Role of zinc insufficiency in fetal alveolar macrophage dysfunction and RSV exacerbation associated with fetal ethanol exposure

Abstract

Background: We previously reported that maternal alcohol use significantly increases the risk of sepsis in premature and term newborns. In the mouse, fetal ethanol exposure results in an immunosuppressed phenotype for the alveolar macrophage (AM) and decreases bacterial phagocytosis. In pregnant mice, ethanol decreased AM zinc homeostasis, which contributed to immunosuppression and impaired AM phagocytosis. In this study, we explored whether ethanol-induced zinc insufficiency extended to the pup AMs and contributed to immunosuppression and exacerbated viral lung infections.

Methods: C57BL/6 female mice were fed a liquid diet with 25% ethanol-derived calories or pair-fed a control diet with 25% of calories as maltose-dextrin. Some pup AMs were treated in vitro with zinc acetate before measuring zinc pools or transporter expression and bacteria phagocytosis. Some dams were fed additional zinc supplements in the ethanol or control diets, and then we assessed pup AM zinc pools, zinc transporters, and the immunosuppressant TGFβ1. On postnatal day 10, some pups were given intranasal saline or respiratory syncytial virus (RSV), and then AM RSV phagocytosis and the RSV burden in the airway lining fluid were assessed.

Results: Fetal ethanol exposure decreased pup AM zinc pools, zinc transporter expression, and bacterial clearance, but in vitro zinc treatments reversed these alterations. In addition, the expected ethanol-induced increase in TGFβ1 and immunosuppression were associated with decreased RSV phagocytosis and exacerbated RSV infections. However, additional maternal zinc supplements blocked the ethanol-induced perturbations in the pup AM zinc homeostasis and TGFβ1 immunosuppression, thereby improving RSV phagocytosis and attenuating the RSV burden in the lung.

Conclusion: These studies suggest that, despite normal maternal dietary zinc intake, in utero alcohol exposure results in zinc insufficiency, which contributes to compromised neonatal AM immune functions, thereby increasing the risk of bacterial and viral infections.

Keywords: Alveolar macrophage; Fetal ethanol exposure; Phagocytic function; Zinc insufficiency; Zinc transporters.

Figure 1.. Fetal ethanol exposure decreased intracellular zinc levels in the AMs from newborn pups but was restored by zinc treatment.

Dams were fed the control or ethanol diet during pregnancy and the AMs from the pups were isolated by lavage on the first day of life. The AM were cultured for 16 h with some AMs treated with media containing 25 μM zinc acetate. After incubation, FluoZin-3AM was added to the media (30 min.) before the cells were washed and fixed for confocal microscopy. RFUs were quantified by computerized analysis of the confocal fluorescent images and the bar heights represent the mean RFU/field ± S.E.M. from at least 6 different litters (A). Representative fluorescent images are shown for each condition (B). CTRL = control group; CTRL + Zn = control AMs treated in vitro with zinc acetate; EtOH = ethanol group; and EtOH + Zn = ethanol AMs treated in vitro with zinc acetate. *** denotes p = 0.05 when compared to the CTRL group and ** denotes p ≤ 0.05 when comparing the ETOH and ETOH + Zn groups.

Figure 2.. Fetal ethanol exposure decreased Zip1 protein expression in the AMs from newborn pups but expression was restored by zinc treatment.

On the first day of life, the AMs from the pups (± fetal ethanol exposure) were isolated by lavage and cultured for 16 h. Zip1 protein expression in the AMs was determined via immunostaining and quantified using confocal fluorescent microscopy plus ImagePro Plus analysis. Bar heights represent mean RFU/field ± S.E.M. from at least 6 different litters per group (A). Representative fluorescent images are shown for each condition (B). CTRL = control group; CTRL + Zn = control AMs treated in vitro with zinc acetate; EtOH = ethanol group; and EtOH + Zn = ethanol AMs treated in vitro with zinc acetate. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.5 EtOH + Zn vs. EtOH.

