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Abstract Despite advances in antimicrobial and anti-inflammatory treatment, inflammation and its consequences remain a major challenge in the field of medicine. Inflammatory reactions can lead to life-threatening conditions such as septic shock, while chronic inflammation has the potential to worsen the condition of body tissues and ultimately lead to significant impairment of their functionality. Although the central nervous system has long been considered immune privileged to peripheral immune responses, recent research has shown that strong immune responses in the periphery also affect the brain, leading to reactive microglia, which belong to the innate immune system and reside in the brain, and neuroinflammation. The inflammatory response is primarily a protective mechanism to defend against pathogens and tissue damage. However, excessive and chronic inflammation can have negative effects on neuronal structure and function. Neuroinflammation underlies the pathogenesis of many neurological and neurodegenerative diseases and can accelerate their progression. Consequently, targeting inflammatory signaling pathways offers potential therapeutic strategies for various neuropathological conditions, particularly Parkinson’s and Alzheimer’s disease, by curbing inflammation. Here the blood–brain barrier is a major hurdle for potential therapeutic strategies, therefore it would be highly advantageous to foster and utilize brain innate anti-inflammatory mechanisms. The tricarboxylic acid cycle-derived metabolite itaconate is highly upregulated in activated macrophages and has been shown to act as an immunomodulator with anti-inflammatory and antimicrobial functions. Mesaconate, an isomer of itaconate, similarly reduces the inflammatory response in macrophages. Nevertheless, most studies have focused on its esterified forms and its peripheral effects, while its influence on the CNS remained largely unexplored. Therefore, this study investigated the immunomodulatory and therapeutic potential of endogenously synthesized itaconate and its isomer mesaconate in lipopolysaccharide (LPS)-induced neuroinflammatory processes. Our results show that both itaconate and mesaconate reduce LPS-induced neuroinflammation, as evidenced by lower levels of inflammatory mediators, reduced microglial reactivity and a rescue of synaptic plasticity, the cellular correlate of learning and memory processes in the brain. Overall, this study emphasizes that both itaconate and mesaconate have therapeutic potential for neuroinflammatory processes in the brain and are of remarkable importance due to their endogenous origin and production, which usually leads to high tolerance.

Abstract Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1β than Pfkl S775A/S775A after LPS treatment. In an in vivo inflammation model, Pfkl S775A/S775A mice show reduced levels of MCP-1 and IL-1β. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.

Abstract The hormonal decline during menopause increases women's risk of chronic diseases. D-limonene, a monoterpene found in the human diet, possesses biological properties related to hypolipemic, antioxidant, anti-inflammatory, and gut microbiota-modulating activities, primarily observed in preclinical studies. Postprandial responses include physiological adaptations to the stress of a nutrient surplus, providing an opportunity to assess metabolic resilience, being a suitable strategy for exploring post-menopause-associated metabolic alterations. Here, we investigated the effects of D-limonene supplementation (2 g/day) on postprandial metabolism in postmenopausal women (n = 9) challenged with a standardized mixed meal in a 3-week single-arm clinical study. Our findings revealed that D-limonene did not induce marked differences in postprandial responses to the dietary challenge. The supplementation with D-limonene induced no alterations in serum lipid/lipoprotein profile or glycemia/insulinemia. D-limonene supplementation did not affect the transient postprandial inflammatory response regarding changes in gene expression of peripheral blood mononuclear cells (PBMC) and circulating inflammatory markers. Nevertheless, D-limonene reduced postprandial levels of lithocholic acid, a gut microbiota-derived bile acid, and regulated the plasma concentrations of selected amino acids, carbohydrate metabolism-derived metabolites, and organic acids. In conclusion, our data do not support the claim that short-term D-limonene supplementation beneficially affects the postprandial metabolism of postmenopausal women. Graphical Abstract

