Targeting the ACOD1-itaconate axis stabilizes atherosclerotic plaques.

Inflammatory macrophages are key drivers of atherosclerosis that can induce rupture-prone vulnerable plaques. Skewing the plaque macrophage population towards a more protective phenotype and reducing the occurrence of clinical events is thought to be a promising method of treating atherosclerotic patients. In the current study, we investigate the immunomodulatory properties of itaconate, an immunometabolite derived from the TCA cycle intermediate cis-aconitate and synthesised by the enzyme Aconitate Decarboxylase 1 (ACOD1, also known as IRG1), in the context of atherosclerosis. Ldlr-/- atherogenic mice transplanted with Acod1-/- bone marrow displayed a more stable plaque phenotype with smaller necrotic cores and showed increased recruitment of monocytes to the vessel intima. Macrophages from Acod1-/- mice contained more lipids whilst also displaying reduced induction of apoptosis. Using multi-omics approaches, we identify a metabolic shift towards purine metabolism, in addition to an altered glycolytic flux towards production of glycerol for triglyceride synthesis. Overall, our data highlight the potential of therapeutically blocking ACOD1 with the aim of stabilizing atherosclerotic plaques.

An integrated toolbox to profile macrophage immunometabolism.

Macrophages are dynamic immune cells that can adopt several activation states. Fundamental to these functional activation states is the regulation of cellular metabolic processes. Especially in mice, metabolic alterations underlying pro-inflammatory or homeostatic phenotypes have been assessed using various techniques. However, researchers new to the field may encounter ambiguity in choosing which combination of techniques is best suited to profile immunometabolism. To address this need, we have developed a toolbox to assess cellular metabolism in a semi-high-throughput 96-well-plate-based format. Application of the toolbox to activated mouse and human macrophages enables fast metabolic pre-screening and robust measurement of extracellular fluxes, mitochondrial mass and membrane potential, and glucose and lipid uptake. Moreover, we propose an application of SCENITH technology for ex vivo metabolic profiling. We validate established activation-induced metabolic rewiring in mouse macrophages and report new insights into human macrophage metabolism. By thoroughly discussing each technique, we hope to guide readers with practical workflows for investigating immunometabolism.

d-2-Hydroxyglutarate is an anti-inflammatory immunometabolite that accumulates in macrophages after TLR4 activation.

Macrophages undergo extensive metabolic rewiring upon activation which assist the cell in roles beyond energy production and synthesis of anabolic building blocks. So-called immunometabolites that accumulate upon immune activation can serve as co-factors for enzymes and can act as signaling molecules to modulate cellular processes. As such, the Krebs-cycle-associated metabolites succinate, itaconate and alpha-ketoglutarate (αKG) have emerged as key regulators of macrophage function. Here, we describe that 2-hydroxyglutarate (2HG), which is structurally similar to αKG and exists as two enantiomers, accumulates during later stages of LPS-induced inflammatory responses in mouse and human macrophages. D-2HG was the most abundant enantiomer in macrophages and its LPS-induced accumulation followed the induction of Hydroxyacid-Oxoacid Transhydrogenase (HOT). HOT interconverts αKG and gamma-hydroxybutyrate into D-2HG and succinic semialdehyde, and we here identified this enzyme as being immune-responsive and regulated during the course of macrophage activation. The buildup of D-2HG may be further explained by reduced expression of D-2HG Dehydrogenase (D2HGDH), which converts D-2HG back into αKG, and showed inverse kinetics with HOT and D-2HG levels. We tested the immunomodulatory effects of D-2HG during LPS-induced inflammatory responses by transcriptomic analyses and functional profiling of D-2HG-pre-treated macrophages in vitro and mice in vivo. Together, these data suggest a role for D-2HG in the negative feedback regulation of inflammatory signaling during late-stage LPS-responses in vitro and as a regulator of local and systemic inflammatory responses in vivo. Finally, we show that D-2HG likely exerts distinct anti-inflammatory effects, which are in part independent of αKG-dependent dioxygenase inhibition. Together, this study reveals an immunometabolic circuit resulting in the accumulation of the immunomodulatory metabolite D-2HG that can inhibit inflammatory macrophage responses.

Macrophages are metabolically heterogeneous within the tumor microenvironment.

Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although metabolism influences macrophage function, studies on the metabolic characteristics of ex vivo tumor-associated macrophage (TAM) subsets are rather limited. Using transcriptomic and metabolic analyses, we now reveal that pro-inflammatory major histocompatibility complex (MHC)-IIhi TAMs display a hampered tricarboxylic acid (TCA) cycle, while reparative MHC-IIlo TAMs show higher oxidative and glycolytic metabolism. Although both TAM subsets rapidly exchange lactate in high-lactate conditions, only MHC-IIlo TAMs use lactate as an additional carbon source. Accordingly, lactate supports the oxidative metabolism in MHC-IIlo TAMs, while it decreases the metabolic activity of MHC-IIhi TAMs. Lactate subtly affects the transcriptome of MHC-IIlo TAMs, increases L-arginine metabolism, and enhances the T cell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as a carbon source and metabolic and functional regulator of TAMs.

Myeloid-Specific Acly Deletion Alters Macrophage Phenotype In Vitro and In Vivo without Affecting Tumor Growth.

Cancer cells rely on ATP-citrate lyase (Acly)-derived acetyl-CoA for lipid biogenesis and proliferation, marking Acly as a promising therapeutic target. However, inhibitors may have side effects on tumor-associated macrophages (TAMs). TAMs are innate immune cells abundant in the tumor microenvironment (TME) and play central roles in tumorigenesis, progression and therapy response. Since macrophage Acly deletion was previously shown to elicit macrophages with increased pro- and decreased anti-inflammatory responses in vitro, we hypothesized that Acly targeting may elicit anti-tumor responses in macrophages, whilst inhibiting cancer cell proliferation. Here, we used a myeloid-specific knockout model to validate that absence of Acly decreases IL-4-induced macrophage activation. Using two distinct tumor models, we demonstrate that Acly deletion slightly alters tumor immune composition and TAM phenotype in a tumor type-dependent manner without affecting tumor growth. Together, our results indicate that targeting Acly in macrophages does not have detrimental effects on myeloid cells.

IDH-Mutant Brain Tumors Hit the Achilles' Heel of Macrophages with R-2-Hydroxyglutarate.

Isocitrate dehydrogenase (IDH) mutations produce high levels of the 'oncometabolite' R-2-hydroxyglutarate (R-2-HG) and play a key role in the initiation and progression of glioma tumors in the brain. A recent study in Nature Cancer by Friedrich et al. describes how IDH-mutant-derived R-2-HG elicits an immunosuppressive phenotype in glioma-associated macrophages. As such, the authors uncovered a new vulnerability that can be exploited for therapy.

Myeloid ATP Citrate Lyase Regulates Macrophage Inflammatory Responses In Vitro Without Altering Inflammatory Disease Outcomes.

Macrophages are highly plastic, key regulators of inflammation. Deregulation of macrophage activation can lead to excessive inflammation as seen in inflammatory disorders like atherosclerosis, obesity, multiple sclerosis and sepsis. Targeting intracellular metabolism is considered as an approach to reshape deranged macrophage activation and to dampen the progression of inflammatory disorders. ATP citrate lyase (Acly) is a key metabolic enzyme and an important regulator of macrophage activation. Using a macrophage-specific Acly-deficient mouse model, we investigated the role of Acly in macrophages during acute and chronic inflammatory disorders. First, we performed RNA sequencing to demonstrate that Acly-deficient macrophages showed hyperinflammatory gene signatures in response to acute LPS stimulation in vitro. Next, we assessed endotoxin-induced peritonitis in myeloid-specific Acly-deficient mice and show that, apart from increased splenic Il6 expression, systemic and local inflammation were not affected by Acly deficiency. Also during obesity, both chronic low-grade inflammation and whole-body metabolic homeostasis remained largely unaltered in mice with Acly-deficient myeloid cells. Lastly, we show that macrophage-specific Acly deletion did not affect the severity of experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis. These results indicate that, despite increasing inflammatory responses in vitro, macrophage Acly deficiency does not worsen acute and chronic inflammatory responses in vivo. Collectively, our results indicate that caution is warranted in prospective long-term treatments of inflammatory disorders with macrophage-specific Acly inhibitors. Together with our earlier observation that myeloid Acly deletion stabilizes atherosclerotic lesions, our findings highlight that therapeutic targeting of macrophage Acly can be beneficial in some, but not all, inflammatory disorders.

