In this report, we describe a role for zinc supplementation in modulating the innate immune response to a common allergen in vivo
which is characterized by cellular infiltration and cytokine release into the airways of mice. In addition, we found that zinc supplementation is sufficient to decrease airway hyperresponsiveness and serum IgE levels suggesting an important therapeutic treatment. Our data suggests that zinc works to temper inflammation at the level of A20 within the NF-κB pathway. A20 is a zinc-fingered protein that is a well-known inhibitor of the NF-κB pathway and was thus a good potential target for the location of zinc's anti-inflammatory effect within this pathway. In our study, we found that zinc supplementation, when added concurrently with GC frass, did not affect A20 levels in the short term (4 h), but resulted in decreased A20 protein levels at 18 h. We postulate that this was most likely due to transcriptional regulation with the enhancement of the NF-κB negative feedback loop and inhibition of continued activation of NF-κB, thus eventually decreasing A20 protein production. It has been shown that zinc can induce increased A20 levels in the presence of a variety of stimuli including LPS [18
] and phorbol myristate acetate (PMA) [19
]. These experiments differed from ours in that media containing zinc was added to cells for 10 days prior to stimulation, but they support our interpretation that zinc regulates A20 to alter NF-κB activity.
Our results indicate a potential mechanism by which zinc regulates NF-κB activity; i.e. by enhancing the activity of A20 thus resulting in de-ubiquitination of RIP1 and leading to its degradation. The ubiquitin editing abilities of A20 were recently examined and better defined. Shembade et. al. [5
] showed that A20 removes the activating chains of ubiquitin from locus K63 on RIP1 and facilitates the addition of inhibitory chains to locus K48, targeting RIP1 for degradation and thus preventing the activation of IKK downstream. They also showed that A20 inhibits the polyubiquitinization and activation of TRAF6, a process that is dependent on Ubc13 enzymatic activity. Ubc13 is also ubiquitinated at K48 by A20 and targeted for degradation, but this occurs late after stimulation. TRAF6 is then rendered inactive, though not degraded [5
]. Utilizing these findings, we were able to evaluate A20 activity using RIP1 and TRAF6 protein levels as a surrogate. With respect to A20 activity, we were able to show that RIP1 was degraded more quickly in the presence of zinc gluconate after stimulation with GC frass, but TRAF6 levels remained unchanged. These findings suggest that the addition of zinc gluconate augments the activity of A20; enabling it to act more rapidly on RIP1 resulting in its degradation and thus ceasing the NF-κB inflammatory cascade more quickly. A limitation of the current study was our evaluation of A20 activity via RIP1 as an indirect assessment; however our studies clearly show that in the presence of zinc, RIP1 degradation is enhanced, suggesting an increased proteosomal targeting due to altered ubiquitin states. Our findings suggest that zinc is acting on the NF-κB pathway at the level of A20 to further enhance its inhibitory effects.
In this study we also wanted to investigate zinc as an anti-inflammatory with potential therapeutic utility for airway inflammation. Asthma is a chronic inflammatory disease of the airways and is characterized by increased mucus production and airway hyperresponsiveness. Several studies have shown a role for increased NF-κB in asthmatics, including increased NF-κB p65 protein abundance, IκBα phosphorylation and IKK activity in peripheral blood mononuclear cells (PBMC) of uncontrolled asthmatics compared to normal individuals [20
] and greater levels of NF-κB p65 and p50 activation in cultured bronchial epithelial cells from untreated asthmatics than controls [21
]. NF-κB was also found to be upregulated in bronchial epithelial cells a murine model of ovalbumin-induced allergic airway inflammation [22
]. The importance of NF-κB signaling in allergic inflammation and mucus production was shown using mice with IKK-deficient Clara epithelial cells [23
]. In this study, we found a significant reduction in airway hyperresponsiveness in our murine model by administering zinc gluconate for three days following the final allergen exposure. Importantly, the levels of serum IgE were substantially decreased following zinc administration. IgE is known to bind to specific Fcε receptors on mast cells thus result in release of histamine and other inflammatory substances to produce an allergic cascade. Zinc supplementation did not appear to have any effect on levels of the Th2 cytokines IL-13, IL-5 and IL-4. Much of the literature on zinc and asthma has focused primarily on levels of dietary zinc; however Lang et. al. [24
] recently showed that zinc supplementation following establishment of allergic inflammation reduced the levels of eosinophils and lymphocytes in the BAL fluid of mice. They failed to find a significant difference in the amount of mucus hyperplasia, determined by PAS staining, following zinc administration however. We also found decreased numbers of eosinophils and neutrophils in the BAL fluid of mice; however unlike neutrophil levels, the decrease in eosinophil levels did not reach statistical significance. It is possible that the amount of times zinc is administered (i.e. 3 times in our protocol compared to twice a week from days 34-52 in the Lang protocol) was responsible for the different results. To our knowledge, this is the first report showing that zinc supplementation can modulate airway responsiveness to cholinergic agents.
The literature on zinc as a potent antioxidant is vast but what role this may have in airway inflammation remains unclear. Recent studies have looked at abnormal distributions of trace minerals, including zinc, as an instigator of oxidative damage and inflammation in asthma [25
]. Prasad et. al. [18
] investigated the role of zinc as an antioxidant and after supplementation to human subjects, noted a decrease in oxidative stress and NF-κB activation in isolated mononuclear cells when compared to placebo. TNFα and IL-1β are inflammatory cytokines known to exert oxidative stress on cells via the generation of reactive oxygen species (ROS). In their study, they also noted a decrease in TNFα and IL-1β cytokine levels in their volunteers who took zinc supplements, suggesting that one way in which zinc functions as an antioxidant is by negatively regulating the gene expression of these inflammatory cytokines to prevent the formation of ROS [18
]. Thus, zinc's role as an antioxidant likely works concurrently with its anti-inflammatory role via similar or overlapping biochemical mechanisms. Similar to Prasad et. al., we were able to show that zinc negatively inhibits NF-κB-induced cytokine production. We acknowledge that some of the positive effects noted on airway hyperresponsivity and inflammation may have been an antioxidant effect of zinc supplementation. However, we continue to promote the supplementation of zinc as a therapeutic to temper the inflammation as well as the oxidative stress induced by allergic stimuli in the airways.