Thekla Cordes

Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

A biomolecular isolation framework for eco-systems biology

Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single samples to facilitate meaningful data integration, analysis and modeling. We have developed a new methodological framework for the reproducible isolation of high-quality genomic DNA, large and small RNA, proteins, and polar and non-polar metabolites from single unique mixed microbial community samples. The methodology is based around reproducible cryogenic sample preservation and cell lysis. Metabolites are extracted first using organic solvents, followed by the sequential isolation of nucleic acids and proteins using chromatographic spin columns. The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest. The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels

Metabolic reprogramming is emerging as a hallmark of the innate immune response, and the dynamic control of metabolites such as succinate serves to facilitate the execution of inflammatory responses in macrophages and other immune cells. Immunoresponsive gene 1 (Irg1) expression is induced by inflammatory stimuli, and its enzyme product cis-aconitate decarboxylase catalyzes the production of itaconate from the tricarboxylic acid cycle. Here we identify an immunometabolic regulatory pathway that links Irg1 and itaconate production to the succinate accumulation that occurs in the context of innate immune responses. Itaconate levels and Irg1 expression correlate strongly with succinate during LPS exposure in macrophages and non-immune cells. We demonstrate that itaconate acts as an endogenous succinate dehydrogenase inhibitor to cause succinate accumulation. Loss of itaconate production in activated macrophages from Irg1−/− mice decreases the accumulation of succinate in response to LPS exposure. This metabolic network links the innate immune response and tricarboxylic acid metabolism to function of the electron transport chain.

Development of a Scale-Down Model of hydrodynamic stress to study the performance of an industrial CHO cell line under simulated production scale bioreactor conditions.

The objective of this study was to develop a Scale-Down Model of a hydrodynamic stress present in large scale production bioreactors to investigate the performance of CHO cells under simulated production bioreactor conditions. Various levels of hydrodynamic stress were generated in 2L bioreactors mimicking those present in different locations of a large scale stirred tank bioreactor. In general, it was observed that tested cells are highly robust against the effect of hydrodynamic stress. However, at elevated hydrodynamic stress equivalent to an average energy dissipation rate, ε, equal to 0.4W/kg, the specific monoclonal antibody productivity, qmAb, decreased by 25% compared to the cultivation conditions corresponding to ε equal to 0.01W/kg. Even stronger decrease of qmAb, in the order of 30%, was observed when ε was periodically oscillating between 0.01 and 0.4W/kg to simulate the repeated passage of cells through the highly turbulent impeller discharge zone of a production scale bioreactor. Despite this effect, no changes in metabolite consumption or byproduct formation were observed. Furthermore, considering the experimental error product quality was independent of the applied ε. To achieve a molecular insight into the observed drop of cellular productivity, a transcriptome analysis using mRNA microarrays was performed. It was found that transcripts related to DNA damage and repair mechanisms were upregulated when high ε was applied for cultivation.

Itaconic Acid: The Surprising Role of an Industrial Compound as a Mammalian Antimicrobial Metabolite

Itaconic acid is well known as a precursor for polymer synthesis and has been involved in industrial processes for decades. In a recent surprising discovery, itaconic acid was found to play a role as an immune-supportive metabolite in mammalian immune cells, where it is synthesized as an antimicrobial compound from the citric acid cycle intermediate cis-aconitic acid. Although the immune-responsive gene 1 protein (IRG1) has been associated to immune response without a mechanistic function, the critical link to itaconic acid production through an enzymatic function of this protein was only recently revealed. In this review, we highlight the history of itaconic acid as an industrial and antimicrobial compound, starting with its biotechnological synthesis and ending with its antimicrobial function in mammalian immune cells.

