Intestinal Microbial Products From Alcohol-Fed Mice Contribute to Intestinal Permeability and Peripheral Immune Activation

Abstract

Background: Alcohol use causes significant disruption of intestinal microbial communities, yet exactly how these dysbiotic communities interact with the host is unclear. We sought to understand the role of microbial products associated with alcohol dysbiosis in mice on intestinal permeability and immune activation in an in vitro model system.

Methods: Microbiota samples from binge-on-chronic alcohol-fed and pair-fed male and female mice were cultured in Gifu Anaerobic Broth for 24 hours under anaerobic conditions. Live/whole organisms were removed, and microbial products were collected and added to human peripheral blood mononuclear cells (PBMCs) or polarized C2BBe1 intestinal epithelial monolayers. Following stimulation, transepithelial electrical resistance (TEER) was measured using a volt/ohm meter and immune activation of PBMC was assessed via flow cytometry.

Results: Microbial products from male and female alcohol-fed mice significantly decreased TEER (mean percentage change from baseline alcohol-fed 0.86 Ω/cm² vs. pair-fed 1.10 Ω/cm² ) compared to microbial products from control mice. Following ex vivo stimulation, immune activation of PBMC was assessed via flow cytometry. We found that microbial products from alcohol-fed mice significantly increased the percentage of CD38+ CD4+ (mean alcohol-fed 17.32% ± 0.683% standard deviation (SD) vs. mean pair-fed 14.2% ± 1.21% SD, p < 0.05) and CD8+ (mean alcohol-fed 20.28% ± 0.88% SD vs. mean pair-fed 12.58% ± 3.59% SD, p < 0.05) T cells.

Conclusions: Collectively, these data suggest that microbial products contribute to immune activation and intestinal permeability associated with alcohol dysbiosis. Further, utilization of these ex vivo microbial product assays will allow us to rapidly assess the impact of microbial products on intestinal permeability and immune activation and to identify probiotic therapies to ameliorate these defects.

Keywords: Alcohol; Dysbiosis; Immune Activation; Intestinal Permeability; Microbial Products.

Figure 1:. Schematic outline of the experimental protocol used in this study.

Cecal and colonic microbiota from alcohol-fed and pair-fed male and female mice were collected, homogenized in sterile PBS and cultured in Gifu Anaerobic for 24 hours under anaerobic conditions. Microbial products were collected by the removal of live and/or whole organisms. Microbial products were then used to assess intestinal permeability and inflammation, as well as peripheral immune activation.

Figure 2:. Binge-on-chronic alcohol feeding alters the intestinal microbial community structure.

Alpha- and beta-diversity of cecal and colonic microbial communities from alcohol-fed and pair-fed mice were assessed using R. (A) Significant differences in α-diversity of the male cecal microbial communities were seen between alcohol-fed mice compared to pair-fed mice. No significance diffrences in α-diversity were seen between alcohol-fed and pair-fed male colonic, or female cecal and colonic microbial communitie in mice (B) Significant differences in β-diversity were seen between; alcohol-fed and pair-fed mice, as well as between male and female mice. No significant difference was observed between the microbial communities in the cecum and colon. Alpha-diversity was determined by multiple general linear regression and beta-diversity was determined by permutational multivariate analysis of variance within R. N=5–10.

Figure 3:. Binge-on-chronic alcohol feeding alters the realative abundance of specific bacterial taxa.

Differentially abundant OTUs was assessed using R and DESeq2. (A) Alcohol-feeding alters the relative abundance of specific OTUs in the male cecal microbiota compared to pair-fed mice. (B) Alcohol-feeding alters the relative abundance of specific OTUs in the male colonic microbiota compared to pair-fed mice. (C) Alcohol-feeding alters the relative abundance of specific OTUs in the female cecal microbiota compared to pair-fed mice. (D) Alcohol-feeding alters the relative abundance of specific OTUs in the female colonic microbiota compared to pair-fed mice. Differentially abundant taxa was determined via negative binomial mixture model using DESeq2 and only statistically significant OTUs were plotted. OTUs with a positive log₂ fold change indicates that the OTU (genus) is more abundant in the pair-fed animals, while OTUs with a negative log₂ fold change indicate that the OTU (genus) is more abundant in the alcohol-fed animals. N=5–10. Each dot represents a unique differentially abundant OTU.

Figure 4:. Sex alters the relative abundance of specific bacterial taxa in alchol-fed and pair-fed mice.

