Alternative (M2) activation of macrophages driven via the α-chain of the receptor for interleukin 4 (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 the fatty acids that support this metabolic program has not been clear. We found that the uptake of triacylglycerol substrates via the scavenger receptor CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation, 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 to this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.

Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation

Etomoxir Actions on Regulatory and Memory T Cells Are Independent of Cpt1a-Mediated Fatty Acid Oxidation.

Natural killer (NK) cells are lymphocytes with important roles in innate and adaptive immune responses to tumours and viral infection. However, in certain chronic diseases, including obesity and cancer, NK cell functional responses are impaired. Recently, research has highlighted the importance of NK cell metabolism in facilitating robust NK cell effector functions. This Review describes our current understanding of mouse and human NK cell metabolism and the key signalling pathways that mediate metabolic responses in NK cells. Furthermore, it explores how defects in metabolism can contribute to the generation of dysfunctional NK cells in chronic disease. Finally, the potential for new therapeutic strategies targeting cellular metabolism is discussed. As in other immune cells, the metabolic pathways in natural killer (NK) cells must be configured to meet the demands of their effector functions. This Review describes the specific metabolic requirements for NK cell responses and how defects in NK cell metabolism may contribute to NK cell dysfunction in chronic disease.

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Immunometabolism and natural killer cell responses.

Etomoxir Inhibits Macrophage Polarization by Disrupting CoA Homeostasis

HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 μM. Concentrations of ETO above 5 μM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 μM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox.

/pdf/the-cpt1a-inhibitor-etomoxir-induces-severe-oxidative-stress-4ti718hj7a.pdf

The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.

/pdf/mitochondrial-atp-fuels-abc-transporter-mediated-drug-efflux-25hqio21vd.pdf

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.

The cellular metabolism of macrophages is an emerging element regulating inflammatory macrophages which are a critical component of tumor microenvironment. The inflammatory macrophage with highly glycolytic phenotype is also known to elevate the glycolytic activity upon pathogenic stimulation such as lipopolysaccharide (LPS). In this study, the dynamic changes in glycolysis were traced in a real-time manner by measuring proton efflux rates (PERs) and oxygen consumption rates (OCR) after an in-situ activation using Seahorse XFe96 analyzer. The PER of human peripheral blood monocyte (PBMC) derived M1 macrophages was increased within an hour after injection of LPS, which corresponding to cytokine release, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In contrast to PBMC-derived M1 macrophage activation, macrophage cell lines of RAW264.7 and J774.A1 required co-stimulation with interferon γ (IFNγ) for the full activation. Interestingly, the LPS and IFNγ co-stimulation modulates glycolytic rates in a bi-phasic manner which was identified only in long term (> 6 hr) monitoring. A series of long term XF analysis using in situ activation revealed that the immediate early glycolytic response fully relies on LPS stimulation while the secondary elevation in PER depends on IFNγ stimulus, which turns on inducible nitric oxide synthase (iNOS) signaling and in turn suppresses mitochondrial respiration. The TNFα production is closely related to the immediate early glycolytic elevation, but independent from IFNγ-induced second elevation. The IFNγ-dependent second glycolysis increase was totally abolished by iNOS inhibitors whereas the immediate early glycolysis elevation was not affected at all. These data imply a temporal orchestration mechanism of LPS and IFNγ signaling in the metabolic regulation and activation of inflammatory macrophages. Citation Format: Yoonseok Kam, Pamela M. Swain, Brian P. Dranka. Bi-phasic metabolic responses to in situ macrophage activation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A67.

Yoonseok Kam

Papers

The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations.

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.

Abstract A67: Bi-phasic metabolic responses to in situ macrophage activation