Cutting Edge: STING Induces ACLY Activation and Metabolic Adaptations in Human Macrophages through TBK1.

The 2'3'-cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of IFN genes (STING) pathway can sense infection and cellular stress by detecting cytosolic DNA. Upon ligand binding, cGAS produces the cyclic dinucleotide messenger cGAMP, which triggers its receptor STING. Active STING initiates gene transcription through the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB and induces autophagy, but whether STING can cause changes in the metabolism of macrophages is unknown. In this study, we report that STING signaling activates ATP-citrate lyase (ACLY) by phosphorylation in human macrophages. Using genetic and pharmacologic perturbation, we show that STING targets ACLY via its prime downstream signaling effector TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1). We further identify that TBK1 alters cellular metabolism upon cGAMP treatment. Our results suggest that STING-mediated metabolic reprogramming adjusts the cellular response to DNA sensing in addition to transcription factor activation and autophagy induction.

Pyruvate dehydrogenase fuels a critical citrate pool that is essential for Th17 cell effector functions.

Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in T helper 17 (Th17) cells still remains to be studied. Here, we show that PDH is essential for the generation of a glucose-derived citrate pool needed for Th17 cell proliferation, survival, and effector function. In vivo, mice harboring a T cell-specific deletion of PDH are less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increases glutaminolysis, glycolysis, and lipid uptake in a mammalian target of rapamycin (mTOR)-dependent manner. However, cellular citrate remains critically low in mutant Th17 cells, which interferes with oxidative phosphorylation (OXPHOS), lipid synthesis, and histone acetylation, crucial for transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restores their metabolism and function, identifying a metabolic feedback loop within the central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.

Itaconate controls its own synthesis via feedback-inhibition of reverse TCA cycle activity at IDH2.

Macrophages undergo extensive metabolic reprogramming during classical pro-inflammatory polarization (M1-like). The accumulation of itaconate has been recognized as both a consequence and mediator of the inflammatory response. In this study we first examined the specific functions of itaconate inside fractionated mitochondria. We show that M1 macrophages produce itaconate de novo via aconitase decarboxylase 1 (ACOD1) inside mitochondria. The carbon for this reaction is not only supplied by oxidative TCA cycling, but also through the reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase (IDH). While macrophages are capable of sustaining a certain degree of itaconate production during hypoxia by augmenting the activity of IDH-dependent reductive carboxylation, we demonstrate that sufficient itaconate synthesis requires a balance of reductive and oxidative TCA cycle metabolism in mouse macrophages. In comparison, human macrophages increase itaconate accumulation under hypoxic conditions by augmenting reductive carboxylation activity. We further demonstrated that itaconate attenuates reductive carboxylation at IDH2, restricting its own production and the accumulation of the immunomodulatory metabolites citrate and 2-hydroxyglutarate. In line with this, reductive carboxylation is enhanced in ACOD1-depleted macrophages. Mechanistically, the inhibition of IDH2 by itaconate is linked to the alteration of the mitochondrial NADP+/NADPH ratio and competitive succinate dehydrogenase inhibition. Taken together, our findings extend the current model of TCA cycle reprogramming during pro-inflammatory macrophage activation and identified novel regulatory properties of itaconate.

SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells.

A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30 min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.

Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages.

Since its discovery in inflammatory macrophages, itaconate has attracted much attention due to its antimicrobial and immunomodulatory activity1-3. However, instead of investigating itaconate itself, most studies used derivatized forms of itaconate and thus the role of non-derivatized itaconate needs to be scrutinized. Mesaconate, a metabolite structurally very close to itaconate, has never been implicated in mammalian cells. Here we show that mesaconate is synthesized in inflammatory macrophages from itaconate. We find that both, non-derivatized itaconate and mesaconate dampen the glycolytic activity to a similar extent, whereas only itaconate is able to repress tricarboxylic acid cycle activity and cellular respiration. In contrast to itaconate, mesaconate does not inhibit succinate dehydrogenase. Despite their distinct impact on metabolism, both metabolites exert similar immunomodulatory effects in pro-inflammatory macrophages, specifically a reduction of interleukin (IL)-6 and IL-12 secretion and an increase of CXCL10 production in a manner that is independent of NRF2 and ATF3. We show that a treatment with neither mesaconate nor itaconate impairs IL-1ß secretion and inflammasome activation. In summary, our results identify mesaconate as an immunomodulatory metabolite in macrophages, which interferes to a lesser extent with cellular metabolism than itaconate.

