Decreased ω-6:ω-3 PUFA ratio attenuates ethanol-induced alterations in intestinal homeostasis, microbiota, and liver injury

Abstract

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies, including liver injury. ω-6 PUFAs exert pro-inflammatory activity, while ω-3 PUFAs promote anti-inflammatory activity that is mediated, in part, through specialized pro-resolving mediators [e.g., resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the ω-6:ω-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. ω-3 FA desaturase-1 (fat-1) mice, which endogenously increase ω-3 PUFA levels, were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-γ, Il-6, and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation, stem cell renewal, and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further, gut microbiome and plasma metabolomics revealed several changes in fat-1 versus WT mice that may contribute to a reduced inflammatory response. Finally, these data correlated with a significant reduction in liver injury. Our study suggests that ω-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury.

Keywords: alcoholic liver disease; bile acid metabolism; diet and dietary lipids; gut microbiome; inflammation; intestine; omega-3 fatty acids; polyunsaturated fatty acid.

Fig. 1.

Effects of EtOH and endogenous conversion of tissue ω-6 to ω-3 PUFAs on intestinal morphology and homeostasis. A–C: Ileum tissue ω-6:ω-3 PUFA ratio and total ω-6 and ω-3 PUFAs (n = 3–5). The full panel of FAs is presented in supplemental Table S2. D: Representative images of stained intestinal sections with H&E or Alcian blue, 200× magnification. E, F: Effect of EtOH on small intestine-derived organoids from naïve WT and fat-1 mice cultured for 4 days, 100× magnification (n = 5). Expression of growth and proliferation markers (G–H), mucus production (I), bacteriocidal activity (J), and tight junction genes (K–L) in intestinal organoids from WT or fat-1 mice fed control or EtOH diets cultured for 10 days (n = 3). *P < 0.05. M: immunohistochemistry/confocal imaging of ZO-1 expression (200× magnification).

Fig. 2.

Differential gene expression in WT versus fat-1 mice. A: Effects of modulation of the ω-6:ω-3 PUFA ratio on EtOH-metabolizing enzymes in the intestine. Data are presented as gene expression levels of the corresponding group. Lowercase letters indicate statistically significant changes between groups (a: fat-1-PF vs. WT-PF; b: WT-PF vs. WT-EtOH; c: fat-1-PF vs. fat-1-EtOH; P < 0.05). B: Plasma EtOH levels (n = 6–10). Venn diagram (C) and graphical representation (D) of total number of genes differentially expressed in the EtOH groups.

Fig. 3.

Increased ω-3 PUFAs reduced LPS-mediated intestinal inflammation in mice chronically fed EtOH. A, B: CAE-stained intestine sections and quantification of CAE-positive cells (n = 6–18 mice). C–G: Ileal pro-inflammatory cytokine expression determined by immunoassay: INF-γ (C), IL-6 (D), Il-1β (E), TNF-α (F), and CXCL1 (G). H: Ileal mRNA levels of pro-inflammatory genes (n = 3–11 mice). I: Quantification of CAE-positive cells in the EtOH+LPS group treated with RvD1 (n = 12–14). J: Effects of RvD1 treatment on EtOH+LPS-induced pro-inflammatory cytokines (n = 9–10). K: Immunohistochemistry for lysozyme expression in the intestine. L, M: Ileal expression of antimicrobial genes (n = 3–5).

Fig. 4.

Effects of endogenous ω-6:ω-3 PUFA modulation on EtOH-associated gut microbiota alterations. A: Pie charts of taxonomic composition of bacterial community at the phylum levels in stool samples from WT and fat-1 experimental mice. Statistically significant changes (P < 0.05) between groups are shown: *WT-PF versus WT-EtOH; §fat1-PF versus fat-1-EtOH; #WT-EtOH versus WT-EtOH-LPS; †fat-1-EtOH versus fat-1-EtOH-LPS. B: Principal component analysis of gut microbiota composition based on unweighted UniFrac. P = 0.001, PERMANOVA analysis. C: Firmicutes:Bacteroidetes ratio in experimental groups. D, E: Cladograms showing the taxa most differentially associated with EtOH or post-EtOH LPS treatment in WT and fat-1 mice. Circle sizes in the cladogram plot are proportional to bacterial abundance. The circles represent phyla, genus, class, order, and family going from the inner to the outer circle. F: Selected bacteria at the genus levels in WT and fat-1 experimental mice. For each analysis, n = 5–10 mice per group.

Fig. 5.

Impact of ω-6:ω-3 PUFA ratio modulation on plasma metabolome alterations associated with EtOH administration and LPS challenge. A: Partial least squares-discriminant analysis. B: Plasma metabolites significantly differentially enriched in experimental groups.

Fig. 6.

Effects of modulation ω-6:ω-3 PUFA ratio on EtOH- and LPS-mediated alterations in fecal BA profiles and related metabolic pathways. A: Fecal primary and secondary BA concentrations (n = 3–7). Expression of genes related to BA detoxification in the ileum (RNA-seq) (B) and in the liver (C) as determined by qRT-PCR in WT and fat-1 mice. Expression of genes involved in BA synthesis (D), transport (E), and reuptake (F) in the liver. G, H: Expression of Nr1h4/Fxr, Nr1i2/Pxr, and Nr1i3/Car in the ileum and liver, respectively (n = 3–5). CA, cholic acid, DCA, deoxycholic acid.

Fig. 7.

BA detoxification gene expression. Expression of genes involved in the metabolic inactivation of BA in the ileum (A) (RNA-seq, n = 3–5) and liver (B) (qPCR, n = 5–10). *P < 0.05, one-way ANOVA.

Fig. 8.

Decreased ω-6:ω-3 PUFA ratio resulted in the improvement of liver injury associated with EtOH and LPS administration. Serum ALT levels (A), representative H&E-stained liver sections (400×) (B), liver TGs (C), correlation between intestinal CXCL1 protein expression and ALT (D), and heat map to illustrate Spearman correlations of the relative abundance of microbiome at the genus levels and parameters of liver injury WT and fat-1 mice in the EtOH+LPS group (n = 5, statistical significance indicated as *P < 0.05) (E). The r values are represented by gradient colors, where red and blue cells indicate positive and negative correlations, respectively; *P < 0.05. F: Model showing potential mechanism by which decreased tissue ω-6 PUFA and increased ω-3 PUFA results in a specific changes in intestinal homeostasis, the gut microbiota, and BA metabolism that coordinate to ameliorate liver injury in mice chronically fed EtOH and challenged with LPS. A–E: n = 3–12 mice per group.