Differentially abundant OTUs was assessed using R and DESeq2 (A) Sex alters the relative abundance of specific OTUs in the alcohol-fed cecal microbiota. (B) Sex alters the relative abundance of specific OTUs in the alcohol-fed colonic microbiota. (C) Sex alters the relative abundance of specific OTUs in the pair-fed cecal microbiota. (D) Sex alters the relative abundance of specific OTUs in the pair-fed colonic microbiota. Differentially abundant taxa was determined via negative binomial mixture model using DESeq2. (A and B) OTUs with a positive log₂ fold change indicates that the OTU (genus) is more abundant in the microbial communities from female alcohol-fed mice, while OTUs with a negative log₂ fold change indicate that the OTU (genus) is more abundant in microbial communities from male alcohol-fed mice. (C and D) OTUs with a positive log₂ fold change indicates that the OTU (genus) is more abundant in the microbial communities from female pair-fed mice, while OTUs with a negative log₂ fold change indicate that the OTU (genus) is more abundant in microbial communities from male pair-fed mice. N=5–10. Each dot represents a unique differentially abundant OTU.

Figure 5:. Transepithelial electrical resistance (TEER) of polarized intestinal epithlieal cells decreases following treatment with alcohol-associated microbial products.

The effect of microbial products on polarized epitheilal TEER was deterimed via a volt/ohm meter. (A) Cecal microbial products from alcohol or pair-fed microbiota harvested from male and female mice were added to the apical chamber of polarized C2BBe1 cells for a total of 20 hrs. and TEER was assessed 20 hrs. post stimulation. (B) Colonic microbial products from alcohol or pair-fed microbiota collected from male and female mice were added to the apical chamber of polarized C2BBe1 cells for a total of 20 hrs. and TEER was assessed 20 hrs. post stimulation. N=5–10, repeated 3x. *indicates P<0.05, by 2-way ANOVA with Sidak’s multiple comparisons test. The same media only controls are shown in panel (A and B) for visualization, however all samples were ran at the same time.

Figure 6:. Treatment with alcohol-associated microbial products increased translocation of dextran to the basal lateral surface of polarized intestinal epithlieal cells.

Microbial products from alcohol or pair-fed intestinal samples harvested from male and female mice were added to the apical chamber of polarized C2BBe1 cells for a total of 20 hrs. and dextran translocation was assessed. Male and female alcohol-associated (A) cecal and (B) colonic microbial products increase translocation of a 3,000 kDa FITC-dextran to the basal lateral surface. Male and female alcohol-associated (C) cecal and (D) colonic microbial products increase translocation of a 40,000 kDa Texas Red-dextran to the basal lateral surface via a fluorescence spectroscopy. N=5–10, repeated 3x. *indicates P < 0.05, by 2-way ANOVA with Sidak’s multiple comparisons test. The same media only controls are shown in panel (A and B) and in panel (C and D) for visualization, however all samples were ran on the same plate.

Figure 7:. Treatment with alcohol-associated microbial products increased secretion of IL-8 from polarized intestinal epithlieal cells.

Microbial products from alcohol or pair-fed intestinal samples harvested from male and female mice were added to the apical chamber of polarized C2BBe1 cells for a total of 20 hrs. and IL-8 levels were assessed (A) IL-8 levels in the apical chamber culture supernatant following treatment with cecal microbial products from alcohol or pair-fed mice. (B) IL-8 levels in the apical chamber culture supernatant following treatment with colonic microbial products. (C) IL-8 levels in the basal lateral chamber culture supernatant following treatment with cecal microbial products from alcohol or pair-fed mice. (D) IL-8 levels in the basal lateral chambers culture supernatant following treatment with colonic microbial products. N=5–10, repeated 3x. *indicates P < 0.05, 2-way ANOVA with Sidak’s multiple comparisons test. The same media only controls are shown in panel (A and B) and in panel (C and D) for visualization, however all samples were ran on the same plate.

Figure 8:. Alcohol-associated microbial products increase PBMC immune activation.

Cecal microbial products from alcohol or pair-fed intestinal samples harvested from male and female mice were added to normal PBMCs for 4 hrs. and immune activation the percentage of (A) CD38+ CD4+ T-cells and (B) CD38+ CD8+ T-cells via a flow cytometry. Colonic microbial products from alcohol or pair-fed intestinal samples collected from male and female mice were added to normal PBMCs for 4 hrs. and immune activation the percentage of (C) CD38+ CD4+ T-cells and (D) CD38+ CD8+ T-cells via a flow cytometry. N=5–10, repeated 3x. *indicates P < 0.05, by 2-way ANOVA with Sidak’s multiple comparisons test. The same unstimulated controls are shown in panel (A and C) and in panel (B and D) for visualization, however all samples were ran at the same time.

Figure 9:. In vivo alcohol-associated microbial products increase PBMC immune activation.

In vivo collected colonic microbial products from alcohol or pair-fed intestinal samples collected from male and female mice were added to normal PBMCs for 4 hrs. and immune activation the percentage of (A) CD38+ CD4+ T-cells and (B) CD38+ CD8+ T-cells via a flow cytometry. N=5, repeated 2x. *indicates P < 0.05, by 2-way ANOVA with Sidak’s multiple comparisons test.