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.

Fat-1 Transgenic Mice With Augmented n3-Polyunsaturated Fatty Acids Are Protected From Liver Injury Caused by Acute-On-Chronic Ethanol Administration

“…These mice were bred in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at the University of Louisville, maintained as heterozygotes, and genotyped as previously described ( Warner et al, 2019 ) using primer sequences to the fat-1 transgene (with Gdf5 as an internal control, Table 1 ). Tissue n3-PUFA enrichment and a decrease in the n6/n3-PUFA ratio in fat-1 mice has been confirmed in our previous reports ( Warner et al, 2019 ; Hardesty et al, 2021 ), and is consistent with that reported by J.X. Kang for these mice ( Kang et al, 2004 ).…”

“…Along with n3-PUFAs, EtOH also induced n6-PUFAs; the exact molecular mechanism responsible for this increase remains to be investigated. A similar phenomenon of elevated n3-PUFA levels was previously observed in the livers of mice chronically fed EtOH ( Hardesty et al, 2021 ; Warner et al, 2021 ), and in the plasma of EtOH-fed rats ( Guiraud et al, 2008 ); however, no change in n3-PUFAs with acute-on-chronic EtOH feeding has also been reported ( Wang et al, 2017 ). This discrepancy may be due to differences in the animal models (e.g., differences in the diet composition, the amount and duration of EtOH administration, etc.…”

“…Our group showed that mice fed a diet high in n6-PUFAs developed more severe manifestations of ALD than those fed a diet high in saturated fats ( Warner et al, 2017 ). We also demonstrated that n3-PUFA endogenous enrichment, with a concomitant decrease in the n6/n3-PUFA ratio (using fat-1 mice that endogenously convert n6-PUFAs to n3-PUFAs), attenuated liver damage in an early-stage ALD mouse model characterized by steatosis and modest liver injury ( Warner et al, 2019 ; Hardesty et al, 2021 ). This protection was afforded by favorable effects on gut barrier function as well as hepatic Wnt signaling ( Warner et al, 2019 ; Hardesty et al, 2021 ).…”

“…We also demonstrated that n3-PUFA endogenous enrichment, with a concomitant decrease in the n6/n3-PUFA ratio (using fat-1 mice that endogenously convert n6-PUFAs to n3-PUFAs), attenuated liver damage in an earlystage ALD mouse model characterized by steatosis and modest liver injury (Warner et al, 2019;Hardesty et al, 2021). This protection was afforded by favorable effects on gut barrier function as well as hepatic Wnt signaling (Warner et al, 2019;Hardesty et al, 2021). Similarly, Huang et al demonstrated decreased acute ethanol (EtOH)-induced liver injury and steatosis, as well as decreased lipogenic gene expression, in fat-1 mice (Huang et al, 2015).…”

“…Plasma alanine aminotransferase activity (ALT, an indirect measure of hepatocyte cell death) was determined using the ALT/GPT reagent as per the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). Liver tissue hematoxylin and eosin (H&E) staining for gross tissue morphology and Oil Red O staining for neutral lipids were performed as described previously ( Hardesty et al, 2021 ). From H&E-stained sections, lipid accumulation was quantified by steatosis scoring, where a score of 0 indicates 0–5% tissue coverage by lipid droplets, 1 = 5–33% coverage, 2 = 33–66% coverage, and 3 = 66–100% coverage ( Bedossa and Consortium, 2014 ).…”