Triacylglycerol synthesis enhances macrophage inflammatory function

Macrophages frequently develop lipid droplets (LDs), composed largely of triacylglycerols (TGs) and cholesterol esters, in diverse disease contexts such as atherosclerosis, tuberculosis, obesity, cancer, and neuroinflammation. While LDs can serve as energy stores via TG lipolysis and subsequent fatty acid oxidation (FAO), inflammatory activation of macrophages diminishes FAO and oxidative phosphorylation while promoting de novo fatty acid synthesis and TG accumulation. The functional significance of TG-rich LD formation under inflammatory stimulation remains unclear, particularly because FAO is suppressed. LDs have been implicated in additional roles including sequestration of toxic lipids, mitigation of ER stress, autophagosome lipid supply, and as platforms for eicosanoid biosynthesis from arachidonic acid. This study investigates how TG synthesis and LD biogenesis contribute to inflammatory macrophage activation, focusing on their impact on inflammatory mediator production and phagocytic function, and probing the mechanistic link to prostaglandin E2 (PGE2) synthesis.

Prior work established that Toll-like receptor agonists drive prolonged TG storage and LD formation in macrophages, and that inflammatory activation reprograms metabolism toward glycolysis (Warburg metabolism) with altered TCA cycle flux and increased fatty acid synthesis. TG synthesis is catalyzed by DGAT1/2, with LDs known to provide functions beyond energy storage, including lipid detoxification, ER homeostasis, and serving as sites for eicosanoid synthesis. Foam cells are prevalent in atherosclerosis and infections such as tuberculosis, with emerging evidence in multiple sclerosis, certain cancers, obesity, and vaping-related lung disease. The ratio of TGs to cholesterol esters within LDs may influence inflammatory phenotypes. In inflammatory macrophages, FAO is downregulated despite increased Cpt1a expression, suggesting complex regulation of fatty acid handling. Autocrine PGE2 has been implicated in promoting pro-IL-1β expression. However, the specific contribution of TG synthesis and LDs to eicosanoid production and inflammatory effector functions had not been fully defined.

• Cells and stimulation: Primary murine bone marrow-derived macrophages (BMDMs) were generated in RPMI-1640 with 10 mM glucose, 2 mM L-glutamine, antibiotics, 10% FCS, and 20 ng/mL CSF-1 for 7 days. Inflammatory activation used 50 ng/mL IFN-γ plus 20 ng/mL LPS for 18 h; alternative activation used 20 ng/mL IL-4.
• Pharmacologic and genetic perturbations: TG synthesis was inhibited with the selective DGAT1 inhibitor T863 (DGAT1i; 50 μM in vitro). DGAT1 expression was suppressed using lentiviral shRNA (pLKO.1 vector; puromycin selection). Where indicated, exogenous PGE2 (10 μM) was added 1 h after stimulation.
• In vivo LPS challenge: C57BL/6 mice (6–8 weeks) received T863 (5 mg/kg, i.p.) or vehicle 30 min before i.p. LPS (8 mg/kg). Body temperature was monitored every 3 h. Serum and peritoneal lavage were collected at 2 h or 10 h.
• Lipidomics and targeted metabolomics: Lipids were extracted using the MTBE method and quantified by LC-MS (Agilent UHPLC-QQQ). Peak areas were quantile-normalized. FA 20:4 (arachidonic acid) within TGs and phospholipids was profiled by LC-MS on a QTOF with identification via LipidBlast. Targeted metabolites (e.g., carnitine, acylcarnitines, itaconate) were quantified by LC-MS with MRM.
• Stable isotope tracing: 13C-palmitate (100 μM, 18 h) tracing assessed incorporation into C16 acylcarnitine and citrate. 13C-glucose (10 mM, 18 h) traced contributions to lactate and citrate. 13C-glycerol (10 mM, 30 min prior to harvest) assessed labeling of glycerol phosphate and glycolytic intermediates by GC-MS.
• Flow cytometry and imaging: LDs assessed by BODIPY 493/503 staining; FA uptake by BODIPY FL C16. Cytokines measured by intracellular staining for pro-IL-1β (FITC) and IL-6 (PE) in F4/80+ cells; Brefeldin A used to block secretion. Mitochondrial parameters: membrane potential (TMRM), mass (MitoTracker Deep Red), ROS (MitoSOX). ER content measured with ER-Tracker dyes. Phagocytosis measured using pHrodo Red Staphylococcus aureus bioparticles. Confocal microscopy imaged LDs; transmission EM evaluated ultrastructure and LD counts.
• RNA sequencing: TruSeq stranded mRNA libraries sequenced (HiSeq 3000). Differential expression analyzed with DESeq2; pathway analysis performed on DGAT1i-induced changes.
• Cytokine ELISAs: IL-1β and IL-6 quantified in supernatants or serum (BioLegend ELISA Max kits).
• Seahorse assays: Basal extracellular acidification rate (ECAR) measured under standard conditions.
• Statistics: Unpaired two-tailed Student’s t tests or one-way ANOVA with Bonferroni correction; data shown as mean ± s.e.m.; p < 0.05 considered significant.

