Human Alcohol-Microbiota Mice have Increased Susceptibility to Bacterial Pneumonia

Abstract

Preclinical studies have shown that chronic alcohol abuse leads to alterations in the gastrointestinal microbiota that are associated with behavior changes, physiological alterations, and immunological effects. However, such studies have been limited in their ability to evaluate the direct effects of alcohol-associated dysbiosis. To address this, we developed a humanized alcohol-microbiota mouse model to systematically evaluate the immunological effects of chronic alcohol abuse mediated by intestinal dysbiosis. Germ-free mice were colonized with human fecal microbiota from individuals with high and low Alcohol Use Disorders Identification Test (AUDIT) scores and bred to produce human alcohol-associated microbiota or human control-microbiota F1 progenies. F1 offspring colonized with fecal microbiota from individuals with high AUDIT scores had increased susceptibility to Klebsiella pneumoniae and Streptococcus pneumoniae pneumonia, as determined by increased mortality rates, pulmonary bacterial burden, and post-infection lung damage. These findings highlight the importance of considering both the direct effects of alcohol and alcohol-induced dysbiosis when investigating the mechanisms behind alcohol-related disorders and treatment strategies.

Keywords: alcohol; gut–lung axis; host defense; klebsiella; microbiome; pneumonia; streptococcus.

Figure 1

Experimental Schema.

Figure 2

Microbial community structure is maintained in the F1 generation of human alcohol-associated microbiota mice, as shown by the 16s sequencing of human donor fecal samples and cecal microbial community from F0 and F1 conventionalized germ-free mice. (A) Beta diversity of human, F0, and F1 human-microbiota associated mice, as determined by distance-based redundancy analysis (dbRDA) on sample-wise Bray–Curtis dissimilarity distances. (B) Relative abundance of microbial taxa in human fecal samples, as well as cecal samples from F0, and F1 human-microbiota associated mice. n = 5–6/group.

Figure 3

Differentially abundant microbial taxa structure in human alcohol-associated microbiota mice. Differentially abundant ASVs as determined by DESeq2. (A) Human donor fecal samples, (B) F0 conventionalized mice, and (C) F1 human-microbiota associated mice. n = 5–6/group.

Figure 4

Human alcohol-associated microbiota mice have increased weight loss following K. pneumoniae infection. F1 microbiota mice were infected with K. pneumoniae, and weight loss was assessed daily for 48 h post infection. Post-infection weight loss in (A) combined male and female mice, (B) male mice, and (C) female mice. (D) Donor-dependent weight change in combined male and female mice post infection. Dots represent the mean and SEM per group (n = 20 control-microbiota F1 mice, and n = 20 alcohol-associated F1 mice). Red lines indicate F1 alcohol-associated microbiota mice. p values are indicated in the figure and were determined by two-way ANOVA with a post hoc Sidak’s multiple comparisons correction.

Figure 5

Human alcohol-associated microbiota mice have increased mortality following K. pneumoniae infection. F1 microbiota mice were infected with K. pneumoniae, and survival was assessed 48 h post infection. Survival post infection in (A) combined male and female mice, (B) male mice, and (C) female mice. (D) Donor-dependent survival in combined male and female mice post infection. Dots represent the mean and SEM per group (n = 20 control-microbiota F1 mice, and n = 20 alcohol-associated F1 mice). Red lines indicate F1 alcohol-associated microbiota mice. p values are indicated in the figure and were determined by the Log-rank (Mantel–Cox) test.

Figure 6

Human alcohol-associated microbiota mice have increased pulmonary bacterial burden and dissemination. F1 Microbiota mice were infected with K. pneumoniae and bacterial burden was assessed. Log transformation burden of K. pneumoniae in the lungs of (A) combined male and female F1 mice, (B) male mice, (C) female mice, and (D) donor-dependent pulmonary burden. Log transformation burden of K. pneumoniae in the spleens of (E) combined male and female F1 mice, (F) male mice, (G) female mice, and (H) donor-dependent pulmonary burden. Bars represent the mean ± SEM and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 7

Human alcohol-associated microbiota mice have increased pulmonary damage. F1 Microbiota mice were infected with K. pneumoniae, and pulmonary damage was assessed. (A) Representative lung H&E images at 20× magnification. Lung inflammatory scores via H&E histology of (B) combined male and female F1 mice and (C) donor-dependent pulmonary damage. Bars represent the mean ± SEM, and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 8

Human alcohol-associated microbiota mice have decreased barrier function. F1 microbiota mice were infected with K. pneumoniae, and the level of BAL protein was determined in the lungs of (A) combined male and female F1 mice, (B) male mice, (C) female mice, and (D) donor-dependent pulmonary BAL protein levels. Bars represent the mean ± SEM and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 9