NTFD—a stand-alone application for the non-targeted detection of stable isotope-labeled compounds in GC/MS data

Summary: Most current stable isotope-based methodologies are targeted and focus only on the well-described aspects of metabolic networks. Here, we present NTFD (non-targeted tracer fate detection), a software for the non-targeted analysis of all detectable compounds derived from a stable isotope-labeled tracer present in a GC/MS dataset. In contrast to traditional metabolic flux analysis approaches, NTFD does not depend on any a priori knowledge or library information. To obtain dynamic information on metabolic pathway activity, NTFD determines mass isotopomer distributions for all detected and labeled compounds. These data provide information on relative fluxes in a metabolic network. The graphical user interface allows users to import GC/MS data in netCDF format and export all information into a tab-separated format. Availability: NTFD is C++- and Qt4-based, and it is freely available under an open-source license. Pre-compiled packages for the installation on Debian- and Redhat-based Linux distributions, as well as Windows operating systems, along with example data, are provided for download at http://ntfd.mit.edu/. Contact: gregstep@mit.edu

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity.

Bridging the gap between non-targeted stable isotope labeling and metabolic flux analysis

BackgroundMetabolism gained increasing interest for the understanding of diseases and to pinpoint therapeutic intervention points. However, classical metabolomics techniques only provide a very static view on metabolism. Metabolic flux analysis methods, on the other hand, are highly targeted and require detailed knowledge on metabolism beforehand.ResultsWe present a novel workflow to analyze non-targeted metabolome-wide stable isotope labeling data to detect metabolic flux changes in a non-targeted manner. Furthermore, we show how similarity-analysis of isotopic enrichment patterns can be used for pathway contextualization of unidentified compounds. We illustrate our approach with the analysis of changes in cellular metabolism of human adenocarcinoma cells in response to decreased oxygen availability. Starting without a priori knowledge, we detect metabolic flux changes, leading to an increased glutamine contribution to acetyl-CoA production, reveal biosynthesis of N-acetylaspartate by N-acetyltransferase 8-like (NAT8L) in lung cancer cells and show that NAT8L silencing inhibits proliferation of A549, JHH-4, PH5CH8, and BEAS-2B cells.ConclusionsDifferential stable isotope labeling analysis provides qualitative metabolic flux information in a non-targeted manner. Furthermore, similarity analysis of enrichment patterns provides information on metabolically closely related compounds. N-acetylaspartate and NAT8L are important players in cancer cell metabolism, a context in which they have not received much attention yet.

ATF4-Induced Metabolic Reprograming Is a Synthetic Vulnerability of the p62-Deficient Tumor Stroma

Tumors undergo nutrient stress and need to reprogram their metabolism to survive. The stroma may play a critical role in this process by providing nutrients to support the epithelial compartment of the tumor. Here we show that p62 deficiency in stromal fibroblasts promotes resistance to glutamine deprivation by the direct control of ATF4 stability through its p62-mediated polyubiquitination. ATF4 upregulation by p62 deficiency in the stroma activates glucose carbon flux through a pyruvate carboxylase-asparagine synthase cascade that results in asparagine generation as a source of nitrogen for stroma and tumor epithelial proliferation. Thus, p62 directly targets nuclear transcription factors to control metabolic reprogramming in the microenvironment and repress tumorigenesis, and identifies ATF4 as a synthetic vulnerability in p62-deficient tumor stroma.

Tracing insights into human metabolism using chemical engineering approaches

Metabolism coordinates the conversion of available nutrients toward energy, biosynthetic intermediates, and signaling molecules to mediate virtually all biological functions. Dysregulation of metabolic pathways contributes to many diseases, so a detailed understanding of human metabolism has significant therapeutic implications. Over the last decade major technological advances in the areas of analytical chemistry, computational estimation of intracellular fluxes, and biological engineering have improved our ability to observe and engineer metabolic pathways. These approaches are reminiscent of the design, operation, and control of industrial chemical plants. Immune cells have emerged as an intriguing system in which metabolism influences diverse biological functions. Application of metabolic flux analysis and related approaches to macrophages and T cells offers great therapeutic opportunities to biochemical engineers.