Triacylglycerol synthesis enhances macrophage inflammatory function

Macrophages are essential immune cells found in most tissues. Foam cells, macrophages containing lipid droplets (LDs) rich in triacylglycerols (TGs) and cholesterol esters (CEs), are observed in numerous disease states. While LDs can serve as energy stores through TG lipolysis and fatty acid (FA) oxidation, pro-inflammatory signals in macrophages lead to decreased FA oxidation and increased TG synthesis, resulting in LD development. Inflammation, while crucial for protection, can cause disease when dysregulated, with IL-1β and other pro-inflammatory cytokines playing a significant role. Macrophage metabolic reprogramming is intrinsically linked to inflammatory activation, characterized by enhanced Warburg metabolism, diminished oxidative phosphorylation (OXPHOS), and altered TCA cycle dynamics. Increased FA synthesis in inflammatory macrophages is associated with TG and CE accumulation within LDs. TG synthesis depends on DGAT1/2 enzymes. LDs, while primarily energy storage organelles in adipocytes, also have diverse functions in other cell types, including toxic lipid sequestration, ER stress prevention, and serving as lipid donors for autophagosome formation. Importantly, LDs act as platforms for eicosanoid production from arachidonic acid (AA). LD-containing macrophages are prevalent in atherosclerosis, tuberculosis, multiple sclerosis, certain cancers, obese adipose tissue, and vaping-related lung disease. The TG:CE ratio in LDs is likely significant, and the pro-inflammatory nature of CE-rich LD macrophages is still debated. This study aims to investigate the significance of LDs in inflammatory macrophages, focusing on the impact of TG synthesis on macrophage function.

Existing research demonstrates a link between inflammatory activation signals and metabolic changes in macrophages. Studies have shown that Toll-like receptor agonists promote triglyceride storage in macrophages, and that inhibition of TG hydrolysis negatively impacts phagocytic capacity. The role of lipid droplets (LDs) in inflammation has been explored, with evidence suggesting they act as platforms for eicosanoid production. However, the precise functional significance of LDs in inflammatory macrophages, particularly the role of triacylglycerol (TG) synthesis, remained unclear. Previous work has highlighted the presence of foam cells in various diseases, and the importance of the TG/CE ratio in LDs is being investigated. This study builds upon these existing findings to directly address the functional role of TG synthesis and LD accumulation in inflammatory macrophage activation.

The study utilized both in vitro and in vivo approaches. In vitro experiments employed bone marrow-derived macrophages (BMDMs) stimulated with lipopolysaccharide (LPS) and interferon-γ (IFNγ) to induce inflammatory activation. The effects of inhibiting TG synthesis were investigated using a selective DGAT1 inhibitor (T863) and DGAT1 shRNA. Lipidomics analysis was performed to assess changes in lipid profiles. Metabolic tracing experiments using ¹³C-labeled substrates (palmitate, glucose, glycerol) were conducted to track carbon metabolism. RNA sequencing was used to analyze gene expression changes. Flow cytometry was used to measure cytokine production (IL-1β, IL-6), phagocytosis, mitochondrial function, ER mass, and lipid droplet content. In vivo studies involved injecting C57BL/6 mice with LPS and T863 to assess the impact of DGAT1 inhibition on inflammation in vivo. Serum cytokine levels and peritoneal macrophage LD formation were analyzed. Electron microscopy was used to visualize mitochondrial morphology and lipid droplet accumulation. Statistical analysis included unpaired two-tailed Student's t tests and one-way ANOVA with Bonferroni's multiple comparison tests.

Inflammatory macrophages exhibit metabolic reprogramming, diverting resources towards TG synthesis and accumulating LDs. Both Dgat1 and Dgat2, the enzymes responsible for TG synthesis, are upregulated. Inhibition of TG synthesis using DGAT1 inhibitor T863 or DGAT1 shRNA significantly reduced LD accumulation, as visualized by microscopy and flow cytometry. This inhibition also led to a marked decrease in the production of pro-inflammatory cytokines IL-1β and IL-6, and a reduction in phagocytic capacity. In vivo experiments demonstrated that T863 treatment reduced LPS-induced systemic inflammation and decreased serum levels of IL-1β and IL-6. Interestingly, inhibition of TG synthesis did not affect core metabolic pathways like glycolysis or itaconate production, but it did increase mitochondrial ROS. The study revealed that a key effect of inhibiting TG synthesis was a significant reduction in PGE2 production. Exogenous PGE2 restored IL-1β and IL-6 production and phagocytic capacity in macrophages with inhibited TG synthesis, suggesting PGE2 acts as a critical downstream mediator of TG synthesis's effects. The accumulation of AA-containing TGs in LDs provides substrate for PGE2 synthesis, and this is disrupted by TG synthesis inhibition.

This study demonstrates a previously unrecognized crucial role for TG synthesis and LD accumulation in the inflammatory response of macrophages. The findings suggest that the metabolic reprogramming observed in inflammatory macrophages is not merely a consequence of activation, but actively contributes to their function. The dependence of inflammatory responses on PGE2 highlights a potential therapeutic target. The observed increase in long-chain acylcarnitines when TG synthesis is inhibited suggests an alternative mechanism for handling excess FAs in the absence of LD formation, although the functional significance of this accumulation needs further exploration. The lack of effect on core metabolic pathways like glycolysis and itaconate production indicates that TG synthesis operates in a parallel pathway important for maximizing inflammatory function. The impact of DGAT1 inhibition on PGE2 production and the ability of exogenous PGE2 to rescue inflammatory function underscore the importance of this pathway in inflammatory macrophage activation.

This research establishes that triacylglycerol synthesis via DGAT1 is essential for optimal inflammatory macrophage activation. Inhibition of TG synthesis leads to decreased LD formation, reduced production of inflammatory mediators (including IL-1β, IL-6, and PGE2), and impaired phagocytosis. The critical role of PGE2 in this process suggests that targeting TG synthesis could be a valuable therapeutic strategy for inflammatory diseases. Future research should investigate the potential off-target effects of the DGAT1 inhibitor and the precise mechanisms through which PGE2 amplifies inflammation.

While the study strongly suggests a causal link between TG synthesis and inflammatory macrophage function, the use of a DGAT1 inhibitor raises the possibility of off-target effects. Further studies with genetic knockout models would strengthen the findings. The in vivo experiments primarily focused on systemic inflammation; further research could examine the impact of DGAT1 inhibition on specific inflammatory tissues. The mechanisms underlying the increased mitochondrial ROS and long-chain acylcarnitine accumulation in the absence of TG synthesis warrant further investigation.