Human alcohol-associated microbiota mice have decreased pulmonary immune cell numbers. F1 microbiota mice were infected with K. pneumoniae and the numbers of BAL immune cells were assessed. Total BAL counts were determined in the lungs of (A) combined male and female F1 mice, (B) male mice, (C) female mice, and (D) donor dependent. Total macrophages post infection were determined in the lungs of (E) combined male and female F1 mice, (F) male mice, (G) female mice, and (H) donor dependent (D). Total neutrophils post infection were determined in the lungs of (I) combined male and female F1 mice, (J) male mice, (K) female mice, and (L) donor dependent. Bars represent the mean ± SEM, and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 10

Human alcohol-associated microbiota mice have altered pulmonary inflammation. F1 microbiota mice were infected with K. pneumoniae, and the levels of BAL cytokines/chemokines were assessed. Total BAL (A) IL-6, (B) TNF-α, (C) CXCL1, and (D) IL-1β were determined in the lungs of combined male and female F1 mice. Total BAL GM-CSF levels were determined in the lungs of (E) combined male and female F1 mice, (F) male mice, (G) female mice, and (H) donor dependent. Bars represent the mean ± SEM, and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 11

Human alcohol-associated microbiota mice exhibit increased weight loss following S. pneumoniae infection. F1 microbiota mice were infected with S. pneumoniae and weight loss was assessed out to 48 h post infection. Post-infection weight loss in (A) combined male and female mice, (B) male mice, and (C) female mice. (D) Donor-dependent weight change in combined male and female mice post infection. Dots represent the mean and SEM per group (n = 18 control-microbiota F1 mice and n = 25 alcohol-associated F1 mice). Red lines indicate F1 alcohol-associated microbiota mice. p values are indicated in the figure and were determined by two-way ANOVA with a post-hoc Sidak’s multiple comparisons correction.

Figure 12

Human alcohol-associated microbiota mice have increased pulmonary bacterial burden and dissemination. F1 microbiota mice were infected with S. pneumoniae and bacterial burden was assessed. Log transformation burden of S. pneumoniae in the lungs of (A) combined male and female F1 mice, (B) male mice, (C) female mice, and (D) donor-dependent pulmonary burden. Log transformation burden of S. pneumoniae in the spleens of (E) combined male and female F1 mice, (F) male mice, (G) female mice, and (H) donor-dependent pulmonary burden. Bars represent the mean ± SEM, and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 13

Human alcohol-associated microbiota mice have increased pulmonary damage. F1 microbiota mice were infected with S. pneumoniae, and pulmonary damage was assessed. (A) Representative lung H&E images at 20× magnification. Lung inflammatory scores of (B) female F1 mice, and (C) donor-dependent pulmonary damage. Bars represent the mean ± SEM, and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 14

Human alcohol-associated microbiota mice have increased pulmonary leak and immune cell numbers. F1 microbiota mice were infected with S. pneumoniae and the level of BAL protein was determined in the lungs of (A) female F1 mice, and (B) donor-dependent pulmonary BAL protein levels. The numbers of BAL immune cells were assessed. Total BAL counts were determined in the lungs of (C) female F1 mice and (D) donor-dependent mice. Post-infection macrophages were enumerated in the lungs of (E) female F1 mice, and (F) donor-dependent mice. Post-infection neutrophils were also enumerated in the lungs of (G) female F1 mice, and (H) donor dependent mice. Bars represent the mean ± SEM, and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 15

Human alcohol-associated microbiota mice have increased pulmonary inflammation. F1 microbiota mice were infected with S. pneumoniae and the levels of BAL cytokines/chemokines were assessed. Total BAL (A,B) IL-6, (C,D) TNF-α, (E,F) CXCL1, and (G,H) IL-1β were determined in the lungs of female F1 mice. Bars represent the mean ± SEM and dots represent individual mice. p values are indicated in the figure and were determined by Welch’s t-test.

Figure 16

Human alcohol-associated microbiota mice have increased drinking preference and anxiety-like behavior. Male and female F1 alcohol-associated microbiota mice were subjected to a 30-min marble-burying assessment prior to and following a 15-day 2-bottle choice alcohol-drinking paradigm. (A) 2-bottle choice alcohol-drinking preference, and (B) marble-burying score. Dots represent the mean and SEM per group (n = 10 (5 male and 5 female) control-microbiota F1 mice, and n = 10 (5 male and 5 female) alcohol-associated F1 mice). p values are indicated with a *, corresponding to p = 0.03 and p = 0.05 for drinking preference and marble-burying, respectively. p values were determined by the Mann–Whitney test.