Abstract Datasets consist of measurement data and metadata. Metadata provides context, essential for understanding and (re-)using data. Various metadata standards exist for different methods, systems and contexts. However, relevant information resides at differing stages across the data-lifecycle. Often, this information is defined and standardized only at publication stage, which can lead to data loss and workload increase. In this study, we developed Metadatasheet, a metadata standard based on interviews with members of two biomedical consortia and systematic screening of data repositories. It aligns with the data-lifecycle allowing synchronous metadata recording within Microsoft Excel, a widespread data recording software. Additionally, we provide an implementation, the Metadata Workbook, that offers user-friendly features like automation, dynamic adaption, metadata integrity checks, and export options for various metadata standards. By design and due to its extensive documentation, the proposed metadata standard simplifies recording and structuring of metadata for biomedical scientists, promoting practicality and convenience in data management. This framework can accelerate scientific progress by enhancing collaboration and knowledge transfer throughout the intermediate steps of data creation.

Objective: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor regulating xenobiotic responses as well as physiological metabolism. Dietary AhR ligands activate the AhR signaling axis, whereas AhR activation is negatively regulated by the AhR repressor (AhRR). While AhR-deficient mice are known to be resistant to diet-induced obesity (DIO), the influence of the AhRR on DIO has not been assessed so far. Methods: In this study, we analyzed AhRR−/− mice and mice with a conditional deletion of either AhRR or AhR in myeloid cells under conditions of DIO and after supplementation of dietary AhR ligands. Moreover, macrophage metabolism was assessed using Seahorse Mito Stress Test and ROS assays as well as transcriptomic analysis. Results: We demonstrate that global AhRR deficiency leads to a robust, but not as profound protection from DIO and hepatosteatosis as AhR deficiency. Under conditions of DIO, AhRR−/− mice did not accumulate TCA cycle intermediates in the circulation in contrast to wild-type (WT) mice, indicating protection from metabolic dysfunction. This effect could be mimicked by dietary supplementation of AhR ligands in WT mice. Because of the predominant expression of the AhRR in myeloid cells, AhRR-deficient macrophages were analyzed for changes in metabolism and showed major metabolic alterations regarding oxidative phosphorylation and mitochondrial activity. Unbiased transcriptomic analysis revealed increased expression of genes involved in de novo lipogenesis and mitochondrial biogenesis. Mice with a genetic deficiency of the AhRR in myeloid cells did not show alterations in weight gain after high fat diet (HFD) but demonstrated ameliorated liver damage compared to control mice. Further, deficiency of the AhR in myeloid cells also did not affect weight gain but led to enhanced liver damage and adipose tissue fibrosis compared to controls. Conclusions: AhRR-deficient mice are resistant to diet-induced metabolic syndrome. Although conditional ablation of either the AhR or AhRR in myeloid cells did not recapitulate the phenotype of the global knockout, our findings suggest that enhanced AhR signaling in myeloid cells deficient for AhRR protects from diet-induced liver damage and fibrosis, whereas myeloid cell-specific AhR deficiency is detrimental.

Abstract Background “Red tides” are harmful algal blooms caused by dinoflagellate microalgae that accumulate toxins lethal to other organisms, including humans via consumption of contaminated seafood. These algal blooms are driven by a combination of environmental factors including nutrient enrichment, particularly in warm waters, and are increasingly frequent. The molecular, regulatory, and evolutionary mechanisms that underlie the heat stress response in these harmful bloom-forming algal species remain little understood, due in part to the limited genomic resources from dinoflagellates, complicated by the large sizes of genomes, exhibiting features atypical of eukaryotes. Results We present the de novo assembled genome (~ 4.75 Gbp with 85,849 protein-coding genes), transcriptome, proteome, and metabolome from Prorocentrum cordatum, a globally abundant, bloom-forming dinoflagellate. Using axenic algal cultures, we study the molecular mechanisms that underpin the algal response to heat stress, which is relevant to current ocean warming trends. We present the first evidence of a complementary interplay between RNA editing and exon usage that regulates the expression and functional diversity of biomolecules, reflected by reduction in photosynthesis, central metabolism, and protein synthesis. These results reveal genomic signatures and post-transcriptional regulation for the first time in a pelagic dinoflagellate. Conclusions Our multi-omics analyses uncover the molecular response to heat stress in an important bloom-forming algal species, which is driven by complex gene structures in a large, high-G+C genome, combined with multi-level transcriptional regulation. The dynamics and interplay of molecular regulatory mechanisms may explain in part how dinoflagellates diversified to become some of the most ecologically successful organisms on Earth.

