(OR23-06-25) Effects of EPA/DHA on Atherosclerosis, Immune Cells, and Inflammation in Female Mice: 12/15-Lipoxygenase-Dependent and -Independent Mechanisms

Objectives: EPA/DHA supplementation may reduce cardiovascular disease, potentially through the conversion of these ω3-fatty acids into pro-resolving lipid mediators by 12/15-lipoxygenase (12/15Lox). This study examines the effects of EPA/DHA on atherosclerosis, immune function, and gut microbiome in female LDL receptor knockout (Ldlr^-/-) mice with and without deletion of the12/15Lox gene (15Lox^-/- and 15Lox^+/+).

Methods: Eight-week old Ldlr^-/-/15Lox^+/+ single knockout (SKO, n=24) and Ldlr^-/-/15Lox^-/- double knockout (DKO, n=24) female mice were randomized to four diet groups (n=6/group): 1) low-fat diet (LFD); 2) LFD + 1% EPA/DHA (w/w); 3) high-fat diet (HFD); and 4) HFD + 1% EPA/DHA. After 16 weeks, mice were euthanized and plasma, aorta, spleen, and gut cecum content were isolated. Immune cells were profiled by flow cytometry, and gut microbiome analysis was performed via shotgun metagenomics sequencing.

Results: In SKO mice, there was a trend toward increased aorta plaque lesion area in the HFD versus LFD groups (6.9±2.5% vs. 3.7±2.0%, mean±SD, p=0.09). In DKO, HFD significantly increased lesion area relative to LFD (6.0±1.7% vs. 2.6±1.4%, p=0.01). EPA/DHA tended to reduce lesion area in HFD-fed SKO mice (3.8±3.0%, p=0.10), but not in DKO mice (4.9±2.5%, p=0.62). In mice fed HFD, EPA/DHA significantly reduced inflammatory splenic monocytes in SKO (19 ±7.2% vs. 10±6.5%, p=0.02), but not DKO mice (13±5.6% vs. 13±7.0%, p=0.99). EPA/DHA significantly lowered plasma triglycerides and LDL cholesterol levels across LFD and HFD, but reduced plasma TNF-α levels only in HFD-fed mice. Notably, plasma levels of 12-HEPE, an EPA-derived metabolite thought to be 12/15Lox-dependent, were elevated by EPA/DHA supplementation in both SKO and DKO mice, both on LFD and HFD. Gut microbiome analyses revealed significant differences in microbial species by genotypes and diet groups.

Conclusions: In female mice, EPA/DHA supplementation lowers diet-induced atherosclerosis in SKO mice but not DKO mice lacking 12/15Lox, possibly mediated by immune cells and gut microbiome as genotype differences were observed in these parameters. However, the conversion of EPA/DHA to downstream metabolites of 12/15Lox likely involves alternative enzymatic pathways to 12/15Lox.

Funding Sources: USDA-NIFA-AFRI 2022-67017-36243