Fig 1.
Schematic outline of the experimental protocol used in this study.
C57BL/6 mice were administrered binge-on-chronic alcohol (10 days chronic and 2x binges). Following alcohol feeding groups of mice were sacrificed 24 hrs following the final binge or infected intratracheally with K. pneumoniae (~1 x 102 CFU in 100 μl of PBS) and sacrificed at 48 hours post infection. 5% EtOH diet was maintained continuously throughout the experiment.
Fig 2.
Binge-on-chronic alcohol feeding causes intestinal epithelial damage.
(A) Blood alcohol levels (mg/dl) of chronic alcohol fed mice following 10 days of diet and 6 hrs following the second binge alcohol administration. (B) Body weights of pair-fed and alcohol-fed mice at baseline and post binge-on-chronic alcohol feeding (10 days chronic + 2x binge). (C) Circulating levels of intestinal fatty acid binding protien (i-FABP) in alcohol- and pair-fed control mice. Bars are the mean ± SEM, *indicates P<0.05, by Mann-Whitney U. N = 10/group.
Fig 3.
Binge-on-chronic alcohol use alters the intestinal microbial and metabolic profile.
(A) Alcohol-treated mice have significantly lower alpha-diversity compared to pair-fed mice as determined by observed species via Qiime. (B) Alcohol-treated mice (red circles) showed significantly different microbial community structures from pair-fed mice (blue circles) as determined by principal coordinate analysis of the weighted UniFrac metric via Qiime. (C) Alcohol-treated mice have altered relative abundance of specific OTUs compared to pair-fed mice. (D) Alcohol-fed (red) mice had significantly different microbial metabolites from pair-fed (blue) mice, as shown by Partial Least Squares Discriminant Analysis. (E) Alcohol-treated mice have altered relative concentration of mirobial metabolites compared to pair-fed mice. *indicates P<0.05, by Mann-Whitney U with both Benjamini-Hochberg and Bonferroni corrections. N = 3-10/group.
Fig 4.
Binge-on-chronic alcohol use increases host susceptibility to Klebsiella pneumoniae in mice.
(A) Klebsiella lung burden (CFU/ml) at 48 hrs. post infection in pair-fed and binge-on-chronic alcohol treated mice. (B) Difference in absolute number of homing receptor positive lung CD4+ T-cells isolated 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-fed and pair-fed mice. (C) Difference in absolute number of homing receptor positive lung CD8+ T-cells 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-fed and pair-fed mice. Bars represent the mean of the cell counts post infection minus the cell counts prior to infection ± SEM. * indicates P < 0.05, by Mann-Whitney U or by ANOVA with Dunn’s correction. N = 10/group.
Fig 5.
Binge-on-chronic alcohol use alters the levels of pulmonary macrophages and dendritic cells.
Difference in absolute number of CD103+ dendritic cells, DC/interstitial macrophages, and aveolar macrophages isolated 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-fed and pair-fed mice. Bars represent the mean of the cell counts post infection minus the cell counts prior to infection ± SEM, * indicates P < 0.05, by ANOVA with Dunn’s correction. N = 10/group.
Fig 6.
Binge-on-chronic alcohol use alters acute pulmonary inflammatory cytokines.
Levels of pulmonary acute inflammatory cytokines were asssesed 48 hrs. post-Klebsiella infection via LEGENDplex 13-ples assay kit. Bars represent the mean cytokine level ± SEM, * indicates P < 0.05, by ANOVA with Dunn’s correction. N = 10/group.
Fig 7.
Schematic outline of the experimental protocol used in this study.
C57BL/6 mice were administrered antibiotics daily for 14 days. Following antibiotic treatment mice were recolonized with cecal microbiota from alcohol-fed or pair-fed mice. Groups of mice were sacraficed 7 days following the final recolonization or infected intratracheally with K. pnuemoniae (~1x102 CFU in 100 μl of PBS) and sacrificed at 48 hours post infection.
Fig 8.
Adoptive transfer maintains microbial community structure.
(A) The microbial communities of alcohol-dysbiosis recolonized mice (blue squares) compared to the microbial communities of alcohol-fed donor mice (red triangles), and the microbial communities of pair-fed recolonized mice (green triangles) compared to the microbial communities of pair-fed donor mice (orange circles). (B) Relative abundance of OTUs are similar between alcohol-microbiota recipient and alcohol-microbiota donor animals, as well as pair-fed microbiota recipient and pair-fed microbiota donor animals.
Fig 9.
Alcohol-dysbiosis significantly increases intestinal barrier damage and inflammation.
(A) Circulating levels of intestinal fatty acid binding protien (i-FABP) in alcohol-dysbiosis and pair-fed recolonized mice. (B) Absolute number of effector (CD44+, CD62L-) CD8+ T-cells in the intestine of alcohol-dysbiosis and pair-fed recolonized mice. Bars are the mean ± SEM, *indicates P<0.05, by Mann-Whitney U. N = 10/group.
Fig 10.
Alcohol-associated dysbiosis increases host susceptibility to Klebsiella pneumoniae in mice.
(A) Klebsiella lung burden (CFU/ml) at 48 hrs. post infection in pair-fed recolonized and alcohol-dysbiosis recolonized treated mice. (B) Difference in absolute number of homing receptor positive lung CD4+ T-cells isolated 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-dysbiosis and pair-fed recolonized mice. (C) Difference in absolute number of homing receptor positive lung CD8+ T-cells 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-dysbiosis and pair-fed recolonized mice. Bars represent the mean of the cell counts post infection minus the cell counts prior to infection ± SEM. * indicates P < 0.05, by Mann-Whitney U or by ANOVA with Dunn’s correction. N = 10/group.
Fig 11.
Alcohol-associated dysbiosis alters the levels of pulmonary macrophages and dendritic cells.
Difference in absolute number of CD103+ dendritic cells, DC/interstitial macrophages, and aveolar macrophages isolated 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-dysbiosis and pair-fed recolonized mice. Bars represent the mean of the cell counts post infection minus the cell counts prior to infection ± SEM, * indicates P < 0.05, by ANOVA with Dunn’s correction. N = 10/group.
Fig 12.
Alcohol-associated dysbiosis alters acute pulmonary inflammatory cytokines.
Levels of pulmonary acute inflammatory cytokines were asssesed 48 hrs. post-Klebsiella infection via LEGENDplex 13-ples assay kit. Bars represent the mean cytokine level ± SEM, * indicates P < 0.05, by ANOVA with Dunn’s correction. N = 10/group.
Fig 13.
Alcohol-associated dysbiosis increases intestinal T-cell sequestration in mice.
(A) Difference in absolute number of homing receptor positive intestinal CD4+ T-cells isolated 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-dysbiosis and pair-fed recolonized mice. (B) Difference in absolute number of homing receptor positive intestinal CD8+ T-cells 48 hrs. post-Klebsiella infection from values obtained pre-infection in alcohol-dysbiosis and pair-fed recolonized mice. Bars represent the mean of the cell counts post infection minus the cell counts prior to infection ± SEM, * indicates P < 0.05, by ANOVA with Dunn’s correction. N = 10/group.
Fig 14.
Working model for the effects of alcohol-dysbiosis on host defense against pulmonary pathogens.
Alcohol promotes intestinal microbial and metabolic dysbiosis, which increases intestinal inflammation and permeability leading to intestinal T-cell sequestration and impaired T-cell trafficking to the respiratory tract, all of which, in combination, increase host susceptibility to respiratory infection with Klebseilla pneumoniae.