Alcohol-associated intestinal dysbiosis impairs pulmonary host defense against Klebsiella pneumoniae

Abstract

Chronic alcohol consumption perturbs the normal intestinal microbial communities (dysbiosis). To investigate the relationship between alcohol-mediated dysbiosis and pulmonary host defense we developed a fecal adoptive transfer model, which allows us to investigate the impact of alcohol-induced gut dysbiosis on host immune response to an infectious challenge at a distal organ, independent of prevailing alcohol use. Male C57BL/6 mice were treated with a cocktail of antibiotics (ampicillin, gentamicin, neomycin, vancomycin, and metronidazole) via daily gavage for two weeks. A separate group of animals was fed a chronic alcohol (or isocaloric dextrose pair-fed controls) liquid diet for 10 days. Microbiota-depleted mice were recolonized with intestinal microbiota from alcohol-fed or pair-fed (control) animals. Following recolonization groups of mice were sacrificed prior to and 48 hrs. post respiratory infection with Klebsiella pneumoniae. Klebsiella lung burden, lung immunology and inflammation, as well as intestinal immunology, inflammation, and barrier damage were examined. Results showed that alcohol-associated susceptibility to K. pneumoniae is, in part, mediated by gut dysbiosis, as alcohol-naïve animals recolonized with a microbiota isolated from alcohol-fed mice had an increased respiratory burden of K. pneumoniae compared to mice recolonized with a control microbiota. The increased susceptibility in alcohol-dysbiosis recolonized animals was associated with an increase in pulmonary inflammatory cytokines, and a decrease in the number of CD4+ and CD8+ T-cells in the lung following Klebsiella infection but an increase in T-cell counts in the intestinal tract following Klebsiella infection, suggesting intestinal T-cell sequestration as a factor in impaired lung host defense. Mice recolonized with an alcohol-dysbiotic microbiota also had increased intestinal damage as measured by increased levels of serum intestinal fatty acid binding protein. Collectively, these results suggest that alterations in the intestinal immune response as a consequence of alcohol-induced dysbiosis contribute to increased host susceptibility to Klebsiella pneumonia.

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.