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.
C 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.
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.
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.
Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) contribute to cancer-related inflammation and tumor progression. While several myeloid molecules have been ascribed a ...regulatory function in these processes, the triggering receptors expressed on myeloid cells (TREMs) have emerged as potent modulators of the innate immune response. While various TREMs amplify inflammation, others dampen it and are emerging as important players in modulating tumor progression-for instance, soluble TREM-1 (sTREM-1), which is detected during inflammation, associates with disease progression, while TREM-2 expression is associated with tumor-promoting macrophages. We hypothesized that TREM-1 and TREM-2 might be co-expressed on tumor-infiltrating myeloid cells and that elevated sTREM-1 associates with disease outcomes, thus representing a possibility for mutual modulation in cancer. Using the 4T1 breast cancer model, we found TREM-1 and TREM-2 expression on MDSC and TAM and that sTREM-1 was elevated in tumor-bearing mice in multiple models and correlated with tumor volume. While TREM-1 engagement enhanced TNF, a TREM-2 ligand was detected on MDSC and TAM, suggesting that both TREM could be functional in the tumor setting. Similarly, we detected TREM-1 and
expression in myeloid cells in the RENCA model of renal cell carcinoma (RCC). We confirmed these findings in human disease by demonstrating the expression of TREM-1 on tumor-infiltrating myeloid cells from patients with RCC and finding that sTREM-1 was increased in patients with RCC. Finally, The Cancer Genome Atlas analysis shows that
expression in tumors correlates with poor outcomes in RCC. Taken together, our data suggest that manipulation of the TREM-1/TREM-2 balance in tumors may be a novel means to modulate tumor-infiltrating myeloid cell phenotype and function.
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
expression ...positively correlates with human coronary arterial calcification (CAC). However, the role of
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
and found that carriage of either one of the eQTL SNP's previously associated with increased
expression conferred higher expression in human inflammatory macrophages (M1) compared to alternatively activated macrophages (M2). Furthermore, we found that
expression in human M1 dysregulated several inflammatory pathways related to leukocyte activation, apoptosis and extracellular matrix degradation. Similarly, murine M1 expressed substantial levels of
, as did oxLDL treated macrophages. Transcriptome analysis confirmed that murine
controls the expression of genes related to inflammation and lipid regulation pathways, suggesting a possible role in atherosclerosis. Analysis of
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
represses a specific set of genes related to carbohydrate, ion and amino acid membrane transport. Metabolomic analysis confirmed that
deficiency may promote a beneficial relationship between iron homeostasis and glucose metabolism. Together, our results suggest that
plays a role in the development of cardiovascular disease, as indicated by
-dependent dysregulation of macrophage inflammatory pathways, macrophage metabolism and promotion of vulnerability features in advanced lesions.
Abstract
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 beta-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. 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-OI, 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.
This research was supported, in part, by the Intramural Research Program of the NIH, National Cancer Institute, CCR, CIL. RNA sequencing and initial data analysis were conducted at the Frederick National Laboratory for Cancer Research at the CCR Sequencing Facility, NCI Frederick. Human specimens were provided by the Clinical Biospecimen Repository and Processing Core of the Pittsburgh Liver Research Center (supported by National Institutes of Health grant 1P30DK120531). Acyl-CoA analysis was supported by R01GM132261 to NWS.
Abstract
Nrf2 is a transcription factor that, under basal conditions, is produced in the cytosol and is marked for degradation by its repressor Keap1. Oxidative stress causes Keap1 to release Nrf2, ...and it accumulates in the nucleus to upregulate certain antioxidant gene targets. Itaconate, the metabolic product of IRG1, has been suggested to play a role in alkylating Keap1, leading to the release of Nrf2. However, there is much ambiguity and even controversy surrounding this role of itaconate. Our study therefore seeks to definitively elucidate the role of itaconate in the regulation and activation of Nrf2. Here we utilize expression analysis and targeted metabolomics in bone marrow-derived macrophages isolated from varying murine backgrounds (Acod1−/−, Nos2−/−, and double knockouts). Despite previously published data suggesting a role for itaconate in Nrf2 activity, our data demonstrate a significant role of Nos2, but not itaconate, in Nrf2 activation, as Acod1−/− mice show little change in the activity of Nrf2 transcription targets, while Nos2−/− mice show severe impairment of Nrf2 gene target activity. For Nrf2 to properly upregulate its gene targets, it must be released from Keap1, phosphorylated, and then allowed to translocate to the nucleus; all three must occur. This may help explain why some studies have found itaconate to be important in Keap1’s regulation of Nrf2, while others demonstrate that this same itaconate mechanism produces comparatively little Nrf2 activity.
