Research article
Increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation

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Abstract

Aging is a complex process associated with physiological changes in numerous organ systems. In particular, aging of the immune system is characterized by progressive dysregulation of immune responses, resulting in increased susceptibility to infectious diseases, impaired vaccination efficacy and systemic low-grade chronic inflammation. Increasing evidence suggest that intracellular zinc homeostasis, regulated by zinc transporter expression, is critically involved in the signaling and activation of immune cells. We hypothesize that epigenetic alterations and nutritional deficits associated with aging may lead to zinc transporter dysregulation, resulting in decreases in cellular zinc levels and enhanced inflammation with age. The goal of this study was to examine the contribution of age-related zinc deficiency and zinc transporter dysregulation on the inflammatory response in immune cells. The effects of zinc deficiency and age on the induction of inflammatory responses were determined using an in vitro cell culture system and an aged mouse model. We showed that zinc deficiency, particularly the reduction in intracellular zinc in immune cells, was associated with increased inflammation with age. Furthermore, reduced Zip 6 expression enhanced proinflammatory response, and age-specific Zip 6 dysregulation correlated with an increase in Zip 6 promoter methylation. Furthermore, restoring zinc status via dietary supplementation reduced aged-associated inflammation. Our data suggested that age-related epigenetic dysregulation in zinc transporter expression may influence cellular zinc levels and contribute to increased susceptibility to inflammation with age.

Introduction

Aging of the immune system results in a progressive dysregulation of immune responses including immunosenescence, where there is a gradual decline in both cellular and humoral immune responses, and increased susceptibility to infectious diseases and compromised vaccination efficacy in the elderly [1]. At the same time, “inflamm-aging,” a low-grade systemic chronic inflammation characterized by constitutively elevated levels of proinflammatory cytokines in blood, is commonly observed in the elderly population [2], [3]. Chronic inflammation has been implicated in the promotion of many age-related diseases including cancer, cardiovascular disease and autoimmune diseases. In addition, increases in inflammatory mediators in the blood are significant predictors of morbidity and mortality in aged individuals [4], [5], [6].

Zinc is an essential micronutrient required for many cellular processes, especially for the normal development and function of the immune system [7], [8], [9]. National surveys indicate that a significant portion of the aged population has inadequate zinc intake [10], [11], [12], [13], and a decline in zinc status, as shown by plasma zinc concentrations, is observed with increasing age [14], [15], [16], [17]. There are remarkable similarities between the hallmarks of zinc deficiency and age-related immunological dysfunction, both characterized by impaired immune responses and systemic chronic inflammation. Thus age-related zinc deficiency may play a significant role in age-associated dysregulation of immune function and may be a contributing factor in age-related inflammation and associated morbidities [18], [19]. Importantly, zinc has anti-inflammatory properties and low zinc status is associated with increased susceptibility to infections and exaggerated inflammatory responses [20], [21], [22], [23]. Recent studies indicate that intracellular zinc homeostasis is critically involved in the signaling events in immune cells, and the regulation of cellular zinc in these immune cells is mediated by changes in the expression of specific zinc transporters [24], [25], [26]. Zinc transporters comprises a family of multiple transmembrane spanning domain proteins that are encoded by two solute-linked carrier (SLC) gene families: SLC30 (ZnT) and SLC39 (Zip) [27], [28]. ZnT and Zip family zinc transporters have opposing roles in regulating cellular zinc homeostasis; ZnT transporters reduce cytosolic zinc bioavailability by promoting zinc efflux and Zip transporters function by increasing cytosolic zinc. In the context of inflammation, stimulation of immune cells with inflammatory stimuli such as lipopolysaccharide (LPS) results in changes in cellular zinc that is mediated by alterations in zinc transporter expression [29]. Thus alterations and/or dysregulation of zinc transporter expression with age could potentially affect zinc homeostasis in immune cells and contribute to immune dysfunction and chronic inflammation [18], [30].

The mechanisms contributing to age-related zinc loss and age-related inflammation are unclear. Accumulating evidence indicates that epigenetic dysregulation is a common feature of aging, characterized by global DNA hypomethylation and gene-specific promoter hypermethylation or hypomethylation, as well as alteration in histone modifications [31], [32], [33], [34]. In the immune system, age-associated epigenetic modifications such as DNA methylation have been shown to affect immune cell activation and may also contribute to the decline of cellular zinc with age, as several zinc transporters have been shown to be susceptible to epigenetic regulation [35], [36], [37], [38]. At the same time, nutrient deficits such as zinc deficiency may further modulate epigenetic regulation [39], [40], [41]. The goal of the current study was to examine the contribution of age-related zinc deficiency and zinc transporter dysregulation on the inflammatory response in immune cells using an in vitro cell culture system and an aged mouse model. We hypothesized that age-related decreases in cellular zinc levels, in part, are mediated by epigenetic alterations that result in zinc transporter dysregulation and contribute to enhanced inflammation with age. Moreover, enhancing zinc status in aged mice should mitigate age-related inflammation.

Section snippets

Cell culture, in vitro zinc depletion and LPS treatments

Human monocytic cell line THP-1 was obtained from American Type Tissue Collection (Manassas, VA, USA). Cells were grown in RPMI 1640 culture medium with 10% fetal bovine serum (FBS) and maintained in humidified incubators with 5% CO2 at 37°C. Zinc-deficient (ZD) media were prepared, as previous published, using a chelation strategy in which zinc was removed from FBS by incubating with 10% Chelex 100 (wt/vol) (Sigma, St. Louis, MO, USA) overnight at 4°C with constant stirring [42]. THP-1 cells

Proinflammatory response is associated with reduced intracellular zinc and is enhanced by zinc deficiency

The effects of zinc deficiency on inflammation were determined in THP-1 cells in vitro. After 10 days of culture in ZD media, THP-1 cells had significantly lower total zinc compared to cells cultured in ZA media (Fig. 1A). In addition, stimulation of THP-1 cells with LPS (Toll-like receptor 4 agonist) significantly reduced intracellular zinc levels in ZA THP1 cells (Fig 1B). The intracellular zinc levels in ZD THP-1 cells remained significantly lower compared to ZA cells, both pre- and post-LPS

Discussion

Declining zinc status during aging may contribute to immune dysfunction and chronic inflammation, and many age-related health problems are conditions that are also associated with poor zinc status [30], [49]. While zinc is known to act as an anti-inflammatory agent, however to date, the precise mechanisms linking zinc, age and inflammation are unclear. Our study is the first to report the potential role of age-related epigenetic control on zinc homeostasis. Specifically, these studies provide

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    Funding support: Oregon Agricultural Experiment Station (OR00735), Environmental Health Science Center at Oregon State University (NIEHS P30 ES00210), Oregon State University General Research Fund, Linus Pauling Institute, and National Institute on Aging, NIH (R01 AG016322).

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