Transcriptomics and Targeted Metabolomics Reveal That <scp>CES1</scp> Ablation in Macrophages Confers a Distinct Inflammatory Phenotype Involving Altered Interleukin‐1β, Citrate, and Lipid Levels

Inflammation is beneficial when the initial threat is neutralized and the response is self-limiting, but when unresolved, it is often deleterious. Macrophages are immune cells that differentiate into classically activated (M1) and alternatively activated (M2) cells that promote and attenuate inflammation, respectively. Carboxylesterase 1 (CES1) is a serine hydrolase that metabolizes neutral lipids, including triacylglycerols (TAGs) and others. THP-1 macrophages with deficient CES1 expression (CES1KD cells) have a distinctly foamy phenotype as compared to CES1-expressing THP-1 macrophages (control cells), which is due to greater quantities of TAG-containing lipid droplets. CES1KD cells also produce more IL-1β and PGE2 than control cells following lipopolysaccharide (LPS) stimulation, with no evidence of pyroptosis. Here, we examined the proinflammatory phenotype of CES1KD cells in more detail by examining their transcriptomic and inflammatory signatures. Based on RNA-seq data, we observed a strong concordance between the differentially expressed genes in baseline (unstimulated) CES1KD cells and those in control cells treated with LPS/IFNγ (r = 0.728). This is consistent with the altered metabolite profiles that were observed in CES1KD cells (elevated levels of citrate, prostaglandins, and TAGs) and increased expression of HIF1α, pro-caspase-1, and pro-IL-1β that together signify a proinflammatory phenotype. Gene Ontology, KEGG, and Reactome analyses of CES1KD cells in their baseline state revealed the enrichment of upregulated pathways involved in antibacterial and antiviral defense and inflammasome signaling comparable to those seen in control cells stimulated with LPS/IFNγ. NLRP3 inflammasome-dependent production of IL-1β was much more pronounced in CES1KD cells and did not require an LPS priming step. We conclude that CES1KD macrophages, even under baseline conditions (M0) exhibit an immunophenotype like that of M1 control cells. These findings suggest that active CES1 may negatively regulate macrophage inflammatory immune responses, and thus enhancing its activity might be a novel strategy to attenuate inflammation.