Abstract Sporadic Parkinson’s Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD.

BackgroundCachexia is a body wasting syndrome that significantly affects well-being and prognosis of cancer patients, without effective treatment. Serum metabolites take part in pathophysiological processes of cancer cachexia, but apart from altered levels of select serum metabolites, little is known on the global changes of the overall serum metabolome, which represents a functional readout of the whole-body metabolic state. Here, we aimed to comprehensively characterize serum metabolite alterations and analyze associated pathways in cachectic cancer patients to gain new insights that could help instruct strategies for novel interventions of greater clinical benefit.MethodsSerum was sampled from 120 metastatic cancer patients (stage UICC IV). Patients were grouped as cachectic or non-cachectic according to the criteria for cancer cachexia agreed upon international consensus (main criterium: weight loss adjusted to body mass index). Samples were pooled by cachexia phenotype and assayed using non-targeted gas chromatography-mass spectrometry (GC-MS). Normalized metabolite levels were compared using t-test (p < 0.05, adjusted for false discovery rate) and partial least squares discriminant analysis (PLS-DA). Machine-learning models were applied to identify metabolite signatures for separating cachexia states. Significant metabolites underwent MetaboAnalyst 5.0 pathway analysis.ResultsComparative analyses included 78 cachectic and 42 non-cachectic patients. Cachectic patients exhibited 19 annotable, significantly elevated (including glucose and fructose) or decreased (mostly amino acids) metabolites associating with aminoacyl-tRNA, glutathione and amino acid metabolism pathways. PLS-DA showed distinct clusters (accuracy: 85.6%), and machine-learning models identified metabolic signatures for separating cachectic states (accuracy: 83.2%; area under ROC: 88.0%). We newly identified altered blood levels of erythronic acid and glucuronic acid in human cancer cachexia, potentially linked to pentose-phosphate and detoxification pathways.ConclusionWe found both known and yet unknown serum metabolite and metabolic pathway alterations in cachectic cancer patients that collectively support a whole-body metabolic state with impaired detoxification capability, altered glucose and fructose metabolism, and substrate supply for increased and/or distinct metabolic needs of cachexia-associated tumors. These findings together imply vulnerabilities, dependencies and targets for novel interventions that have potential to make a significant impact on future research in an important field of cancer patient care.

Cruciferous-rich diets, particularly broccoli, have been associated with reduced risk of developing cancers of various sites, cardiovascular disease and type-2 diabetes. Sulforaphane (SF), a sulfur-containing broccoli-derived metabolite, has been identified as the major bioactive compound mediating these health benefits. Sulforaphane is a potent dietary activator of the transcription factor Nuclear factor erythroid-like 2 (NRF2), the master regulator of antioxidant cell capacity responsible for inducing cytoprotective genes, but its role in glucose homeostasis remains unclear. In this study, we set to test the hypothesis that SF regulates glucose metabolism and ameliorates glucose overload and its resulting oxidative stress by inducing NRF2 in human hepatoma HepG2 cells. HepG2 cells were exposed to varying glucose concentrations: basal (5.5 mM) and high glucose (25 mM), in the presence of physiological concentrations of SF (10 μM). SF upregulated the expression of glutathione (GSH) biosynthetic genes and significantly increased levels of reduced GSH. Labelled glucose and glutamine experiments to measure metabolic fluxes identified that SF increased intracellular utilisation of glycine and glutamate by redirecting the latter away from the TCA cycle and increased the import of cysteine from the media, likely to support glutathione synthesis. Furthermore, SF altered pathways generating NADPH, the necessary cofactor for oxidoreductase reactions, namely pentose phosphate pathway and 1C-metabolism, leading to the redirection of glucose away from glycolysis and towards PPP and of methionine towards methylation substrates. Finally, transcriptomic and targeted metabolomics LC-MS analysis of NRF2-KD HepG2 cells generated using CRISPR-Cas9 genome editing revealed that the above metabolic effects are mediated through NRF2. These results suggest that the antioxidant properties of cruciferous diets are intricately connected to their metabolic benefits.

