EPA Induces an Anti-Inflammatory Transcriptome in T Cells, Implicating a Triglyceride-Independent Pathway in Cardiovascular Risk Reduction

Abstract

Twice-daily intake of purified eicosapentaenoic acid (EPA) reduces atherosclerotic cardiovascular disease risk in patients with high triglycerides, but its exact mechanism remains unclear. We exposed non-activated CD4⁺ T cells to 100μM EPA, oleic acid, palmitic acid, or control, and conducted RNA and ATAC-sequencing after 48 hours. EPA exposure downregulated immune response-related genes like HLA-DRA, CD69, and IL2RA, and upregulated oxidative stress prevention genes like NQO1. Transcription factor footprinting showed decreased GATA3 and PU.1, and increased REV-ERB. These effects were specific to EPA, suggesting it induces an anti-inflammatory transcriptomic landscape in CD4⁺ T cells, contributing to its observed cardiovascular benefits.

Keywords: T cells; atherosclerosis; eicosapentaenoic acid; oleic acid; palmitic acid; transcriptomics.

Graphical abstract

Figure 1

Experimental Set-Up and In Vitro Model Verification (A) Experimental set-up for RNA and ATAC sequencing of EPA-, OA-, and PA-exposed nonactivated CD4⁺ T cells; n = 8. (B) Dot plot showing the relative expression of CPT1A after 48 hours of fatty acid exposure as a confirmation of the in vitro model by means of real-time quantitative polymerase chain reaction. Values are colored by fatty acid. The mean fold change (± SEM) demonstrated that CPT1A was up-regulated for EPA (12.4 ± 1.9; P < 0.001), OA (19.5 ± 3.0; P < 0.001), and PA (11.3 ± 2.2; P < 0.003); n = 8. EPA = eicosapentaenoic acid; OA = oleic acid; PA = palmitic acid.

Figure 2

EPA, OA, and PA Exposure in Nonactivated CD4⁺ T Cells Induces Changes in Transcriptomics (A) Volcano plot showing the gene expression of nonactivated CD4⁺ T cells exposed to EPA, OA, or PA. All 19,991 protein-coding genes are shown for each fatty acid. DEGs are colored by fatty acid and denoted by a larger size. Nonsignificant genes are shown in grey and denoted by a smaller size. Log2 fold change is used to show the direction of gene expression. (B) Venn diagram showing the unique response of nonactivated CD4⁺ T cells to each fatty acid. Values are colored by fatty acid. There are 6 DEGs overlapping for all 3 fatty acids, 18 DEGs overlapping for EPA and OA, 4 DEGs overlapping for EPA and PA, and 7 DEGs overlapping for OA and PA. EPA = eicosapentaenoic acid; OA = oleic acid; PA = palmitic acid.

Figure 3

Up- and Down-Regulated Pathways and Transcription Factors in EPA-Exposed Nonactivated CD4⁺ T Cells (A) Pathway enrichment analysis of all down-regulated EPA DEGs generated with the use of clusterProfiler using 10 human pathway databases. Top 10 enrichments are shown. (B) Pathway enrichment analysis of all up-regulated EPA DEGs generated with the use of clusterProfiler using 10 human pathway databases. Top 10 enrichments are shown. (C) Known motif analysis on promotors of down- vs up-regulated EPA ATAC peaks. Enrichment of transcription factor (TF)–binding motifs was performed with the use of HOMER. Four motifs are shown with supplementing information on P value, percentage of genes in up-regulated gene set in down-regulated gene set, transcription factor name, −log(P value), and percentage in sequence. (D) Known motif analysis on promotors of up- vs down-regulated EPA ATAC peaks. Enrichment of transcription factor binding motifs was performed with the use of HOMER. Four motifs are shown with supplementing information on P value, percentage of genes in up-regulated gene set and in down-regulated gene set, transcription factor name, −log(P value), and percentage in sequence. EPA = eicosapentaenoic acid; TF = transcription factor.