact of zinc deficiency. At the molecular level, NF-kappaB p65 DNA-binding activity was enhanced by zinc deficiency in response to polymicrobial sepsis. Furthermore, expression of the NF-kappaB-targeted genes IL-1beta, TNFalpha, ICAM-1, and the acute phase response gene SAA1/2 were elevated by zinc deficiency. Unexpectedly, the amount of NF-kappaB p65 mRNA and protein was increased in the lung including alveolar epithelia of zinc-deficient mice. These events occurred with a significant and concomitant increase in caspase-3 activity within 24 h of sepsis onset in zinc-deficient mice relative to control group. Short-term zinc supplementation reversed these effects. Reconstitution of zinc deficiency in lung epithelial cultures resulted in similar findings in response to TNFalpha. Taken together, zinc deficiency systemically enhances the spread of infection and NF-kappaB activation in vivo in response to polymicrobial sepsis, leading to enhanced inflammation, lung injury, and, as reported previously, mortality. Zinc supplementation immediately before initiation of sepsis reversed these effects thereby supporting the plausibility of future studies that explore zinc supplementation strategies to prevent sepsis-mediated morbidity and mortality.

Zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis.

OBJECTIVE: Zinc deficiency is common among populations at high risk for sepsis mortality, including elderly, alcoholic, and hospitalized patients. Zinc deficiency causes exaggerated inflammatory responses to endotoxin but has not been evaluated during bacterial sepsis. We hypothesized that subacute zinc deficiency would amplify immune responses and oxidant stress during bacterial sepsis {lsqb;i.e., cecal ligation and puncture (CLP){rsqb; resulting in increased mortality and that acute nutritional repletion of zinc would be beneficial. DESIGN: Prospective, randomized, controlled animal study. SETTING: University medical center research laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: Ten-week-old, male, C57BL/6 mice were randomized into three dietary groups: 1) control diet, 2) zinc-deficient diet for 3 weeks, and 3) zinc-deficient diet for 3 weeks followed by oral zinc supplementation for 3 days (n = 35 per diet). Mice were then assigned to receive either CLP or sham operation (n = 15 each per diet). CLP and sham-operated treatment groups were further assigned to a 7-day survival study (n = 10 per treatment per diet) or were evaluated at 24 hours (n = 5 per treatment per diet) for signs of vital organ damage. MEASUREMENTS AND MAIN RESULTS: Sepsis mortality was significantly increased with zinc deficiency (90% vs. 30% on control diet). Zinc-deficient animals subject to CLP had higher plasma cytokines, more severe organ injury, including increased oxidative tissue damage and cell death, particularly in the lungs and spleen. None of the sham-operated animals died or developed signs of organ damage. Zinc supplementation normalized the inflammatory response, greatly diminished tissue damage, and significantly reduced mortality. CONCLUSIONS: Subacute zinc deficiency significantly increases systemic inflammation, organ damage, and mortality in a murine polymicrobial sepsis model. Short-term zinc repletion provides significant, but incomplete protection despite normalization of inflammatory and organ damage indices.

Zinc modulates cytokine-induced lung epithelial cell barrier permeability.

Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-alpha, interferon-gamma, and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and beta-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury.

Zinc modulates airway epithelium susceptibility to death receptor-mediated apoptosis.

The accelerated loss of lung epithelium through activation of extrinsic apoptosis is believed to play a causative role in lung pathogenesis. Previous investigations have shown that zinc is required to sustain lung epithelial cell viability under stress conditions and that depletion of intracellular zinc predisposes cells to apoptosis. In this investigation, we determined whether intracellular zinc deficiency enhanced the susceptibility of primary, differentiated cultures of human lung epithelium to death receptor-mediated apoptosis, leading to barrier dysfunction. Cultures obtained from multiple donors were exposed to stimuli that provoke death receptor-mediated apoptosis and depleted of intracellular zinc with a zinc-specific chelating agent. Transepithelial resistance, paracellular transport, caspase-8 and caspase-3 activity, and apoptosis were measured. Activation of extrinsic apoptosis or zinc chelation alone resulted in a nominal increase in caspase function and apoptosis without major evidence of barrier disruption. Activation of extrinsic apoptosis in addition to zinc depletion resulted in an abrupt decrease in transepithelial resistance, a substantial increase in apoptosis, and an increased paracellular leak. Cultures were rescued by supplementation with zinc sulfate. Further analysis revealed that exogenous zinc facilitates cell survival through activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. We conclude that intracellular zinc is a vital factor in lung epithelium that protects cells from death receptor-mediated apoptosis and barrier dysfunction.