Epidemiological studies identified air pollution as one of the prime causes for human morbidity and mortality, due to harmful effects mainly on the cardiovascular and respiratory systems. Damage to the lung leads to several severe diseases such as fibrosis, chronic obstructive pulmonary disease and cancer. Noxious environmental aerosols are comprised of a gas and particulate phase representing highly complex chemical mixtures composed of myriads of compounds. Although some critical pollutants, foremost particulate matter (PM), could be linked to adverse health effects, a comprehensive understanding of relevant biological mechanisms and detrimental aerosol constituents is still lacking. Here, we employed a systems toxicology approach focusing on wood combustion, an important source for air pollution, and demonstrate a key role of the gas phase, specifically carbonyls, in driving adverse effects. Transcriptional profiling and biochemical analysis of human lung cells exposed at the air–liquid-interface determined DNA damage and stress response, as well as perturbation of cellular metabolism, as major key events. Connectivity mapping revealed a high similarity of gene expression signatures induced by wood smoke and agents prompting DNA-protein crosslinks (DPCs). Indeed, various gaseous aldehydes were detected in wood smoke, which promote DPCs, initiate similar genomic responses and are responsible for DNA damage provoked by wood smoke. Hence, systems toxicology enables the discovery of critical constituents of complex mixtures i.e. aerosols and highlights the role of carbonyls on top of particulate matter as an important health hazard.

Follicular and marginal zone B (FoB and MZB, respectively) cells have divergent metabolic characteristics. Here the authors show that deficiency of glutamate cysteine ligase (Gclc), the enzyme for glutathione synthesis, differentially impacts FoB and MZB homeostasis, while specifically impeding FoB activation and downstream antiviral immunity.

The Micrarchaeota lineage includes poorly characterized archaea with reduced genomes that likely depend on host interactions for survival. Here, the authors report a stable co-culture of a member of the Micrarchaeota and its host, and use multi-omic and physiological analyses to shed light on this symbiosis.

Summary: 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.

Background & Aims: Inflammation is the hallmark of chronic liver disease. Metabolism is a key determinant to regulate the activation of immune cells. Here, we define the role of sirtuin 1 (SIRT1), a main metabolic regulator, in controlling the activation of macrophages during cholestatic liver disease and in response to endotoxin. Methods: We have used mice overexpressing SIRT1, which we treated with intraperitoneal lipopolysaccharides or induced cholestasis by bile duct ligation. Bone marrow–derived macrophages were used for mechanistic in vitro studies. Finally, PEPC-Boy mice were used for adoptive transfer experiments to elucidate the impact of SIRT1-overexpressing macrophages in contributing to cholestatic liver disease. Results: We found that SIRT1 overexpression promotes increased liver inflammation and liver injury after lipopolysaccharide/GalN and bile duct ligation; this was associated with an increased activation of the inflammasome in macrophages. Mechanistically, SIRT1 overexpression associated with the activation of the mammalian target of rapamycin (mTOR) pathway that led to increased activation of macrophages, which showed metabolic rewiring with increased glycolysis and broken tricarboxylic acid cycle in response to endotoxin in vitro. Activation of the SIRT1/mTOR axis in macrophages associated with the activation of the inflammasome and the attenuation of autophagy. Ultimately, in an in vivo model of cholestatic disease, the transplantation of SIRT1-overexpressing myeloid cells contributed to liver injury and fibrosis. Conclusions: Our study provides novel mechanistic insights into the regulation of macrophages during cholestatic disease and the response to endotoxin, in which the SIRT1/mTOR crosstalk regulates macrophage activation controlling the inflammasome, autophagy and metabolic rewiring.

