J Nutr Biochem. 2013 (Jan); 24 (1): 353359 ~ FULL TEXT
Carmen P. Wong, Kathy R. Magnusson, and Emily Hoa
School of Biological and Population Health Sciences,
Oregon State University,
OR 97331, USA.
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.
From the FULL TEXT Article
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 . At the same time, inflammaging, 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 .
Zinc is an essential micronutrient required for many cellular processes, especially for the normal development and function of the immune system . National surveys indicate that a significant portion of the aged population has inadequate zinc intake , and a decline in zinc status, as shown by plasma zinc concentrations, is observed with increasing age . 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 . 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 . 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 . 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 . 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 . At the same time, nutrient deficits such as zinc deficiency may further modulate epigenetic regulation . 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.
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 new evidence that suggests that methylation of specific zinc transporters with age may be one of the contributing factors resulting in zinc transporter dysregulation, and age-related zinc deficiency and inflammation. In this study, we demonstrated that zinc deficiency, particularly the reduction in intracellular zinc within immune cells, was associated with increased inflammation with increased age of the animal. We further showed that reduced Zip 6 mRNA expression enhanced proinflammatory responses, and age-specific Zip 6 dysregulation correlated with an increase in Zip 6 promoter methylation. Moreover, restoring zinc status via dietary supplementation reduced aged-associated inflammation. Our animal 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.
Recent studies indicate that intracellular zinc homeostasis is critically involved in the signaling events in immune cells . For example, T-cell receptor signaling and Th1 cell differentiation are controlled by activation-induced zinc influx during T-cell activation, and in DC, a reduction in intracellular zinc is required for DC maturation and antigen presentation [25, 29, 50]. In our in vitro study, LPS-stimulated proinflammatory response was accompanied by a significant reduction in intracellular zinc in THP-1 macrophages (Fig. 1). We further showed that reduced intracellular zinc in immune cells of aged mice was associated with increased inflammatory response (Fig. 2). Our data suggested that a decrease in cellular zinc was required for the induction of a proinflammatory response that was dysregulated with age and reaffirmed the importance of zinc homeostasis in controlling immune cell activation.
Zinc transporters play an important role in regulating cellular zinc homeostasis. Members of Zip and ZnT zinc transporter families exhibit tissue and cell-specific expression and possess differential responsiveness to dietary zinc, as well as to physiologic stimuli, including cytokines [27, 51]. The expression of zinc transporters has been profiled in immune cell types, and their regulation plays an important role in specific immune cell activation. Intracellular zinc homeostasis in different leukocyte subsets is regulated by distinct patterns of zinc exporter expression [52, 53]. Zinc transporter expression is important in controlling the activation and maturation of various immune cells [25, 29]. In particular, Zip 6 and ZnT 1 have been shown to be key transporters that control free zinc levels in immune cells. Intracellular zinc levels and Zip 6 are involved in the maturation of DC, and a reduction in cellular zinc or Zip 6 expression leads to enhanced antigen presentation and DC-mediated immune responses . We hypothesized that alteration and/or dysregulation of zinc transporter expression with age can potentially lead to age-associated decline in zinc status, aberrant immune activation and inflammation. We confirmed the role of Zip 6 in mediating the inflammatory response, where loss of Zip 6 mRNA expression by siRNA or with age resulted in an enhanced inflammatory response (Fig. 3).
Epigenetic alterations during aging are emerging as factors that can influence age-related processes, such as chronic inflammation. In the context of the immune system, epigenetic modifications including DNA methylation and histone modifications have been shown to control immune function . For example, Agrawal et al.  recently demonstrated that epigenetic modifications in aged human DNA increase its immunogenicity, resulting in increased DC activation, and may be a potential mechanism leading to age-associated increases in autoimmune and proinflammatory responses. In addition to age-related epigenetic modifications, specific nutrients can also modulate epigenetic regulation and alter disease susceptibility [39, 54, 55]. In particular, there is strong evidence for a role of zinc in DNA methylation [56, 57]. Zinc deficiency may cause methyl deficiency [58, 59], similar to other methyl donors like folate, resulting in altered methylation patterns, abnormal gene expression and developmental defects. Interestingly, several zinc transporters have been reported to be susceptible to epigenetic regulation [37, 38]. We hypothesized that age-related epigenetic modification of zinc transporters, particularly Zip 6, may contribute to Zip 6 dysregulation and the decline of zinc status with age. In our study, we determined changes in methylation status with age in immune cells and observed global DNA hypomethylation in the spleens of aged mice compared to young mice (Fig. 3). At the same time, DNA methylation status of CpG islands in the promoter region of Zip 6 was increased in the splenocytes of aged mice relative to young mice. Similar promoter hypermethylations were also observed in ZnT 1 and ZnT 5 (data not shown). Thus, age-related epigenetic alterations may represent one mechanism that contributes to age-related deficits in cellular zinc levels and enhanced inflammation. Preliminary results in zinc supplemented old mice with improved zinc status did not reveal changes in Zip 6 promoter methylation compared to old mice fed a ZA diet (data not shown). However, the methylation assay used in this report may not be sensitive enough to detect subtle changes in DNA methylation profile under dietary zinc supplementation conditions. Detailed characterization of the specific CpG residues within various zinc transporter promoter regions that are differentially methylated with age and zinc status is currently ongoing using methylation assays with improved sensitivity. Secondly, it is also possible that age-related epigenetic dysregulation of Zip6 is not reversible, but zinc supplementation simply overcomes losses in cellular zinc and exerts anti-inflammatory effects, despite continued dysregulation of methylation and zinc transporter expression. Taken together, our data suggested that age-related epigenetic alterations may contribute to deficits in cellular zinc levels in immune cells and enhance inflammation. Results from our animal studies will aid in providing rationale for future human studies that will help understand whether improving zinc status in the elderly will be helpful in correcting age-related immune defects and prevent chronic inflammation.
To date, the precise factors contributing to age-related zinc deficiency remain poorly defined. Results from the current study lend support to the hypothesis that age-related epigenetic modifications such as DNA methylation may be one of the contributing factors that lead to the dysregulation of zinc regulatory proteins such as zinc transporters. This may lead to impaired zinc utilization and age-associated decline in zinc status. The resulting zinc loss, particularly in immune cells, may subsequently contribute to immune dysfunction and enhanced inflammatory response. We further showed that improving zinc status in the aged mice via dietary zinc supplementation can overcome age-related chronic inflammation. Future studies will focus on further characterizing how age-related epigenetic modifications, either with age itself or in combination with age-related zinc deficiency, could impact key regulatory mechanisms that exacerbate intracellular zinc loss, immune dysregulation and chronic inflammation. Ultimately, these studies aid in the identification of factors that may influence zinc status and contribute to the promotion of inflammation-mediated disorders with age.