Zinc supplementation alters airway inflammation and airway hyperresponsiveness to a common allergen

Abstract

Background: Zinc supplementation can modulate immunity through inhibition of NF-κB, a transcription factor that controls many immune response genes. Thus, we sought to examine the mechanism by which zinc supplementation tempers the response to a common allergen and determine its effect on allergic airway inflammation.

Methods: Mice were injected with zinc gluconate prior to German cockroach (GC) feces (frass) exposure and airway inflammation was assessed. Primary bone marrow-derived neutrophils and DMSO-differentiated HL-60 cells were used to assess the role of zinc gluconate on tumor necrosis factor (TNF)α expression. NF-κB:DNA binding and IKK activity were assessed by EMSA and in vitro kinase assay. Protein levels of A20, RIP1 and TRAF6 were assessed by Western blot analysis. Establishment of allergic airway inflammation with GC frass was followed by administration of zinc gluconate. Airway hyperresponsiveness, serum IgE levels, eosinophilia and Th2 cytokine production were assessed.

Results: Administration of zinc gluconate prior to allergen exposure resulted in significantly decreased neutrophil infiltration and TNFα cytokine release into the airways. This correlated with decreased NF-κB activity in the whole lung. Treatment with zinc gluconate significantly decreased GC frass-mediated TNFα production from bone-marrow derived neutrophils and HL-60 cells. We confirmed zinc-mediated decreases in NF-κB:DNA binding and IKK activity in HL-60 cells. A20, a natural inhibitor of NF-κB and a zinc-fingered protein, is a potential target of zinc. Zinc treatment did not alter A20 levels in the short term, but resulted in the degradation of RIP1, an important upstream activator of IKK. TRAF6 protein levels were unaffected. To determine the application for zinc as a therapeutic for asthma, we administered zinc following the establishment of allergic airway inflammation in a murine model. Zinc supplementation decreased airway hyperresponsiveness and serum IgE levels, but had no effect on Th2 cytokine expression.

Conclusions: This report suggests that the mechanism by which zinc supplementation alters NF-κB activity is via the alteration of A20 activity. In addition, this study provides evidence that supplementation of zinc to asthmatics may alter airway reactivity and serum IgE levels, suggesting zinc supplementation as a potential treatment for asthmatics.

Figure 1

Mouse treatment protocols. A. Zinc supplementation followed by GC frass exposure. B. Allergen challenge with GC frass followed by zinc supplementation.

Figure 2

Zinc gluconate decreased airway inflammation in vivo. Naïve BALB/c mice were given an i.p. injection of PBS or zinc gluconate (0.1, 1, or 10 mg/kg) once a day for 3 days prior to a single i.t. inhalation of GC frass (40 μg/40 μl). 18 h later, BAL fluid was harvested, infiltrated cells counted and BAL fluid was analyzed by ELISA. In all cases means ± SEM are shown (n = 6 mice per group). A. Total cells in BAL fluid (different from PBS *p < 0.001; different from GC frass treated **p < 0.05). B. TNFα ELISA (different from PBS * < 0.001; different from GC frass treated without zinc supplementation **p < 0.05, as determined by ANOVA).

Figure 3

Zinc supplementation decreased NF-κB-DNA binding in whole lung. Mice were given daily i.p. injections of PBS or zinc gluconate (10 mg/kg) for 3 days prior to a single i.t. inhalation of PBS or GC frass (40 μg/40 μl). 4 h later, whole lungs were removed and snap frozen. Lungs were homogenized, nuclear extracts isolated and analyzed by EMSA. NF-κB-DNA binding is shown, top arrows designate supershift of p65 and p50. Data shown are representative from a single mouse (n = 3 mice per group).

Figure 4

Zinc supplementation decreased neutrophil-derived cytokine production via decreased NF-κB activation. A. Bone marrow-derived neutrophils were isolated and treated with GC frass (1 μg/ml) or LPS (1 μg/ml) with or without zinc gluconate (1 μM) and pyrithione (10 μM) for 18 h. Supernatants were clarified and analyzed by ELISA for TNFα production. Data are means ± SEM (n = 3 separate experiments) and statistical differences assessed by ANOVA (*p < 0.05). B. HL-60 cells were treated as in A and analyzed by ELISA for TNFα production. Data are expressed as mean ± SEM for four experiments (*p < 0.001). C. Zinc supplementation decreased NF-κB-DNA binding. HL-60 cells were stimulated with GC frass or LPS in the presence of zinc and pyrithione for 4 h. Nuclear extracts were analyzed for NF-κB-DNA binding by EMSA. D. Zinc supplementation decreased IKK activity. HL-60 cells were cultured with or without zinc gluconate and pyrithione prior to treatment with GC frass for 1 h. IKK activation was assessed by in vitro kinase assay using recombinant IκBα as a substrate. PBS treated (P) and GC frass treated (F) cells cultured with and without zinc.

Figure 5

A20 protein levels following GC frass with or without zinc supplementation. HL-60 cells were treated with GC frass (1 μg/ml) or LPS (1 μg/ml) with or without zinc gluconate (1 μM) and pyrithione (10 μM) for 18 h (A) or for either 2 or 4 h (B). Cell lysates were harvested, separated by gel electrophoresis and probed with an antibody against A20 or β-actin. Each experiment was performed 3 times.

Figure 6

RIP1 and TRAF6 protein levels following GC frass with or without zinc supplementation. HL-60 cells were treated with GC frass (1 μg/ml) or LPS (1 μg/ml) with or without zinc gluconate (1 μM) and pyrithione (10 μM) for times as indicated (0-4 hr). Cell lysates were harvested, separated by gel electrophoresis and probed with antibodies against RIP1, TRAF6 or β-actin. This experiment was performed 3 times.

Figure 7

Zinc supplementation decreased experimentally-induced allergic asthma in mice. BALB/c mice were sensitized on days 0, 7, and 14 with an intratracheal inhalation of PBS or GC frass (40 μg/40 μl). On day 14, 15, and 16 selected mice were administered i.p. injections of zinc gluconate (10 mg/kg) or PBS. On day 17, mice were anesthetized and acetylcholine was injected after establishment of a stable airway pressure. Blood was collected and BAL fluid was harvested. Lungs were isolated, and cultured for 3 days in the presence of ConA for cytokine analysis by ELISA. In all cases, means ± SEM (n = 6 mice per group) were reported and statistical significance was determined by one-way ANOVA. A. AHR was measured as airway pressure time index (APTI) in cm-H₂O × sec -1 (compared to PBS *p > 0.001; compared to GC frass **p = 0.034). B. Serum IgE levels (compared to PBS *p > 0.001; compared to GC frass **p > 0.001). C. IL-13 levels (compared to PBS; *p > 0.001). D. IL-5 levels (compared to PBS; *p > 0.001).