Myeloid-derived suppressor cells (MDSCs) are a compendium of immature myeloid cells that exhibit potent T-cell suppressive capacity and expand during pathological conditions such as cancer and chronic infections. Although well-characterized in cancer, the physiology of MDSCs in the infection setting remains enigmatic. Here, we integrated single-cell RNA sequencing (scRNA-seq) and functional metabolic profiling to gain deeper insights into the factors governing the generation and maintenance of MDSCs in chronic Staphylococcus aureus infection. We found that MDSCs originate not only in the bone marrow but also at extramedullary sites in S. aureus-infected mice. scRNA-seq showed that infection-driven MDSCs encompass a spectrum of myeloid precursors in different stages of differentiation, ranging from promyelocytes to mature neutrophils. Furthermore, the scRNA-seq analysis has also uncovered valuable phenotypic markers to distinguish mature myeloid cells from immature MDSCs. Metabolic profiling indicates that MDSCs exhibit high glycolytic activity and high glucose consumption rates, which are required for undergoing terminal maturation. However, rapid glucose consumption by MDSCs added to infection-induced perturbations in the glucose supplies in infected mice hinders the terminal maturation of MDSCs and promotes their accumulation in an immature stage. In a proof-of-concept in vivo experiment, we demonstrate the beneficial effect of increasing glucose availability in promoting MDSC terminal differentiation in infected mice. Our results provide valuable information of how metabolic alterations induced by infection influence reprogramming and differentiation of MDSCs.

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.

Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1β. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.

Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer.

BackgroundErythritol is a predictive biomarker of cardiometabolic diseases and is produced from glucose metabolism through the pentose phosphate pathway (PPP). Little is known regarding the regulation of endogenous erythritol synthesis in humans.ObjectiveIn the present study, we investigated the stimuli that promote erythritol synthesis in human lung carcinoma cells and characterized potential points of regulation along the PPP.MethodsHuman A549 lung carcinoma cells were chosen for their known ability to synthesize erythritol. A549 cells were treated with potential substrates for erythritol production, including glucose, fructose, and glycerol. Using siRNA knockdown, we assessed the necessity of enzymes G6PD, TKT, TALDO, and SORD for erythritol synthesis. We also used position-specific 13C-glucose tracers to determine whether the carbons for erythritol synthesis are derived directly from glycolysis or through the oxidative PPP. Finally, we assessed if erythritol synthesis responds to oxidative stress using chemical and genetic models.ResultsIntracellular erythritol was directly associated with media glucose concentration. In addition, siRNA knockdown of TKT or SORD inhibited erythritol synthesis, whereas siG6PD did not. Both chemically induced oxidative stress and constitutive activation of the antioxidant response transcription factor NRF2 elevated intracellular erythritol.ConclusionOur findings indicate that in A549 cells, erythritol synthesis is proportional to flux through the PPP and is regulated by non-oxidative PPP enzymes.

The anti-inflammatory cytokine interleukin-37 (IL-37) belongs to the IL-1 family but is not expressed in mice. We used a human IL-37 (hIL-37tg) expressing mouse, which has been subjected to various models of local and systemic inflammation as well as immunological challenges. Previous studies reveal an immunomodulatory role of IL-37, which can be characterized as an important suppressor of innate immunity. Here, we examined the functions of IL-37 in the central nervous system and explored the effects of IL-37 on neuronal architecture and function, microglial phenotype, cytokine production and behavior after inflammatory challenge by intraperitoneal LPS-injection. In wild-type mice, decreased spine density, activated microglial phenotype and impaired long-term potentiation (LTP) were observed after LPS injection, whereas hIL-37tg mice showed no impairment. In addition, we crossed the hIL-37tg mouse with an animal model of Alzheimer’s disease (APP/PS1) to investigate the anti-inflammatory properties of IL-37 under chronic neuroinflammatory conditions. Our results show that expression of IL-37 is able to limit inflammation in the brain after acute inflammatory events and prevent loss of cognitive abilities in a mouse model of AD.

