Ileum Gene Expression in Response to Acute Systemic Inflammation in Mice Chronically Fed Ethanol: Beneficial Effects of Elevated Tissue n-3 PUFAs

Abstract

Chronic alcohol consumption leads to disturbances in intestinal function which can be exacerbated by inflammation and modulated by different factors, e.g., polyunsaturated fatty acids (PUFAs). The mechanisms underlying these alterations are not well understood. In this study, RNA-seq analysis was performed on ileum tissue from WT and fat-1 transgenic mice (which have elevated endogenous n-3 PUFAs). Mice were chronically fed ethanol (EtOH) and challenged with a single lipopolysaccharide (LPS) dose to induce acute systemic inflammation. Both WT and fat-1 mice exhibited significant ileum transcriptome changes following EtOH + LPS treatment. Compared to WT, fat-1 mice had upregulated expression of genes associated with cell cycle and xenobiotic metabolism, while the expression of pro-inflammatory cytokines and pro-fibrotic genes was decreased. In response to EtOH + LPS, fat-1 mice had an increased expression of genes related to antibacterial B cells (APRIL and IgA), as well as an elevation in markers of pro-restorative macrophages and γδ T cells that was not observed in WT mice. Our study significantly expands the knowledge of regulatory mechanisms underlying intestinal alterations due to EtOH consumption and inflammation and identifies the beneficial transcriptional effects of n-3 PUFAs, which may serve as a viable nutritional intervention for intestinal damage resulting from excessive alcohol consumption.

Keywords: acute systemic inflammation; alcohol; intestine; polyunsaturated fatty acids; transcriptome.

Figure 1

Chronic EtOH consumption followed by LPS challenge leads to global ileal gene changes in both WT and fat-1 mice. (A) WT and fat-1 mice were either pair-fed (WT n = 4, fat-1 n = 4), EtOH-fed (WT n = 3, fat-1 n = 5), or EtOH-fed + a one-time injection of LPS 24 h before sacrifice (WT-EtOH + LPS n = 4, fat-1 EtOH + LPS n = 4).(B) Gross RNA-seq data from WT EtOH + LPS vs. WT EtOH mice, fat-1 EtOH + LPS vs. fat-1 EtOH mice, and fat-1 EtOH + LPS vs. WT EtOH + LPS mice. Nodes in the red gradient were increased for the given comparison and nodes in the blue gradient were decreased for the given comparison, with the total number of genes listed to the side. (C) The number of gene expression changes in response to EtOH + LPS, either exclusive or common to genotype. (D) Plot of log₂ (Fold-change ranked genes) for the WT EtOH + LPS vs. WT EtOH comparison (red increased, blue decreased). (E) Plot of log₂ (Fold-change ranked genes) for the fat-1 EtOH + LPS vs. fat-1 EtOH comparison (red increased, blue decreased). (F) Heatmap of GO processes for the WT EtOH + LPS vs. WT EtOH comparison. (G) Heatmap of GO processes for the fat-1 EtOH + LPS vs. fat-1 EtOH comparison.

Figure 2

Similarity in transcriptional responses of ileum tissue to EtOH + LPS in WT and fat-1 mice. (A) Cluster analysis of ileum genes increased by EtOH + LPS vs. EtOH in both WT and fat-1 mice. Node size indicates relative connectivity. (B) Cluster analysis of ileum genes decreased by EtOH + LPS vs. EtOH in both WT and fat-1 mice.

Figure 3

Exclusive transcriptional responses increased due to EtOH + LPS in WT and fat-1 mice. (A) Cluster analysis of the expression of genes increased exclusively in WT mice in response to EtOH + LPS vs. EtOH. (B) Cluster analysis of ileum genes increased exclusively in fat-1 mice in response to EtOH + LPS vs. EtOH. Node size indicates relative connectivity. Node color indicates relative log₂(Fold-Change) of genes.