GLP-2 Is Locally Produced From Human Islets and Balances Inflammation Through an Inter-Islet-Immune Cell Crosstalk.

Glucagon-like peptide-1 (GLP-1) shows robust protective effects on ß-cell survival and function and GLP-1 based therapies are successfully applied for type-2 diabetes (T2D) and obesity. Another cleavage product of pro-glucagon, Glucagon-like peptide-2 (GLP-2; both GLP-1 and GLP-2 are inactivated by DPP-4) has received little attention in its action inside pancreatic islets. In this study, we investigated GLP-2 production, GLP-2 receptor (GLP-2R) expression and the effect of GLP-2R activation in human islets. Isolated human islets from non-diabetic donors were exposed to diabetogenic conditions: high glucose, palmitate, cytokine mix (IL-1ß/IFN-γ) or Lipopolysaccharide (LPS) in the presence or absence of the DPP4-inhibitor linagliptin, the TLR4 inhibitor TAK-242, the GLP-2R agonist teduglutide and/or its antagonist GLP-2(3-33). Human islets under control conditions secreted active GLP-2 (full-length, non-cleaved by DPP4) into the culture media, which was increased by combined high glucose/palmitate, the cytokine mix and LPS and highly potentiated by linagliptin. Low but reproducible GLP-2R mRNA expression was found in all analyzed human islet isolations from 10 donors, which was reduced by pro-inflammatory stimuli: the cytokine mix and LPS. GLP-2R activation by teduglutide neither affected acute or glucose stimulated insulin secretion nor insulin content. Also, teduglutide had no effect on high glucose/palmitate- or LPS-induced dysfunction in cultured human islets but dampened LPS-induced macrophage-dependent IL1B and IL10 expression, while its antagonist GLP-2(3-33) abolished such reduction. In contrast, the expression of islet macrophage-independent cytokines IL6, IL8 and TNF was not affected by teduglutide. Medium conditioned by teduglutide-exposed human islets attenuated M1-like polarization of human monocyte-derived macrophages, evidenced by a lower mRNA expression of pro-inflammatory cytokines, compared to vehicle treated islets, and a reduced production of itaconate and succinate, marker metabolites of pro-inflammatory macrophages. Our results reveal intra-islet production of GLP-2 and GLP-2R expression in human islets. Despite no impact on ß-cell function, local GLP-2R activation reduced islet inflammation which might be mediated by a crosstalk between endocrine cells and macrophages.

Fumarate is a terminal electron acceptor in the mammalian electron transport chain.

For electrons to continuously enter and flow through the mitochondrial electron transport chain (ETC), they must ultimately land on a terminal electron acceptor (TEA), which is known to be oxygen in mammals. Paradoxically, we find that complex I and dihydroorotate dehydrogenase (DHODH) can still deposit electrons into the ETC when oxygen reduction is impeded. Cells lacking oxygen reduction accumulate ubiquinol, driving the succinate dehydrogenase (SDH) complex in reverse to enable electron deposition onto fumarate. Upon inhibition of oxygen reduction, fumarate reduction sustains DHODH and complex I activities. Mouse tissues display varying capacities to use fumarate as a TEA, most of which net reverse the SDH complex under hypoxia. Thus, we delineate a circuit of electron flow in the mammalian ETC that maintains mitochondrial functions under oxygen limitation.