Abstract
Metabolism is a key modulator of macrophage differentiation, polarization and effector responses. While M1 favors a glycolytic configuration resembling the “Warburg effect”, M2 is fueled by ...oxidative metabolism. Here we describe a previously unappreciated metabolic phenotype controlled by The Triggering Receptor Expressed on Myeloid Cells (TREM)-2, a key player in inflammation and innate immunity. Trem2−/− macrophages show lower TCA cycle metabolites compared to wild type, but maintenance of glycolytic intermediates. Interestingly, while we show Trem2 abrogation is beneficial in M1 and protective against MCSF withdrawal, we find Trem2−/− cells to have disrupted lipid utilization as they accumulate both long chain free fatty acids (FFA) and carnitine-conjugated lipids. Because lipid metabolism is key for M2 responses, we hypothesized that Trem2 supports a metabolic program to maintain M2 activation. Indeed, in M2 cell, Trem2 sustains mitochondrial respiratory capacity, CD36 expression and lipoprotein and FFA uptake. Lipidomics confirmed Trem2−/− M2 cells had decreased FFA and enhanced triglycerides. Moreover, Trem2−/− M2 cells show a distinct metabolic profile with enhanced glutamine utilization, suggesting compensatory mechanisms in lieu of FFA for mitochondrial respiration. Transcriptome profiling decisively shows dysregulated genes related to defense response and glycerolipid metabolism, linking the metabolic findings to effector responses. Finally, in a model of in vivo Th2 response, Trem2−/− mice failed to control infection and exhibit metabolic dysregulation. Our findings suggest Trem2 is required for reprograming of macrophages by supporting the energetic requirements for M2 function.
Abstract
The Triggering Receptor Expressed on Myeloid cells-like 4 (Treml4) is a member of the TREM receptor family which are recognized fine-tuners of the inflammatory response. We have previously ...found that Treml4 expression correlates to increased risk of Human Coronary Arterial Calcification and Acute Coronary Syndrome. Moreover, we show that carriage of either one of the eQTL SNPs previously identified to permit Treml4 expression, confers the highest treml4 expression in human inflammatory macrophages when compared to alternative activated macrophages. However, the exact role of Treml4 in the pathogenesis of coronary diseases remains incompletely defined. To elucidate the role of Treml4 in atherosclerosis, we investigated whether treml4 deficiency affected pathogenesis in the Apolipoprotein E knockout mouse (apoe−/−). We found that aortas from apoe−/− mice had increased treml4 expression when compared to wild type and that treatment with either LDL or oxLDL upregulated macrophage treml4 expression in vitro. After a western diet, overall plaque burden and calcification in the aortic sinus of apoe−/−/treml4−/− mice remained unchanged compared to controls. However, we found that treml4 deficiency resulted in less complex lesions as indicated by decreased foam cell content, plaque necrosis and collagen deposition as well as dysregulation of matrix metalloproteinases expression within the plaques. Moreover, less collagenous metaplasia was observed in the brachiocephalic artery of apoe−/−/treml4−/− mice. Taken together, our results suggest that Treml4 contributes to the inflammatory mechanisms and extracellular matrix regulation associated with lesion complexity without affecting lesion burden or calcification.
Abstract
Classical pro-inflammatory activation of macrophages is characterized by profound intracellular metabolic changes, with increased glycolytic usage of carbon, away from Oxidative ...Phosphorylation (OXPHOS). We previously demonstrated that Nitric Oxide (NO) levels induced in Bone Marrow Derived Macrophages (BMDMs) from Wild Type (WT) mice are necessary and sufficient for the repression of OXPHOS. Here we demonstrate that NO is also responsible for the “break” in the mitochondrial TCA cycle and citrate accumulation during LPS/IFNγ stimulation; macrophages that lack NO maintain indeed substantial levels of Oxygen Consumption Rates (OCR) and TCA cycle intermediates. Carbon tracing experiments in the presence of U13C-glucose show almost undetected labelled α-ketoglutarate from citrate in WT but a conserved pattern of heavy carbon fate in Nos2−/− macrophages. Moreover we found that mitochondrial respiration elicited through citrate was decreased in WT M1 macrophages, but isocitrate was a full substrate for complex I-dependent OCR suggesting suppression of metabolism at mitochondrial Aconitase (ACO2). Consistent with this data, we found ACO2 enzymatic activity blunted in WT vs Nos2−/−. In addition we observed that M1 macrophages reroute pyruvate away from Pyruvate Dehydrogenase (PDH) in an NO dependent manner since only WT show halted flux through PDH. Surprisingly, we demonstrate this mechanism to be independent on the activation of Hif1α and its suggested effect on limiting acetyl-coA for the TCA. With these data together we hypothesize that NO orchestrates macrophage metabolism during inflammation inhibiting OXPHOS by blocking Krebs Cycle, therefore depriving of substrates the mitochondrial electron transport chain.
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