Figure 3.. Fetal ethanol exposure decreased ZnT1 protein expression in the AMs from newborn pups but was restored by zinc treatment.

Protein expression of zinc transporter, ZnT1, in alveolar macrophages isolated from pups was determined via immunostaining and quantified using fluorescent microscope and ImagePro Plus analysis. Bar heights represent mean RFU/field ± S.E.M. from at least 5 different litters per group (A). Representative fluorescent images are shown for each condition (B). CTRL = control group; CTRL + Zn = control AMs treated in vitro with zinc acetate; EtOH = ethanol group; and EtOH + Zn = ethanol AMs treated in vitro with zinc acetate. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.05 EtOH + Zn vs. EtOH.

Figure 4.. Fetal ethanol exposure decreased ZnT4 protein expression in the AMs from newborn pups but expression was restored by zinc treatment.

Protein expression of zinc transporter ZnT4 was determined and quantified similar to other transporters. Bar heights represent mean RFU/field ± S.E.M. from at least 5 different litters per group (A). Representative fluorescent images are shown for each condition (B). CTRL = control group; CTRL + Zn = control AMs treated in vitro with zinc acetate; EtOH = ethanol group; and EtOH + Zn = ethanol AMs treated in vitro with zinc acetate. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.05 EtOH + Zn vs. EtOH.

Figure 5.. The bacterial phagocytic index in the AMs from the newborn pups was decreased by fetal ethanol exposure but in vitro zinc treatments restored clearance.

Inactivated TRITC-labeled Staphylococcus aureus bacteria was added to the cells and incubated for another 4 h. Internalization of TRITC-labeled S. aureus was determined using confocal fluorescent analysis. Phagocytic index (PI) was calculated as the percentage of cells with internalized fluorescence × the mean RFU per field. Bar heights represent mean PI relative to the control ± S.E.M. from at least 5 separate litters (A). Representative fluorescent images are shown for each condition (B). CTRL = control group; CTRL + Zn = AMs treated in vitro with zinc acetate; EtOH = ethanol group; and EtOH + Zn = ethanol AMs treated in vitro with zinc acetate. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.05 EtOH + Zn vs. EtOH.

Figure 6.. Fetal ethanol exposure decreased the relative gene expression of Zip1 and ZnT1 in the AMs from the newborn pups but these decreases were blocked by maternal zinc supplements.

Dams were fed the control or ethanol diets ± zinc acetate supplements (100 mg/L). On day 10 of life, the AMs from the pups (± fetal ethanol exposure) were isolated by lavage and the relative gene expression of the zinc transporters Zip1 and ZnT1 was determined. The average relative gene expression of Zip1 and ZnT1 normalized to GUSB were determined by the comparative method (2^−ΔΔCt), with the target gene expression in control AM set as 1 in each case. Each sample was analyzed in duplicate and each value represents the mean ± SEM of at least four separate litters. CTRL = pup AM from control dams; CTRL + Zn = pup AM from control dams with dietary zinc supplements; EtOH = pup AM from ethanol-fed dams; and EtOH + Zn = pup AM from ethanol-fed dams with dietary zinc supplements. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.05 EtOH + Zn vs. EtOH.

Figure 7.. Maternal zinc supplements maintained the neonatal AM zinc pools despite fetal ethanol exposure.

Dams were fed the control or ethanol diets ± zinc acetate supplements (100 mg/L) during pregnancy. On day 10 of life, the AMs from the pups (± fetal ethanol exposure) were isolated by lavage and FluoZin-3AM was added to the incubation media (45 min; 37°C). After a 30 min incubation with medium free of the fluorophore, the cells were fixed and fluorescence determined by confocal fluorescent microscopy. RFUs were quantified by computerized analysis and expressed relative to the values for the control group. Bar heights represent the mean RFU/field ± S.E.M. from at least 4 different litters (A). Representative fluorescent images are shown for each condition (B). CTRL = pup AM from control dams; CTRL + Zn = pup AM from control dams with dietary zinc supplements; EtOH = pup AM from ethanol-fed dams; and EtOH + Zn = pup AM from ethanol-fed dams with dietary zinc supplements. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.05 EtOH + Zn vs. EtOH.