Abstract L-3,4-Dihydroxyphenylalanin (l-DOPA or levodopa) is currently the most used drug to treat symptoms of Parkinson’s disease (PD). After crossing the blood–brain barrier, it is enzymatically converted to dopamine by neuronal cells and restores depleted endogenous neurotransmitter levels. l-DOPA is prone to auto-oxidation and reactive intermediates of its degradation including reactive oxygen species (ROS) have been implicated in cellular damage. In this study, we investigated how oxygen tension effects l-DOPA stability. We applied oxygen tensions comparable to those in the mammalian brain and demonstrated that 2% oxygen almost completely stopped its auto-oxidation. l-DOPA even exerted a ROS scavenging function. Further mechanistic analysis indicated that l-DOPA reprogrammed mitochondrial metabolism and reduced oxidative phosphorylation, depolarized the mitochondrial membrane, induced reductive glutamine metabolism, and depleted the NADH pool. These results shed new light on the cellular effects of l-DOPA and its neuro-toxicity under physiological oxygen levels that are very distinct to normoxic in vitro conditions.

Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires’ disease (LD). The bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD.

Untargeted metabolomics is an important tool in studying health and disease and is employed in fields such as biomarker discovery and drug development, as well as precision medicine. Although significant technical advances were made in the field of mass-spectrometry driven metabolomics, instrumental drifts, such as fluctuations in retention time and signal intensity, remain a challenge, particularly in large untargeted metabolomics studies. Therefore, it is crucial to consider these variations during data processing to ensure high-quality data. Here, we will provide recommendations for an optimal data processing workflow using intrastudy quality control (QC) samples that identifies errors resulting from instrumental drifts, such as shifts in retention time and metabolite intensities. Furthermore, we provide an in-depth comparison of the performance of three popular batch-effect correction methods of different complexity. By using different evaluation metrics based on QC samples and a machine learning approach based on biological samples, the performance of the batch-effect correction methods were evaluated. Here, the method TIGER demonstrated the overall best performance by reducing the relative standard deviation of the QCs and dispersion-ratio the most, as well as demonstrating the highest area under the receiver operating characteristic with three different probabilistic classifiers (Logistic regression, Random Forest, and Support Vector Machine). In summary, our recommendations will help to generate high-quality data that are suitable for further downstream processing, leading to more accurate and meaningful insights into the underlying biological processes.

Abstract Performing long-term cell observations is a non-trivial task for conventional optical microscopy, since it is usually not compatible with environments of an incubator and its temperature and humidity requirements. Lensless holographic microscopy, being entirely based on semiconductor chips without lenses and without any moving parts, has proven to be a very interesting alternative to conventional microscopy. Here, we report on the integration of a computational parfocal feature, which operates based on wave propagation distribution analysis, to perform a fast autofocusing process. This unique non-mechanical focusing approach was implemented to keep the imaged object staying in-focus during continuous long-term and real-time recordings. A light-emitting diode (LED) combined with pinhole setup was used to realize a point light source, leading to a resolution down to 2.76 μm. Our approach delivers not only in-focus sharp images of dynamic cells, but also three-dimensional (3D) information on their (x, y, z)-positions. System reliability tests were conducted inside a sealed incubator to monitor cultures of three different biological living cells (i.e., MIN6, neuroblastoma (SH-SY5Y), and Prorocentrum minimum). Altogether, this autofocusing framework enables new opportunities for highly integrated microscopic imaging and dynamic tracking of moving objects in harsh environments with large sample areas.

