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.

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.

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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.

Changes in bulk transcriptional profiles of heterogeneous samples often reflect changes in proportions of individual cell types. Several robust techniques have been developed to dissect the composition of such mixed samples given transcriptional signatures of the pure components or their proportions. These approaches are insufficient, however, in situations when no information about individual mixture components is available. This problem is known as the  complete deconvolution problem, where the composition is revealed without any a priori knowledge about cell types and their proportions. Here, we identify a previously unrecognized property of tissue-specific genes – their mutual linearity – and use it to reveal the structure of the topological space of mixed transcriptional profiles and provide a noise-robust approach to the complete deconvolution problem. Furthermore, our analysis reveals systematic bias of all deconvolution techniques due to differences in cell size or RNA-content, and we demonstrate how to address this bias at the experimental design level.