Figure 8.. Fetal ethanol exposure increased the relative gene and protein expression of the immunosuppressant TGFβ1 in the AMs from the newborn pups but was blocked by maternal zinc supplements.

Dams were fed the control or ethanol diets ± zinc acetate supplements (100 mg/L) during pregnancy. On day 10 of life, the AMs from the pups (± fetal ethanol exposure) were isolated by lavage and the relative gene expression of TGFβ1 was determined (A). The average relative gene expression of TGFβ1 normalized to GUSB was determined by the comparative method (2^−ΔΔCt), with the target gene expression in control AM set as 1 in each case. Each analysis was performed in duplicate and each value represents the mean ± SEM of at least four separate litters. Protein expression of TGFβ1 was determined and quantified similar to other transporters. Bar heights represent mean RFU/field ± S.E.M. from at least 5 different litters per group (B). Representative fluorescent images for protein expression are shown for each condition (C). CTRL = pup AM from control dams; CTRL + Zn = pup AM from control dams with dietary zinc supplements; EtOH = pup AM from ethanol-fed dams; and EtOH + Zn = pup AM from ethanol-fed dams with dietary zinc supplements. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.01 EtOH + Zn vs. EtOH.

Figure 9.. The phagocytic index for RSV was decreased in the AMs from the ethanol-exposed pups but this decrease was blocked by maternal zinc supplements.

Dams were fed the control or ethanol diets ± zinc acetate supplements (100 mg/L) during pregnancy. On post-natal day 10, some pups (± ethanol and ± maternal zinc supplement) were given intranasal injections of saline or RSV (20 μl; each nasal nare; 2 × 10⁵ PFU) and then returned to their respective dams. After 48 h, the pups were sacrificed and the lungs lavaged. After the cells in the lavage were removed by centrifugation and cultured on slides, they were fixed with 3.7% paraformaldehyde, permeabilized with ice-cold methanol, and then incubated with the primary antibody (1 h; a 1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After washing the cells, AM were incubated with the secondary antibody (anti-goat IgG; a 1:200 dilution; 45 min). RSV in the cell was determined using confocal fluorescent analysis. Phagocytic index (PI) was calculated as the percentage of cells with internalized fluorescence × the mean RFU per field. Bar heights represent mean PI relative to the control ± S.E.M. from at least 6 separate litters (A). Representative fluorescent images are shown for each condition (B). CTRL = pup AM from control dams; CTRL + Zn = pup AM from control dams with dietary zinc supplements; EtOH = pup AM from ethanol-fed dams; and EtOH + Zn = pup AM from ethanol-fed dams with dietary zinc supplements. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.01 EtOH + Zn vs. EtOH.

Figure 10.. Fetal ethanol exposure exacerbated lung RSV growth but this increase was blocked by maternal zinc supplements.

Dams were fed the control or ethanol diets ± zinc acetate supplements (100 mg/L) during pregnancy. On post-natal day 10, some pups (± ethanol and ± maternal zinc supplement) were given intranasal injections of saline or RSV (20 μl; each nasal nare; 2 × 10⁵ PFU) and then returned to their respective dams. After 48 h, the pups were sacrificed and the lungs lavaged. After the cells in the lavage were removed by centrifugation, the cell-free supernatant was then plated for viral PFU determination (6 different litters). CTRL = pup AM from control dams; CTRL + Zn = pup AM from control dams with dietary zinc supplements; EtOH = pup AM from ethanol-fed dams; and EtOH + Zn = pup AM from ethanol-fed dams with dietary zinc supplements. *** denotes p ≤ 0.05 when compared to the CTRL group and ** denotes p ≤ 0.01 EtOH + Zn vs. EtOH.