Insulin secretion following ingestion of a carbohydrate load affects a multitude of metabolic pathways that simultaneously change direction and quantity of interorgan fluxes of sugars, lipids and amino acids. In the present study, we aimed at identifying markers associated with differential responses to an OGTT a population of healthy adults. By use of three metabolite profiling platforms, we assessed these postprandial responses of a total of 202 metabolites in plasma of 72 healthy volunteers undergoing comprehensive phenotyping and of which half enrolled into a weight-loss program over a three-month period. A standard oral glucose tolerance test (OGTT) served as dietary challenge test to identify changes in postprandial metabolite profiles. Despite classified as healthy according to WHO criteria, two discrete clusters (A and B) were identified based on the postprandial glucose profiles with a balanced distribution of volunteers based on gender and other measures. Cluster A individuals displayed 26% higher postprandial glucose levels, delayed glucose clearance and increased fasting plasma concentrations of more than 20 known biomarkers of insulin resistance and diabetes previously identified in large cohort studies. The volunteers identified by canonical postprandial responses that form cluster A may be called pre-pre-diabetics and defined as “at risk” for development of insulin resistance. Moreover, postprandial changes in selected fatty acids and complex lipids, bile acids, amino acids, acylcarnitines and sugars like mannose revealed marked differences in the responses seen in cluster A and cluster B individuals that sustained over the entire challenge test period of 240 min. Almost all metabolites, including glucose and insulin, returned to baseline values at the end of the test (at 240 min), except a variety of amino acids and here those that have been linked to diabetes development. Analysis of the corresponding metabolite profile in a fasting blood sample may therefore allow for early identification of these subjects at risk for insulin resistance without the need to undergo an OGTT.

On an organismal level, metabolism needs to react in a well-orchestrated manner to metabolic challenges such as nutrient uptake. Key metabolic hubs in human blood are pyruvate and lactate, both of which are constantly interconverted by very fast exchange fluxes. The quantitative contribution of different food sources to these metabolite pools remains unclear. Here, we applied in vivo stable isotope labeling to determine postprandial metabolic fluxes in response to two carbohydrate sources of different complexity. Depending on the ingested carbohydrate source, glucose or wheat flour, the net direction of the lactate dehydrogenase, and the alanine amino transferase fluxes were adjusted in a way to ensure sufficient availability, while, at the same time, preventing an overflow in the respective metabolite pools. The systemic lactate pool acts as a metabolic buffer which is fueled in the early- and depleted in the late-postprandial phase and thus plays a key role for systemic metabolic homeostasis.

Summary: Pathogenic viruses induce metabolic changes in host cells to secure the availability of biomolecules and energy to propagate. Influenza A virus (IAV) and severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) both infect the human airway epithelium and are important human pathogens. The metabolic changes induced by these viruses in a physiologically relevant human model and how this affects innate immune responses to limit viral propagation are not well known. Using an ex vivo model of pseudostratified primary human airway epithelium, we here demonstrate that infection with both IAV and SARS-CoV-2 resulted in distinct metabolic changes including increases in lactate dehydrogenase A (LDHA) expression and LDHA-mediated lactate formation. Interestingly, LDHA regulated both basal and induced mitochondrial anti-viral signaling protein (MAVS)-dependent type I interferon (IFN) responses to promote IAV, but not SARS-CoV-2, replication. Our data demonstrate that LDHA and lactate promote IAV but not SARS-CoV-2 replication by inhibiting MAVS-dependent induction of type I IFN in primary human airway epithelium.

