Fig 1.
Alcohol and bacterial load in blood.
(A) Blood alcohol concentration (BAC). C57BL/6 mice were administered alcohol (4.4g/kg) or PBS intraperitoneally (i.p.). Blood was collected for BAC determination at 30, 60, 360, and 480 min post alcohol administration. Trend line represents the average of two independent determinations. n = 3 per determination. (B) Mice were administered alcohol or PBS intraperitoneally (i.p.) and 30 min later mice were inoculated intranasally with B. thailandensis at doses of (3 x 105), (2 x 106), or B. vietnamiensis (2 x 106). Blood was collected at 2 h post infection and Burkholderia species were grown on LB media plates to determine colony forming units (CFU). Bars represent the average CFU per treatment with SEM. Horizontal lines and asterisks (*) represent statistical comparison of PBS (Non-Alcohol) control and alcohol treatment determined by Student’s t-test, n = 4. **p ≤ 0.01, ***p ≤ 0.001.
Fig 2.
Bacterial load in the lungs, spleen and brain of binge-alcohol mice intranasally infected with different Burkholderia species and doses.
Mice were administered alcohol (4.4g/kg) or PBS (i.p.). At 30 min. post binge alcohol mice were infected with B. thailandensis (3 x 105) (A-B), B. thailandensis (2 x 106) (C-D), or B. vietnamiensis (2 x 106) (E-F). Tissues were collected 24 h (A, D, C) or 72 h (B, D, F) later and bacterial tissue burden was determined (CFU/tissue). Asterisks (*) represent statistical comparisons between alcohol treatment and (Non-Alcohol) control per tissue type determined by one-way ANOVA. Bars represent average CFU (n = 4) with SEM. **, p ≤ 0.01, ***, p ≤ 0.001, ****, p ≤ 0.0001.
Fig 3.
Pro-inflammatory cytokines in lung and spleen of binge alcohol mice intranasally infected with different Burkholderia species and doses.
Mice were treated as described in Fig 2 and infected with B. thailandensis (3 x 105) (A-B), B. thailandensis (2 x 106) (C-D), and B. vietnamiensis (2 x 106) (E-F). GM-CSF, TNF- α, IL-10 concentrations were measured in lung homogenates (A, C, E). TNF- α and IL-10 concentrations were measured in spleen homogenates (B, D, F), n = 4. Asterisks (*) represent statistical comparisons between alcohol treatment and (Non-Alcohol) control per cytokine determined by two-way ANOVA. Bars represent average concentration (n = 4) with SEM indicated. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001.
Fig 4.
Lung and brain blood barrier permeability in binge alcohol mice.
Mice were administered alcohol (4.4 g/kg) or PBS (i.p.). At 30 min. post binge alcohol, mice were injected with Evans blue dye (EB). Mice were sacrificed 2 h post EB administration, perfused, and tissues collected. EB dye was extracted from the (A) Lung and (B) Brain tissues using formamide. The concentrations of EB per gram of tissue were determined by absorbance at 610nm. Bars represent the average concentration with SEM (n = 3). Horizontal line and asterisks (*) represent statistical comparison of PBS (Non-Alcohol) control and alcohol treatment determined by a Student’s t-test, (B) **p ≤ 0.01.
Fig 5.
Lung epithelial and brain endothelial cell permeability with and without alcohol treatment.
(A) Lung epithelial (Eph4) and (B) brain endothelial (bEnd.3) cell transepithelial resistance (TEER) was measured in cell monolayers grown in F12 media in 0.4- micron pore diameter membrane inserts. Media was supplemented with 0.0% or 0.2% v/v alcohol and TEER was measured at 1 and 8 h post alcohol administration. In panel (C), the permeability was determined in both cells by adding FITC-Dextran (10 KDa) to the apical side and measured in the baso-lateral side at 2 h post alcohol administration. Bars represent the average TEER across the permeable membrane per treatment with SEM. Horizontal lines and asterisks (*) represent statistical comparison of PBS (Non-Alcohol) control and alcohol treatment determined by Student’s t-test at each time point, (A-C) **p ≤ 0.01, ***p ≤ 0.0001.
Fig 6.
Bacterial invasion and survival in non-phagocytic lung and brain cells with and without alcohol treatment.
(A) Lung epithelial and (B) brain endothelial cells were grown to confluency in F12 cell culture media and co-cultured with B. thailandensis or B. vietnamiensis (MOI 1:10) for 3 h in media supplemented with 0.0% or 0.2% v/v alcohol. Extracellular bacteria were removed by washes X4 and antibiotic treatment for 2 h. Cells were lysed and viable bacteria recovered. Asterisks (*) represent statistical comparisons between alcohol treatment and (Non-Alcohol) control determined by one-way ANOVA. Bars represent average CFU with SEM. **, p ≤ .01; ***, p ≤ .001; ****, p ≤ 0.0001.