Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes.

The immune system plays key roles in determining the fate of developing cancers by not only functioning as a tumour promoter facilitating cellular transformation, promoting tumour growth and sculpting tumour cell immunogenicity1–6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer induced immunosuppression. In many individuals, immunosuppression is mediated by Cytotoxic T-Lymphocyte Associated Antigen-4 (CTLA-4) and Programmed Death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal antibody (mAb) based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10–13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Herein, we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T cell rejection antigens following αPD-1 and/or αCTLA-4 therapy of mice bearing progressively growing sarcomas and show that therapeutic synthetic long peptide (SLP) vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Whereas, mutant tumour antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with αPD-1- and/or αCTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens (TSMA) are not only important targets of checkpoint blockade therapy but also can be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

SUMMARY Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the pro-inflammatory IL-1β-HIF-1α axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions by inhibiting succinate dehydrogenase-mediated oxidation of succinate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1−/− mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages.

The ligation of Toll-like receptors (TLRs) leads to rapid activation of dendritic cells (DCs). However, the metabolic requirements that support this process remain poorly defined. We found that DC glycolytic flux increased within minutes of exposure to TLR agonists and that this served an essential role in supporting the de novo synthesis of fatty acids for the expansion of the endoplasmic reticulum and Golgi required for the production and secretion of proteins that are integral to DC activation. Signaling via the kinases TBK1, IKKε and Akt was essential for the TLR-induced increase in glycolysis by promoting the association of the glycolytic enzyme HK-II with mitochondria. In summary, we identified the rapid induction of glycolysis as an integral component of TLR signaling that is essential for the anabolic demands of the activation and function of DCs.

To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor/normal pairs. Recurrent alterations in lung SqCCs were more similar to other squamous carcinomas than to lung ADCs. Novel significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. Novel amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase/Ras/Raf alterations revealed mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least 5 predicted neoepitopes. While targeted therapies for lung ADC and lung SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.

Alternative (M2) macrophage activation driven through interleukin 4 receptor α (IL-4Rα) is important for immunity to parasites, wound healing, the prevention of atherosclerosis and metabolic homeostasis. M2 polarization is dependent on fatty acid oxidation (FAO), but the source of fatty acids to support this metabolic program has not been clear. We show that the uptake of triacylglycerol substrates via CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation (OXPHOS), enhanced spare respiratory capacity (SRC), prolonged survival and expression of genes that together define M2 activation. Inhibition of lipolysis suppressed M2 activation during infection with a parasitic helminth, and blocked protective responses against this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.

Glia have been implicated in Alzheimer’s disease (AD) pathogenesis. Variants of the microglia receptor TREM2 increase AD risk and activation of “disease-associated microglia” (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene expression changes associated with AD pathology and TREM2 in 5XFAD mice and human AD by snRNA-seq. We confirmed the presence of Trem2 -dependent DAM and identified a novel Serpina3n + C4b + reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less palpable in TREM2 R47H and R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.

Summary Elevated risk of developing Alzheimer’s disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth factor limitation or ER stress. Combined metabolomics and RNA-seq linked this anomalous autophagy to defective mTOR signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.