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.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite2. Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines2, including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17–IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI–IκBζ regulatory axis could be an important new strategy for the treatment of IL-17–IκBζ-mediated autoimmune diseases.

Mitochondria are important targets for cancer chemotherapy and other disease treatments. Gaining access to this organelle can be difficult, as the inner membrane is a barrier limiting diffusive transport. A mitochondrial molecular carrier would be a boon to the development of organelle-specific therapeutics. Here, we report a significant advance in the development of mitochondrial transporters-synthetic cell-permeable peptides that are able to enter mitochondria. Efficient uptake of these mitochondria-penetrating peptides (MPPs) is observed in a variety of cell types, and organellar specificity is attained with sequences that possess specific chemical properties. The MPPs identified are cationic, but also lipophilic; this combination of characteristics facilitates permeation of the hydrophobic mitochondrial membrane. The examination of a panel of MPPs illustrates that mitochondrial localization can be rationally controlled and finely tuned by altering lipophilicity and charge.

Cell-penetrating peptides (CPPs) have found numerous applications in biology and medicine since the first synthetic cell-permeable sequence was identified two decades ago. Numerous types of drugs have been transported into cells using CPPs, including small-molecule pharmaceuticals, therapeutic proteins, and antisense oligonucleotides. Improved agents for medical imaging have been generated by conjugation with CPPs, with the appended peptides promoting cellular uptake and in some cases, cell-type specificity. Organelle-specific CPPs have also been generated, providing a means to target specific subcellular sites. This review highlights achievements in this area and illustrates the numerous examples where peptide chemistry was exploited as a means to provide new tools for biology and medicine.

Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (T17) cells towards induced regulatory T (iT) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating T17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards T17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of T17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iT cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between T17 and iT cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against T17-mediated autoimmune diseases.

Highlights d Developed 13 C-infusion method for studying T cell metabolism in vivo d T cell glucose use and bioenergetics differ between cell culture and mouse models d Glucose metabolism in T cells changes dynamically over an immune response d Glucose-dependent serine biosynthesis supports T cell proliferation in vivo

Upon activation, macrophages undergo extensive metabolic rewiring 1 , 2 . Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process 3 . Itaconate inhibits succinate dehydrogenase (SDH) 4 , 5 , has electrophilic properties 6 , and is associated with a change in cytokine production 4 . Here, we compare the metabolic, electrophilic, and immunologic profiles of macrophages treated with unmodified itaconate and a panel of commonly used itaconate derivatives to examine its role. Using wild type and Irg1 −/− macrophages, we show that neither dimethyl itaconate (DI), 4-octyl itaconate (4OI), nor 4-monoethyl itaconate (4EI) are converted into intracellular itaconate, while exogenous itaconic acid readily enters macrophages. We find that only DI and 4OI induce a strong electrophilic stress response, in contrast to itaconate and 4EI. This correlates with their immunosuppressive phenotype: DI and 4OI inhibit IκBζ and pro-IL-1β induction, as well as IL-6, IL-10, and IFN-β secretion in an Nrf2-independent manner. In contrast, itaconate treatment only suppressed IL-1β secretion but not pro-IL-1β levels, and, surprisingly, strongly enhanced LPS-induced IFN-β secretion. Consistently, Irg1 −/− macrophages produced lower levels of interferon and reduced transcriptional activation of this pathway. Our work establishes itaconate as an immunoregulatory, rather than strictly immunosuppressive metabolite, and highlights the importance of using unmodified itaconate in future studies.

Mitochondria are the energy factories of the cell and also serve as a checkpoint regulating programmed cell death. Not surprisingly, dysfunctional mitochondria are implicated in a variety of diseases ranging from metabolic disorders to cancer. Treatment of these diseases through the delivery of targeted drugs, however, is impeded by the difficulty of penetrating the membranes that define this organelle. The properties of this barrier serve as a major obstacle to drug delivery and a lack of effective transporters has hindered the advancement of mitochondrial medicine. Recently, however, synthetic transporters that show promise for the mito-specific delivery of bioactive cargos have begun to emerge. This review summarizes the most exciting of these developments and discusses their applications.