Alcohol enhances type 1 interferon-α production and mortality in young mice infected with <i>Mycobacterium tuberculosis</i>

<div><p>In the current study, we used a mouse model and human blood samples to determine the effects of chronic alcohol consumption on immune responses during <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infection. Alcohol increased the mortality of young mice but not old mice with <i>Mtb</i> infection. CD11b+Ly6G+ cells are the major source of IFN-α in the lungs of <i>Mtb</i>-infected alcohol-fed young mice, and IFN-α enhances macrophage necroptosis in the lungs. Treatment with an anti-IFNAR-1 antibody enhanced the survival of <i>Mtb</i>-infected alcohol-fed young mice. In response to <i>Mtb</i>, peripheral blood mononuclear cells (PBMCs) from alcoholic young healthy individuals with latent tuberculosis infection (LTBI) produced significantly higher amounts of IFN-α than those from non-alcoholic young healthy LTBI+ individuals and alcoholic and non-alcoholic old healthy LTBI+ individuals. Our study demonstrates that alcohol enhances IFN-α production by CD11b+Ly6G+ cells in the lungs of young <i>Mtb</i>-infected mice, which leads to macrophage necroptosis and increased mortality. Our findings also suggest that young alcoholic LTBI+ individuals have a higher risk of developing active TB infection.</p></div

Absolute number of lung leukocyte populations in <i>Mtb</i>-infected alcohol and control diet-fed mice.

<p>Young control and alcohol diet-fed mice were infected with 50–100 CFU of aerosolized <i>Mtb</i>. At three months p.i., lungs from uninfected control and alcohol diet-fed mice and from <i>Mtb</i>-infected control and alcohol diet-fed mice were isolated. The absolute numbers of various leukocyte populations, namely, <b>a</b>. CD3+ <b>b</b>. CD4+ <b>c</b>. CD8+ <b>d</b>. CD11b+Ly6G+ <b>e</b>. CD11c+ <b>f</b>. CD11b+ <b>g</b>. CD11b+Ly6G- and <b>h</b>. CD3-NK1.1+ cells, per 10⁶ total lung cells were determined by flow cytometry. The data are representative of two independent experiments. Five mice per group were used for each independent experiment. The mean values, p-values and SEs are shown.</p

IFN-α reduces the survival of <i>Mtb</i>-infected alcohol diet-fed young mice.

<p>Control and alcohol diet-fed young mice were infected with 50–100 CFU of aerosolized <i>Mtb</i>. At three months p.i., some of the alcohol-fed <i>Mtb</i>-infected young mice were treated with either an anti-IFNAR-1 mAb or IgG1 isotype-matched control mAb (0.3 mg per mouse, starting 3 months p.i. every 4 days for 3 months). <b>a.</b> Survival rates of <i>Mtb</i>-infected alcohol-fed young mice treated with the anti-IFNAR-1 mAb or IgG1 isotype-matched control mAb. The data from two independent experiments were pooled. Five mice per group were used for each independent experiment. The survival curves were compared using the log-rank test (P<0.001). <b>b to d.</b> At three months p.i., <i>Mtb</i>-infected alcohol diet-fed mice were treated with either the anti-IFNAR-1 mAb or IgG1 isotype-matched control mAb. Lungs were isolated and formalin-fixed. Paraffin-embedded tissue sections were prepared, and hematoxylin and eosin staining was performed. <b>b.</b> A representative figure is shown. <b>c.</b> The % lung lesions and <b>d.</b> lesion area were calculated. The data from two independent experiments were pooled. Three mice per group were used for each independent experiment (n = 6). The mean values, p-values and SEs are shown.</p

IFN-α production in <i>Mtb</i>-infected young alcoholic mice is associated with the expression of molecules involved in necroptosis.

