Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming.

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.

Itaconic acid underpins hepatocyte lipid metabolism in non-alcoholic fatty liver disease in male mice.

Itaconate, the product of the decarboxylation of cis-aconitate, regulates numerous biological processes. We and others have revealed itaconate as a regulator of fatty acid ß-oxidation, generation of mitochondrial reactive oxygen species and the metabolic interplay between resident macrophages and tumors. In the present study, we show that itaconic acid is upregulated in human non-alcoholic steatohepatitis and a mouse model of non-alcoholic fatty liver disease. Male mice deficient in the gene responsible for itaconate production (immunoresponsive gene (Irg)-1) have exacerbated lipid accumulation in the liver, glucose and insulin intolerance and mesenteric fat deposition. Treatment of mice with the itaconate derivative, 4-octyl itaconate, reverses dyslipidemia associated with high-fat diet feeding. Mechanistically, itaconate treatment of primary hepatocytes reduces lipid accumulation and increases their oxidative phosphorylation in a manner dependent upon fatty acid oxidation. We propose a model whereby macrophage-derived itaconate acts in trans upon hepatocytes to modulate the liver's ability to metabolize fatty acids.

Nitric Oxide Modulates Metabolic Processes in the Tumor Immune Microenvironment.

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.

Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages.

Glutamine synthetase (GS) generates glutamine from glutamate and controls the release of inflammatory mediators. In macrophages, GS activity, driven by IL10, associates to the acquisition of M2-like functions. Conditional deletion of GS in macrophages inhibits metastasis by boosting the formation of anti-tumor, M1-like, tumor-associated macrophages (TAMs). From this basis, we evaluated the pharmacological potential of GS inhibitors in targeting metastasis, identifying glufosinate as a specific human GS inhibitor. Glufosinate was tested in both cultured macrophages and on mice bearing metastatic lung, skin and breast cancer. We found that glufosinate rewires macrophages toward an M1-like phenotype both at the primary tumor and metastatic site, countering immunosuppression and promoting vessel sprouting. This was also accompanied to a reduction in cancer cell intravasation and extravasation, leading to synchronous and metachronous metastasis growth inhibition, but no effects on primary tumor growth. Glufosinate treatment was well-tolerated, without liver and brain toxicity, nor hematopoietic defects. These results identify GS as a druggable enzyme to rewire macrophage functions and highlight the potential of targeting metabolic checkpoints in macrophages to treat cancer metastasis.

Metabolic but not transcriptional regulation by PKM2 is important for natural killer cell responses.

Natural Killer (NK) cells have an important role in immune responses to viruses and tumours. Integrating changes in signal transduction pathways and cellular metabolism is essential for effective NK cells responses. The glycolytic enzyme Pyruvate Kinase Muscle 2 (PKM2) has described roles in regulating glycolytic flux and signal transduction, particularly gene transcription. While PKM2 expression is robustly induced in activated NK cells, mice lacking PKM2 in NK cells showed no defect in NK cell metabolism, transcription or antiviral responses to MCMV infection. NK cell metabolism was maintained due to compensatory PKM1 expression in PKM2-null NK cells. To further investigate the role of PKM2, we used TEPP-46, which increases PKM2 catalytic activity while inhibiting any PKM2 signalling functions. NK cells activated with TEPP-46 had reduced effector function due to TEPP-46-induced increases in oxidative stress. Overall, PKM2-regulated glycolytic metabolism and redox status, not transcriptional control, facilitate optimal NK cells responses.

TREML4 Promotes Inflammatory Programs in Human and Murine Macrophages and Alters Atherosclerosis Lesion Composition in the Apolipoprotein E Deficient Mouse.