Metabolic Cancer-Macrophage Crosstalk in the Tumor Microenvironment.

Tumors consist of a wide variety of cells, including immune cells, that affect tumor progression. Macrophages are abundant innate immune cells in the tumor microenvironment (TME) and are crucial in regulating tumorigenicity. Specific metabolic conditions in the TME can alter the phenotype of tumor-associated macrophages (TAMs) in a direction that supports their pro-tumor functions. One of these conditions is the accumulation of metabolites, also known as oncometabolites. Interactions of oncometabolites with TAMs can promote a pro-tumorigenic phenotype, thereby sustaining cancer cell growth and decreasing the chance of eradication. This review focuses on the metabolic cancer-macrophage crosstalk in the TME. We discuss how cancer cell metabolism and oncometabolites affect macrophage phenotype and function, and conversely how macrophage metabolism can impact tumor progression. Lastly, we propose tumor-secreted exosome-mediated metabolic signaling as a potential factor in tumorigenesis. Insight in these processes may contribute to the development of novel cancer therapies.

Succinate Is an Inflammation-Induced Immunoregulatory Metabolite in Macrophages.

Immunometabolism revealed the crucial role of cellular metabolism in controlling immune cell phenotype and functions. Macrophages, key immune cells that support progression of numerous inflammatory diseases, have been well described as undergoing vast metabolic rewiring upon activation. The immunometabolite succinate particularly gained a lot of attention and emerged as a crucial regulator of macrophage responses and inflammation. Succinate was originally described as a metabolite that supports inflammation via distinct routes. Recently, studies have indicated that succinate and its receptor SUCNR1 can suppress immune responses as well. These apparent contradictory effects might be due to specific experimental settings and particularly the use of distinct succinate forms. We therefore compared the phenotypic and functional effects of distinct succinate forms and receptor mouse models that were previously used for studying succinate immunomodulation. Here, we show that succinate can suppress secretion of inflammatory mediators IL-6, tumor necrosis factor (TNF) and nitric oxide (NO), as well as inhibit Il1b mRNA expression of inflammatory macrophages in a SUCNR1-independent manner. We also observed that macrophage SUCNR1 deficiency led to an enhanced inflammatory response without addition of exogenous succinate. While our study does not reveal new mechanistic insights into how succinate elicits different inflammatory responses, it does indicate that the inflammatory effects of succinate and its receptor SUCNR1 in macrophages are clearly context dependent.

Intestinal Macrophages Balance Inflammatory Expression Profiles via Vitamin A and Dectin-1-Mediated Signaling.

Tissue resident intestinal macrophages are known to exhibit an anti-inflammatory phenotype and produce little pro-inflammatory cytokines upon TLR ligation, allowing symbiotic co-existence with the intestinal microbiota. However, upon acute events such as epithelial damage and concomitant influx of microbes, these macrophages must be able to quickly mount a pro-inflammatory response while more inflammatory macrophages are recruited from the blood stream simultaneously. Here, we show that dietary intake of vitamin A is required for the maintenance of the anti-inflammatory state of tissue resident intestinal macrophages. Interestingly, these anti-inflammatory macrophages were characterized by high levels of Dectin-1 expression. We show that Dectin-1 expression is enhanced by the vitamin A metabolite retinoic acid and our data suggests that Dectin-1 triggering might provide a switch to induce a rapid production of pro-inflammatory cytokines. In addition, Dectin-1 stimulation resulted in an altered metabolic profile which is linked to a pro-inflammatory response. Together, our data suggests that presence of vitamin A in the small intestine enhances an anti-inflammatory phenotype as well as Dectin-1 expression by macrophages and that this anti-inflammatory phenotype can rapidly convert toward a pro-inflammatory state upon Dectin-1 signaling.

Let's Enter the Wonderful World of Immunometabolites.

Over the past decade, cancer metabolism research taught us that metabolites are much more than intermediate or end products of metabolism. As such, the name 'oncometabolite' emerged. Immunometabolism research has developed tremendously over the past few years and, in analogy to the cancer metabolism field, the term 'immunometabolite' has been used for different metabolites and purposes. Here, we propose a definition for the term 'immunometabolite' and provide some historical background and future perspectives on this matter. By doing so, we aim to increase interest in this fast-expanding field and to encourage further research.