• Inflammatory activation (IFN-γ + LPS) upregulated genes for TG synthesis and utilization, notably Dgat1 (dominant over Dgat2) and Gpat3, and led to robust accumulation of total TGs and multiple TG species alongside LD formation (BODIPY 493/503 increase).
• Free fatty acids and diacylglycerols decreased in activated macrophages, consistent with utilization for TG synthesis; lipidomics showed early (≥2 h) accumulation of PCs, PIs, PEs, LPCs, SMs, CEs, and HEXCERs.
• Despite suppressed FAO, Cpt1a expression increased, with decreased carnitine and increased long-chain acylcarnitines. 13C-palmitate tracing showed increased labeling of C16 acylcarnitine but reduced labeling of citrate, confirming diminished FAO/TCA entry.
• DGAT1 inhibition (T863) or Dgat1 shRNA reduced total TGs and specific TG species, decreased LDs (microscopy, flow cytometry), decreased BODIPY FL C16 uptake, and increased DAG substrates. Free FAs were unchanged.
• Loss of DGAT1 function reduced inflammatory output: significant decreases in pro-IL-1β and IL-6 production (protein and mRNA), and reduced expression of additional inflammatory genes (e.g., Ccl3, Ccl4, Nlrp1b). Phagocytic capacity was diminished.
• In vivo, T863 pretreatment attenuated LPS-induced systemic inflammation: reduced hypothermia, reduced LD staining in resident and recruited peritoneal macrophages, and decreased serum IL-1β (2 h) and IL-6 (10 h).
• Core metabolic programs of inflammatory activation were largely unaffected by DGAT1 loss: glucose carbon contribution to lactate and citrate, basal ECAR, and itaconate pools were unchanged. Mitochondrial membrane potential and mass were maintained or increased, while mitochondrial ROS rose; no induction of mitochondrial UPR genes. ER content increased further with DGAT1 inhibition without evidence of ER stress gene activation.
• Transcriptomics of DGAT1-inhibited cells identified downregulation of pathways linked to eicosanoid and prostaglandin secretion. PGE2 production increased upon inflammatory activation but was significantly suppressed by DGAT1 inhibition.
• Activated macrophages accumulated arachidonic acid (FA 20:4)-containing TG species; DGAT1 inhibition prevented this AA-TG reservoir.
• Exogenous PGE2 restored and enhanced pro-IL-1β and IL-6 production in DGAT1-inhibited or shDgat1-transduced cells and rescued phagocytic competence. The fold increase induced by PGE2 was greater under DGAT1 inhibition, indicating that impaired PGE2 synthesis explains much of the reduced inflammatory function when TG synthesis is blocked.

The study shows that TG synthesis and LD biogenesis are integral to the inflammatory program of macrophages. Inflammatory cues drive a transcriptional program favoring TG synthesis (predominantly via DGAT1), FA sequestration into LDs, and accumulation of long-chain acylcarnitines due to increased Cpt1a activity in the context of curtailed FAO. Functionally, LDs provide a reservoir of arachidonic acid within AA-containing TGs, facilitating efficient PGE2 production. PGE2, in turn, acts in an autocrine manner to amplify pro-IL-1β expression and promote broader inflammatory cytokine output and phagocytic function. Blocking TG synthesis disrupts LD formation, depletes AA-TG pools, lowers PGE2, and thereby dampens inflammatory activation; exogenous PGE2 rescues these defects, pinpointing PGE2 insufficiency as a critical mediator of the phenotype. Notably, the impairment in inflammatory function occurs without major alterations in glycolysis, citrate flux, or itaconate, indicating that TG/LD-dependent eicosanoid biology is a distinct and necessary arm of inflammatory activation. The in vivo attenuation of LPS-induced systemic inflammation by DGAT1 inhibition supports the physiological relevance and highlights TG synthesis/LDs as potential therapeutic targets in hyperinflammatory conditions.

This work establishes that triacylglycerol synthesis, largely via DGAT1, and the consequent lipid droplet biogenesis are essential for maximal inflammatory macrophage function by enabling PGE2 production from arachidonic acid stored in AA-containing TGs. Inhibiting TG synthesis reduces LDs, PGE2, IL-1β and IL-6 production, and phagocytosis, and mitigates LPS-induced systemic inflammation in vivo. The findings position LDs as functional organelles central to eicosanoid-mediated amplification of inflammation and suggest that targeting TG synthesis/LD formation could modulate pathological inflammation. Future studies should dissect the precise enzymatic steps and LD-associated machinery coupling AA-TG mobilization to PGE2 synthesis, evaluate macrophage-specific DGAT1 genetic models in vivo, and explore the roles and fate of accumulated long-chain acylcarnitines during inflammatory activation.

• Pharmacological inhibition with T863 may have off-target effects and acts systemically in vivo; macrophage-intrinsic specificity was not genetically confirmed in vivo.
• Experiments primarily used murine BMDMs and an IFN-γ + LPS activation model; generalizability to human macrophages and diverse inflammatory contexts requires validation.
• The study did not employ macrophage-specific DGAT1 knockout mice to definitively attribute in vivo effects to macrophages.
• While increased long-chain acylcarnitines were observed upon DGAT1 inhibition, their functional significance and potential extracellular roles were not directly tested.
• Mechanistic details of how AA is mobilized from AA-TG within LDs to PGE2 synthesis (enzymatic players, compartmentalization) were inferred but not fully delineated.