<p><b>a.</b> Young (one to two months of age) and old (17 to 22 months of age) mice were fed control and alcohol diets for one month as detailed in the methods section; then, they were infected with 50–100 CFU of aerosolized <i>Mtb</i> H37Rv, and control and alcohol diet feeding was continued. At six months p.i., lung homogenates from uninfected control and alcohol diet-fed mice and <i>Mtb</i>-infected control and alcohol diet-fed mice were collected, and RIP-1 and RIP-3 gene expression was determined by quantitative real-time PCR. Control and alcohol diet-fed young mice were infected with 50–100 CFU of aerosolized <i>Mtb</i>. At three months p.i., lungs from uninfected control and alcohol diet-fed mice as well as from <i>Mtb</i>-infected control and <i>Mtb</i>-infected alcohol diet-fed mice were isolated and formalin fixed. Paraffin-embedded tissue sections were prepared and analyzed by confocal microscopy for <b>b</b>. RIP-1+ and RIP-3+ cells (red). Representative images of staining patterns were taken of multiple fields at 40X and 63X with oil immersion. The immunofluorescence intensities for these groups were calculated. <b>c.</b> RIP-1- and RIP-3 (red)-expressing F4/80 macrophages (green). <b>d.</b> Lung paraffin-embedded tissue sections were analyzed by confocal microscopy to determine IFN-α (Green), Ly6G (Magenta or Far-red) and RIP1/3 (red) colocalization. Scale bar: 10 μm. The rightmost panel shows a higher magnification for representation, and the yellow squares represent IFN-α-expressing Ly6G<i>+</i> cells. <b>e.</b> Representative images of RIP-1 and RIP-3 expression in lung F4/80 macrophages from anti-IFNAR-1 antibody-treated and isotype control antibody (IgG1)-treated mice are shown. Five mice per group were used for each group. The mean values, p-values and SEs are shown.</p

Alcohol enhances IFN-α production in young mice infected with <i>Mtb</i>.

<p>Young mice were fed control and alcohol diets as detailed in the methods section; then, they were infected with 50–100 CFU of aerosolized <i>Mtb</i> H37Rv, and control and alcohol diet feeding was continued. <b>a.</b> At three months p.i., lung homogenates from uninfected control and alcohol diet-fed and <i>Mtb</i>-infected control and alcohol diet-fed mice were collected, and cytokine and chemokine levels were determined by multiplex ELISA. <b>b-d.</b> At three months p.i., lungs from uninfected control and alcohol diet-fed mice as well as from <i>Mtb</i>-infected control and <i>Mtb</i>-infected alcohol diet-fed mice were isolated and formalin fixed. Paraffin-embedded tissue sections were prepared, and hematoxylin and eosin staining was performed. Inflamed lung areas were compared between the groups. <b>b.</b> A representative figure is shown. <b>c.</b> The lesion area and <b>d</b>. % lung lesions were calculated. The data are representative of two independent experiments. Five mice per group were used for each independent experiment. The mean values, p-values and SEs are shown.</p

Alcoholism enhances <i>Mtb</i>-induced IFN-α production by PBMCs from young LTBI+ individuals.

<p>Blood samples were obtained from 17 non-alcoholic and 20 alcoholic pulmonary tuberculosis patients who were 18–75 years of age. On the basis of age, LTBI+ individuals were categorized in to young (n = 24, age <45) and old (n = 11, age >50 years) groups. PBMCs from alcoholic and non-alcoholic young and old LTBI+ individuals were cultured with or without γ-irradiated <i>Mtb</i> (10 μg/ml). The levels of IFN-α were determined by ELISA. The mean values, p-values and SEs are shown.</p

Young alcoholic mice are susceptible to <i>Mtb</i> infection.

<p>Young (one to two months of age) and old (17 to 22 months of age) mice were fed control and alcohol diets for one month as detailed in the methods section; then, they were infected with 50–100 CFU of aerosolized <i>Mtb</i> H37Rv, and control and alcohol diet feeding was continued. <b>a.</b> Survival was determined. Survival curves were compared using the log-rank test (P<0.05). <b>b.</b> Bacterial burden in lungs at 3 months p.i. The data are representative of two pooled independent experiments (5 mice per group were used for each independent experiment). The mean values, p-values and SEs are shown.</p