Figure 4

Exclusive transcriptional responses decreased due to EtOH + LPS in WT and fat-1 mice. (A) Cluster analysis of ileum genes decreased exclusively in WT mice in response to EtOH + LPS vs. EtOH. (B) Cluster analysis of ileum genes decreased exclusively in fat-1 mice in response to EtOH + LPS vs. EtOH. Node size indicates relative connectivity. Node color indicates relative log₂(Fold-Change) of genes.

Figure 5

Differential transcriptional responses in fat-1 EtOH + LPS vs. WT EtOH + LPS-treated mice. (A) Cluster analysis of ileum genes differentially expressed between fat-1 EtOH + LPS vs. WT EtOH + LPS-treated mice. Node size indicates relative connectivity. Node color indicates relative log₂ (Fold-Change) of genes. (B) Plot of log₂(Fold-change ranked genes) for the fat-1 EtOH + LPS vs. WT EtOH + LPS comparison (red increased, blue decreased). (C) Heatmap of GO processes for the fat-1 EtOH + LPS vs. WT EtOH + LPS comparison.

Figure 6

Increased n-3 PUFAs enhanced the ileum expression of Btnl-mediated T cell and pro-restorative macrophage gene signatures. (A) Heatmap fold change values for ileum Btnl and γδ T cell gene signatures for the WT EtOH + LPS vs. WT EtOH comparison and the fat-1 EtOH + LPS vs. fat-1 EtOH comparison. (B) Ileum Btnl and γδ T cell gene signature expression for WT EtOH + LPS and fat-1 EtOH + LPS-treated mice. (C) Heatmap fold-change values for T_h1, T_reg, and T_h17 cell gene markers for the WT EtOH + LPS vs. WT EtOH comparison and the fat-1 EtOH + LPS vs. fat-1 EtOH comparison. (D) Gene expression of T_h1, T_reg, and T_h17cell gene markers in WT EtOH + LPS vs. fat-1 EtOH + LPS-treated mice. (E) Heatmap fold change values for ileum pro-restorative macrophage markers. (F) Pro-restorative macrophage gene expression for WT EtOH + LPS and fat-1 EtOH + LPS-treated mice. (G) Graphical representation of BTNLs, γδ T cells, and pro-restorative macrophages being enhanced in the ileum by n-3 PUFA enrichment. Statistical significance (p < 0.05) is denoted by an *.

Figure 7

Increased n-3 PUFAs enhanced APRIL-signaling gene expression and IgA + B-Cell markers. (A) Heatmap fold-change values for APRIL signaling genes and IgA genes for the WT EtOH + LPS vs. WT EtOH comparison and the fat-1 EtOH + LPS vs. fat-1 EtOH comparison. (B) APRIL signaling and IgA gene expression for WT EtOH + LPS and fat-1 EtOH + LPS-treated mice. (C) Graphical representation of enhanced APRIL signaling and IgA + B cells in the ileum of mice with n-3 PUFA enrichment. Statistical significance (p < 0.05) is denoted by an *.

Figure 8

Increased n-3 PUFAs attenuated the EtOH + LPS mediated intestinal fibrosis. (A) Heatmap fold-change values for pro-fibrotic receptors and markers for the WT EtOH + LPS vs. WT EtOH comparison and the fat-1 EtOH + LPS vs. fat-1 EtOH comparison. (B) Gene expression of pro-fibrotic receptors in WT EtOH + LPS and fat-1 EtOH + LPS-treated mice. (C) Gene expression of pro-fibrotic markers in WT EtOH + LPS and fat-1 EtOH + LPS-treated mice. (D) Representative Images of Sirius red-stained ileal sections at 200× from WT EtOH + LPS and fat-1 EtOH + LPS-treated (scale bar is 40 μm). (E) Quantification of Sirius red staining area relative to total ileum area for WT EtOH + LPS vs. fat-1 EtOH + LPS mice. (F) Graphical representation of diminished intestinal fibrosis associated with enhanced n-3 PUFAs. Statistical significance (p < 0.05) is denoted by an *.