Chronic alcohol consumption increases the severity of murine influenza virus infections

Abstract

Respiratory infections with both seasonal as well as potential pandemic Influenza viruses represent a significant burden on human health. Furthermore, viruses such as Influenza are increasingly recognized as important etiologic agents in community acquired pneumonia. Within the U.S. alone, approximately 12.9 million people are heavy drinkers and chronic abuse of alcohol is known to increase the risk and severity of community acquired pneumonia. Given the lack of knowledge regarding Influenza disease in this population, we determined the effects of chronic alcohol consumption on Influenza virus infection. Herein, we report that mice exposed to chronic ethanol have sharp increases in morbidity, mortality, and pulmonary virus titers relative to controls. These increases in influenza severity correspond with inhibited pulmonary influenza-specific CD8 T cell responses. Further, chronic ethanol consumption results in an enhanced pulmonary lesion severity, similar to that recently described for pandemic influenzas. Together, our results suggest that chronic alcohol consumption may increase the risk for severe influenza virus infections by altering the pulmonary inflammatory environment and CD8 T cell response.

Figure 1

Chronic EtOH consumption increases the severity of influenza virus infections. EtOH (circle) and water (triangles) conditioned groups of BALB/c mice were infected with a 0.01LD₅₀ dose of influenza virus and then assessed for morbidity as measured by weight loss (A), mortality (B). One representative experiment of 2 (8–10 mice/group) is shown in (A, B).

Figure 2

Chronic EtOH consumption increases the magnitude and duration of influenza virus infections. EtOH (black bars) and water (open bars) conditioned groups of BALB/c mice were infected with a 0.01LD₅₀ dose of influenza virus and then assessed for pulmonary virus titer (A), and influenza nucleocapsid protein (NP) loads (B). Data are the mean ± sem of 5–9 mice/group.

Figure 3

Chronic EtOH inhibits pulmonary Influenza-specific CD8+ T cell immune responses. Four week water (open bars) or EtOH (closed bars) conditioned BALB/c mice were infected with influenza A virus. On day 8 p.i., the number of total pulmonary CD8 T cells (A) as well as the number (B, C) and percentage (D, E) of influenza-specific CD8⁺ T cells were determined by flow cytometry. Cells were identified by binding of influenza-specific MHC-tetramers (B, D) or by influenza-peptide (HA₂₀₄, HA₅₂₉, NP₁₄₇ or control RSV M2₈₀) induced intracellular IFNγ staining (C, E). One representative experiment of 2 (minimum of 4 mice/group, mice analyzed individually) is shown. Data are the mean ± sem. *=p<0.05

Figure 4

Inhibition of pulmonary Influenza-specific CD8⁺ T cell immune responses increases with the chronicity of EtOH consumption. Water (open bars) or EtOH (closed bars) conditioned C57Bl/6 mice were infected with influenza A virus. On day 8 p.i., the number of total pulmonary CD8 T cells (A, F) as well as the percentage (B, C, F, G) of influenza-specific CD8⁺ T cells were determined by flow cytometry (B, F: tetramers; C, G: influenza HA₂₀₄, HA₅₂₉, NP₁₄₇ vs. control RSV M2₈₀ peptide induced intracellular IFNγ staining). (D). Comparison of the pulmonary T cell response. The number of influenza-specific CD8 T cells from EtOH mice were each normalized to their corresponding water control, with the water value equal to 100. One representative experiment of 2–3 (minimum of 4 mice/group, mice analyzed individually) is shown. Data are the mean ± sem. *=p<0.05

Figure 5

Chronic EtOH consumption does not alter pulmonary CD8 T cell IFNγ production in response to PMA/ionomycin. 8 wk EtOH and water conditioned C57Bl/6 mice (5/group) were infected with influenza A virus. On day 8-post infection the lungs were removed and the ability of CD8⁺ T cells to produce IFNγ in response to PMA/ionomycin stimulation was determined by intracellular cytokine staining. (A) Representative plots showing the % of IFNγ⁺ cells of CD8⁺ gated cells. (B) Data are the mean % of IFNγ⁺ cells ± sem of CD8⁺ gated cells from the indicated groups of mice. One representative experiment of 2 (minimum of 4 mice/group, mice analyzed individually) is shown.

Figure 6

Chronic EtOH consumption increases the severity of the pulmonary lesions associated with influenza infections. On day 8 post influenza infection, 8 wk water-control C57Bl/6 mice (A,C) have localized multifocal consolidation (C, arrows) whereas ethanol mice (B,D) have more widespread and coalescing consolidation (D, *). Water mice had characteristic influenza lesions including necrotizing bronchiolitis (E) and moderate interstitial pneumonia (G). Ethanol mice had more pronounced inflammation including increased necrosis and numbers of inflammatory cells (F, arrow) and pulmonary edema (H, *). Sections were negative for bacterial involvement as modified gram stains were negative for bacteria (data not shown). 8 sections including 4 depths per lung were scored independently from 3 mice per group.

Figure 7

Chronic alcohol consumption increases neutrophil recruitment to the lungs. Lung, representative samples from 8-week water (A) and EtOH (B) conditioned C57Bl/6 mice eight days post influenza inoculation. Morphologically, the EtOH mice exhibited increased neutrophil (arrow) infiltration especially noted in a perivascular orientation with extension into alveolar air spaces (arrowhead) versus the typical mononuclear infiltration seen in water controls. (C) Quantification of neutrophils per high power field. Random foci in the lung were counted (60× hpf × 5), and the average neutrophils per high power field (hpf) tallied and the means for each group normalized to Day 5 water with a value of “1”. Water versus ethanol groups had significant differences in neutrophil infiltration (p<0.01, treatment effect, two way ANOVA) with significant posttest differences at Day 8 (p<0.05). 8 sections including 4 depths per lung were scored independently from 3 mice per group.