Fetal exposure to ethanol has long-term effects on the severity of influenza virus infections

Abstract

Alcohol use by pregnant women is a significant public health issue despite well-described risks to the fetus including physical and intellectual growth retardation and malformations. Although clinical studies are limited, they suggest that in utero alcohol exposure also results in significant immune deficiencies in naive neonates. However, little is known about fetal alcohol exposure (FAE) effects on adult infections. Therefore, to determine the long-term effects of FAE on disease susceptibility and the adult immune system, we infected FAE adult mice with influenza virus. In this study, we demonstrate that mice exposed to ethanol during gestation and nursing exhibit enhanced disease severity as well as increased and sustained pulmonary viral titers following influenza virus infection. Secondary exposure to alcohol as an adult further exacerbates these effects. Moreover, we demonstrate that FAE mice have impaired adaptive immune responses, including decreased numbers of virus-specific pulmonary CD8 T cells, a decreased size and frequency of pulmonary B cell foci, and reduced production of influenza-specific Ab following influenza infection. Together, our results suggest that FAE induces significant and long-term defects in immunity and susceptibility to influenza virus infection and that FAE individuals could be at increased risk for severe and fatal respiratory infections.

FIGURE 1

Model of fetal alcohol exposure. Female mice were placed on 5% EtOH prior to breeding, then 10% for the duration of breeding and gestation. Following birth, the EtOH concentration was increased to 12% until weaning. After weaning, pups were placed on water until their weight reached a minimum of 17 grams (∼8-10 weeks old), then they were divided into two groups one of which was returned to EtOH (a final concentration of 20%) (EtOH:EtOH). The other group remained on water (EtOH:Water). After 6 weeks at 20% EtOH, mice were then influenza virus infected. Pups from water fed dams (Water:Water) were used as controls.

FIGURE 2

FAE results in increased influenza associated morbidity and mortality. Water:Water, EtOH:Water and EtOH:EtOH mice were infected with a sublethal dose of mouse-adapted influenza A virus and monitored daily for morbidity (left) and mortality (right). Data are pooled from 2 separate experiments, n=13-17 mice/group. *p<0.05 morbidity: EtOH:Water mice compared to Water:Water controls; mortality, EtOH:water and EtOH:EtOH groups vs. Water:Water mice.

FIGURE 3

FAE results in increased pulmonary viral titers. (A) Water:Water, EtOH:Water and EtOH:EtOH mice were infected with influenza virus and their lungs examined on d 8 p.i. for pulmonary viral titers by an MDCK cell endpoint dilution infection assay. Data are pooled from two separate experiments and represented as mean ± SEM; n=8-15/group, *p<0.05 compared to Water:Water controls. (B) Groups of mice were infected with influenza virus as in A and their lungs inflated and fixed on d 4, 8, 10 and 14 p.i. Lungs were then sectioned and stained for influenza NP antigen as described in the Material and Methods. Data are representative of one to two separate experiments, n=4-7/group

FIGURE 4

Reduced pulmonary influenza-specific CD8 T cell responses in FAE mice. Water:Water, EtOH:Water and EtOH:EtOH mice were infected with influenza virus and their lungs were examined on d 8 p.i. for numbers of total (i.e. CD8a+ cells, left) and influenza-specific CD8 T cells by influenza-PA₂₂₄ and NP₃₆₆ peptide:MHC I tetramer binding (center) ex vivo or intracellular staining for IFNγ production (right) following a 6 hr incubation with PA₂₂₄ and NP₃₆₆ peptides as described in the Materials and Methods. Data are pooled from three separate experiments and represented as mean ± SEM; n=12-13/group; *p<0.05 compared to Water:Water controls

FIGURE 5

Reduced B cell immunity in FAE mice. (A) Lungs from influenza-infected Water:Water, EtOH:Water and EtOH:EtOH mice were inflated, fixed and stained for B220⁺ cells on d 10 and 14 p.i. Data are representative of one to two separate experiments, n=4-7/group (B) Water:Water, EtOH: Water and EtOH:EtOH mice were infected with influenza and the titer of influenza-specific antibody in the serum was determined by hemagglutination inhibition assay as described in the Materials and Methods on days 8 and 14 p.i. *p<0.05 compared to Water:Water controls. The HAI dilution represents the highest serum dilution capable of preventing influenza-virus hemagglutination of red blood cells.

FIGURE 6

Effects of FAE on influenza infection in older mice. 6 month old BALB/c EtOH:Water and Water:Water mice were infected with mouse-adapted influenza (A/JAPAN/305/57) and monitored daily for morbidity (A) and mortality (B). n=11-13/group; *p<0.05 compared to Water:Water controls