ABSTRACT: Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity chronic and autoimmune diseases. These ameliorative effects have been conventionally linked in part to downregulation of expression of proinflammatory cytokine and chemokine genes and more recently, associated with inhibition of Type 1 interferon (IFN1)-regulated gene expression. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives of how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24 h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4 h, and then subjected to scRNAseq using 10X Chromium System. At 0 h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (Sqstm1, Hmox1, Chchd10) and metal homeostasis (Mt1, Mt2, Ftl1, Fth1) suggesting cells are polarized towards a more anti-inflammatory phenotype. At 1 h post-treatment, DHA inhibited LPS-induced cholesterol synthesis genes (Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4 h post-treatment, LPS-treated FLMs reflected a robust proinflammatory response including upregulation of cytokine (Il1a, Il1b, Tnf) and chemokine (Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated (Irf7, Mx1, Oasl1, Ifit1) genes, many of which were suppressed by DHA. Using SCENIC analysis to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Moreover, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs, suggesting that DHA more potently targets the IFN1 response over the NF-kB response. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages
