A Broken Krebs Cycle in Macrophages

Macrophages undergo metabolic rewiring during polarization but details of this process are unclear. In this issue of Immunity, Jha et al., 2015Jha A.K. Huang S.C.-C. Sergushichev A. Lampropoulou V. Ivanova Y. Loginicheva E. Chmielewski K. Stewart K.M. Ashall J. Everts B. et al.Immunity. 2015; 42 (this issue): 419-430Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar report a systems approach for unbiased analysis of cellular metabolism that reveals key metabolites and metabolic pathways required for distinct macrophage polarization states. Macrophages undergo metabolic rewiring during polarization but details of this process are unclear. In this issue of Immunity, Jha et al., 2015Jha A.K. Huang S.C.-C. Sergushichev A. Lampropoulou V. Ivanova Y. Loginicheva E. Chmielewski K. Stewart K.M. Ashall J. Everts B. et al.Immunity. 2015; 42 (this issue): 419-430Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar report a systems approach for unbiased analysis of cellular metabolism that reveals key metabolites and metabolic pathways required for distinct macrophage polarization states. Technological advances in metabolomics are being brought to bear on the immune response. Striking findings are being made that provide new insights into metabolic processes occurring inside immune cells—insights that have consequences for cellular phenotypes (O'Neill and Hardie, 2013O'Neill L.A.J. Hardie D.G. Nature. 2013; 493: 346-355Crossref PubMed Scopus (803) Google Scholar, Ganeshan and Chawla, 2014Ganeshan K. Chawla A. Annu. Rev. Immunol. 2014; 32: 609-634Crossref PubMed Scopus (474) Google Scholar). Notable examples include enhanced glycolysis (the so-called Warburg Effect) being a feature of inflammatory cells, such as M1 macrophages and CD4+ T helper 17 (Th17) lymphocytes. Cells that participate more in immunoregulation or the resolution of inflammation, such as M2 macrophages or regulatory T cells, have fatty acid oxidation and an intact Krebs cycle as hallmark metabolic features. It is still not fully understood why these differences occur, or indeed how the metabolic rewiring is regulated. Rapidity of response might be an important reason for the metabolic differences (Ganeshan and Chawla, 2014Ganeshan K. Chawla A. Annu. Rev. Immunol. 2014; 32: 609-634Crossref PubMed Scopus (474) Google Scholar). In M1 macrophages, glycolysis would allow for rapid ATP production to fuel activation in acute inflammation or anti-bacterial defense, whereas in M2 macrophages oxidative phosphorylation occurs in the more long-term process of resolution and repair or in anti-parasitic immunity. Jha et al., 2015Jha A.K. Huang S.C.-C. Sergushichev A. Lampropoulou V. Ivanova Y. Loginicheva E. Chmielewski K. Stewart K.M. Ashall J. Everts B. et al.Immunity. 2015; 42 (this issue): 419-430Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar now provide us with the most detailed account yet of metabolic changes in macrophage polarization, and they combine metabolomics with transcriptomics to reveal distinct modules in M1 and M2 macrophages. In M2 macrophages, they uncover a module leading to the metabolite UDP-GlcNAc, which is needed for N-glycosylation. In M1 macrophages, two breaks are evident in the Krebs cycle (such that in M1 macrophages it is not a cycle at all but instead is fragmented). One break occurs at isocitrate dehydrogenase (IDH) and the other after succinate, with a novel variant of a pathway (termed the aspartate-arginosuccinate shunt) being evident. The metabolites accumulating are shown to be important for various functions of M1 and M2 macrophages, providing yet more compelling evidence for the importance of immunometabolism in immunity and inflammation. To better understand the integration of metabolomic changes and changes in the transcriptome, the authors carried out a non-targeted systems-level analysis of transcriptional and metabolic changes in the macrophage polarization process. Remarkably, the authors obtained data on more than 2,000 distinct metabolites by mass spectrometry and, by using systems-based algorithms, uncovered modules specific for M2 and M1 macrophages. In M2 macrophages, increased amino-sugar and nucleotide sugar metabolites were evident, characterized by high amounts of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) and a corresponding increase in enzymes producing these metabolites. This result was consistent with previous studies demonstrating that highly glycosylated lectin/mannose receptors are markers for M2 macrophages (Sica and Mantovani, 2012Sica A. Mantovani A. J. Clin. Invest. 2012; 122: 787-795Crossref PubMed Scopus (3917) Google Scholar). The functional consequence of elevated UDP-GlcNAc and ensuing protein glycosylation was demonstrated using inhibitors of N-glycosylation that decreased the expression of several M2 markers, notably CD206 and CD301. The second metabolic feature uncovered was increased glutamine metabolism. An intact Krebs cycle was evident, with a third of all carbons in the Krebs cycle intermediates coming from glutamine. Half of the nitrogen in UDP-GlcNAc was also shown to come from glutamine. Deprivation of glutamine inhibited M2 polarization, with the chemokine CCL22, which is another marker of M2 macrophages, being especially glutamine dependent, although the mechanistic link was not elucidated. In M1 macrophages, a very different metabolomics profile was observed. First, two breaks were evident in the Krebs cycle. The expression of the enzyme isocitrate dehydrogenase, which converts citrate to alpha-ketoglutarate, was decreased 7-fold relative to M0 macrophages. This led to a build-up in citrate, which was redirected for the production of itaconic acid. This metabolite has been shown to have anti-microbial properties (Michelucci et al., 2013Michelucci A. Cordes T. Ghelfi J. Pailot A. Reiling N. Goldmann O. Binz T. Wegner A. Tallam A. Rausell A. et al.Proc. Natl. Acad. Sci. USA. 2013; 110: 7820-7825Crossref PubMed Scopus (594) Google Scholar). Citrate will also be withdrawn for fatty acid biosynthesis, another hallmark of the M1 macrophage. The second break-point in the Krebs cycle occurred after succinate. In the Krebs cycle, succinate is converted to fumarate, which is then converted to malate. A moderate accumulation in succinate was observed, consistent with previous studies (Tannahill et al., 2013Tannahill G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. et al.Nature. 2013; 496: 238-242Crossref PubMed Scopus (2147) Google Scholar). However, a large increase in malate was observed, indicating that the conversion of succinate to fumarate was inefficient. The increase in malate was explained by an enhanced arginosuccinate shunt, which fed into fumarate and then malate production and also generated nitric oxide by interfacing with the NO cycle. Inhibition of aspartate aminotransferase, a key enzyme in the shunt, suppressed NO production and also interleukin-6 (IL-6) production, attesting to the importance of the arginosuccinate shunt for M1 macrophage function. This intellectually challenging and detailed analysis of metabolic changes occurring in macrophage polarization therefore provides us with some important new insights that are functionally relevant (Figure 1). In M2 macrophages, UDP-GlcNAc, required for protein glycosylation, is enhanced. If this process is inhibited, a decrease in expression of M2 markers occurs, although precisely how GlcNAc concentrations link to such expression was not elucidated. The authors speculate that N-glycosylation might be needed to correctly fold and traffic proteins such as CD206 and CD301. Other receptors characteristic of M2 macrophages, such as lectin/mannose receptors, are heavily glycosylated and presumably rely on elevated GlcNAc. For M1 macrophages, two breaks in the Krebs cycle are evident, at IDH and succinate dehydrogenase. The IDH break allows for citrate accumulation, leading to fatty acid synthesis (which in turn participates in such responses as prostaglandin production) and can also feed into NO production (Infantino et al., 2011Infantino V. Convertini P. Cucci L. Panaro M.A. Di Noia M.A. Calvello R. Palmieri F. Iacobazzi V. Biochem. J. 2011; 438: 433-436Crossref PubMed Scopus (255) Google Scholar). Citrate also leads to the production of the anti-microbial metabolite itaconate, which has been shown to inhibit the glyoxylate shunt pathway in pathogens (e.g., in M. tuberculosis) but not mammalian cells (Michelucci et al., 2013Michelucci A. Cordes T. Ghelfi J. Pailot A. Reiling N. Goldmann O. Binz T. Wegner A. Tallam A. Rausell A. et al.Proc. Natl. Acad. Sci. USA. 2013; 110: 7820-7825Crossref PubMed Scopus (594) Google Scholar). The break at IDH is therefore critical for several functions of M1 macrophages. The second break occurs after succinate, which can be withdrawn to activate hypoxia inducible factor 1-alpha (HIF1alpha), leading to increased expression of HIF1alpha genes (Tannahill et al., 2013Tannahill G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. et al.Nature. 2013; 496: 238-242Crossref PubMed Scopus (2147) Google Scholar). In addition, however, there is induction of the arginosuccinate shunt, which will ultimately replenish citrate. Instead of replenishing citrate via a normal Krebs cycle, the M1 macrophage uses the arginosuccinate shunt because of the break after succinate. In fact, if Krebs himself had been using M1 macrophages in his experiments, he would have found a broken cycle. This study, along with several others in the area of immunometabolism, is therefore providing new knowledge on metabolic pathways, which importantly link directly to the specific functioning of immune cells. The paper is being published as a Resource, allowing others to mine the extensive data to generate additional clues into metabolic control of macrophage polarization. Further work is likely to reveal even more insights, which will hopefully lead to improved understanding of immunity in health and disease, which might prove useful in the clinic. This work was supported by an ERC advanced grant (E12435) and an SFI Investigator Award (G20598). Network Integration of Parallel Metabolic and Transcriptional Data Reveals Metabolic Modules that Regulate Macrophage PolarizationJha et al.ImmunityMarch 17, 2015In BriefPolarization of macrophages involves a metabolic and transcriptional rewiring that is only partially understood. Artyomov and colleagues used an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline to identify metabolic modules that support macrophage polarization and function. Full-Text PDF Open Archive