The Triggering Receptor Expressed on Myeloid cells-like 4 (TREML4) is a member of the TREM receptor family, known modulators of inflammatory responses. We have previously found that TREML4 expression positively correlates with human coronary arterial calcification (CAC). However, the role of TREML4 in the pathogenesis of cardiovascular disease remains incompletely defined. Since macrophages play a key role in inflammatory conditions, we investigated if activated macrophages selectively expressed TREML4 and found that carriage of either one of the eQTL SNP's previously associated with increased TREML4 expression conferred higher expression in human inflammatory macrophages (M1) compared to alternatively activated macrophages (M2). Furthermore, we found that TREML4 expression in human M1 dysregulated several inflammatory pathways related to leukocyte activation, apoptosis and extracellular matrix degradation. Similarly, murine M1 expressed substantial levels of Treml4, as did oxLDL treated macrophages. Transcriptome analysis confirmed that murine Treml4 controls the expression of genes related to inflammation and lipid regulation pathways, suggesting a possible role in atherosclerosis. Analysis of Apoe-/-/Treml4-/- mice showed reduced plaque burden and lesion complexity as indicated by decreased stage scores, macrophage content and collagen deposition. Finally, transcriptome analysis of oxLDL-loaded murine macrophages showed that Treml4 represses a specific set of genes related to carbohydrate, ion and amino acid membrane transport. Metabolomic analysis confirmed that Treml4 deficiency may promote a beneficial relationship between iron homeostasis and glucose metabolism. Together, our results suggest that Treml4 plays a role in the development of cardiovascular disease, as indicated by Treml4-dependent dysregulation of macrophage inflammatory pathways, macrophage metabolism and promotion of vulnerability features in advanced lesions.

Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase.

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.

Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression.

Neutrophils are a vital component of immune protection, yet in cancer they may promote tumour progression, partly by generating reactive oxygen species (ROS) that disrupts lymphocyte functions. Metabolically, neutrophils are often discounted as purely glycolytic. Here we show that immature, c-Kit+ neutrophils subsets can engage in oxidative mitochondrial metabolism. With limited glucose supply, oxidative neutrophils use mitochondrial fatty acid oxidation to support NADPH oxidase-dependent ROS production. In 4T1 tumour-bearing mice, mitochondrial fitness is enhanced in splenic neutrophils and is driven by c-Kit signalling. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and T cell suppression when glucose utilisation is restricted. Consistent with these findings, peripheral blood neutrophils from patients with cancer also display increased immaturity, mitochondrial content and oxidative phosphorylation. Together, our data suggest that the glucose-restricted tumour microenvironment induces metabolically adapted, oxidative neutrophils to maintain local immune suppression.

Pharmacologic or Genetic Targeting of Glutamine Synthetase Skews Macrophages toward an M1-like Phenotype and Inhibits Tumor Metastasis.

Glutamine-synthetase (GS), the glutamine-synthesizing enzyme from glutamate, controls important events, including the release of inflammatory mediators, mammalian target of rapamycin (mTOR) activation, and autophagy. However, its role in macrophages remains elusive. We report that pharmacologic inhibition of GS skews M2-polarized macrophages toward the M1-like phenotype, characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis, which could be partly related to HIF1α activation. As a result of these metabolic changes and HIF1α accumulation, GS-inhibited macrophages display an increased capacity to induce T cell recruitment, reduced T cell suppressive potential, and an impaired ability to foster endothelial cell branching or cancer cell motility. Genetic deletion of macrophagic GS in tumor-bearing mice promotes tumor vessel pruning, vascular normalization, accumulation of cytotoxic T cells, and metastasis inhibition. These data identify GS activity as mediator of the proangiogenic, immunosuppressive, and pro-metastatic function of M2-like macrophages and highlight the possibility of targeting this enzyme in the treatment of cancer metastasis.

SLC25A26 overexpression impairs cell function via mtDNA hypermethylation and rewiring of methyl metabolism.

Cancer cells down-regulate different genes to give them a selective advantage in invasiveness and/or metastasis. The SLC25A26 gene encodes the mitochondrial carrier that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix, required for mitochondrial methylation processes, and is down-regulated in cervical cancer cells. In this study we show that SLC25A26 is down-regulated due to gene promoter hypermethylation, as a mechanism to promote cell survival and proliferation. Furthermore, overexpression of SLC25A26 in CaSki cells increases mitochondrial SAM availability and promotes hypermethylation of mitochondrial DNA, leading to decreased expression of key respiratory complex subunits, reduction of mitochondrial ATP and release of cytochrome c. In addition, increased SAM transport into mitochondria leads to impairment of the methionine cycle with accumulation of homocysteine at the expense of glutathione, which is strongly reduced. All these events concur to arrest the cell cycle in the S phase, induce apoptosis and enhance chemosensitivity of SAM carrier-overexpressing CaSki cells to cisplatin.

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.