Pneumonia is a common cause of morbidity and mortality and is most often caused by bacterial pathogens. COVID-19 is characterized by lung infection with potential progressive organ failure. The systemic consequences of both disease on the systemic blood metabolome are not fully understood. The aim of this study was to compare the blood metabolome of both diseases and we hypothesize that plasma metabolomics may help to identify the systemic effects of these diseases. Therefore, we profiled the plasma metabolome of 43 cases of COVID-19 pneumonia, 23 cases of non-COVID-19 pneumonia, and 26 controls using a non-targeted approach. Metabolic alterations differentiating the three groups were detected, with specific metabolic changes distinguishing the two types of pneumonia groups. A comparison of venous and arterial blood plasma samples from the same subjects revealed the distinct metabolic effects of pulmonary pneumonia. In addition, a machine learning signature of four metabolites was predictive of the disease outcome of COVID-19 subjects with an area under the curve (AUC) of 86 ± 10%. Overall, the results of this study uncover systemic metabolic changes that could be linked to the etiology of COVID-19 pneumonia and non-COVID-19 pneumonia.

Individuals carrying a pathogenic germline variant in the breast cancer predisposition gene BRCA1 (gBRCA1+) are prone to developing breast cancer. Apart from its well-known role in DNA repair, BRCA1 has been shown to powerfully impact cellular metabolism. While, in general, metabolic reprogramming was named a hallmark of cancer, disrupted metabolism has also been suggested to drive cancer cell evolution and malignant transformation by critically altering microenvironmental tissue integrity. Systemic metabolic effects induced by germline variants in cancer predisposition genes have been demonstrated before. Whether or not systemic metabolic alterations exist in gBRCA1+ individuals independent of cancer incidence has not been investigated yet. We therefore profiled the plasma metabolome of 72 gBRCA1+ women and 72 age-matched female controls, none of whom (carriers and non-carriers) had a prior cancer diagnosis and all of whom were cancer-free during the follow-up period. We detected one single metabolite, pyruvate, and two metabolite ratios involving pyruvate, lactate, and a metabolite of yet unknown structure, significantly altered between the two cohorts. A machine learning signature of metabolite ratios was able to correctly distinguish between gBRCA1+ and controls in ~82%. The results of this study point to innate systemic metabolic differences in gBRCA1+ women independent of cancer incidence and raise the question as to whether or not constitutional alterations in energy metabolism may be involved in the etiology of BRCA1-associated breast cancer.

The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage.

Summary: Since the discovery of mutations in isocitrate dehydrogenase 1 (IDH1) in gliomas and other tumors, significant efforts have been made to gain a deeper understanding of the consequences of this oncogenic mutation. One aspect of the neomorphic function of the IDH1 R132H enzyme that has received less attention is the perturbation of cellular redox homeostasis. Here, we describe a biosynthetic pathway exhibited by cells expressing mutant IDH1. By virtue of a change in cellular redox homeostasis, IDH1-mutated cells synthesize excess glutamine-derived proline through enhanced activity of pyrroline 5-carboxylate reductase 1 (PYCR1), coupled to NADH oxidation. Enhanced proline biosynthesis partially uncouples the electron transport chain from tricarboxylic acid (TCA) cycle activity through the maintenance of a lower NADH/NAD+ ratio and subsequent reduction in oxygen consumption. Thus, we have uncovered a mechanism by which tumor cell survival may be promoted in conditions associated with perturbed redox homeostasis, as occurs in IDH1-mutated glioma. : Hollinshead et al. demonstrate a role for PYCR1 in control of mitochondrial redox homeostasis. Expression of IDH1 R132H mutation leads to increased NADH-coupled proline biosynthesis, mediated by PYCR1. The resulting metabolic phenotype partially uncouples mitochondrial NADH oxidation from respiration, representing an oxygen-sparing metabolic phenotype. Keywords: glioma, IDH1, redox, metabolism, proline

Metabolome analyses of body fluids are challenging due pre-analytical variations, such as pre-processing delay and temperature, and constant dynamical changes of biochemical processes within the samples. Therefore, proper sample handling starting from the time of collection up to the analysis is crucial to obtain high quality samples and reproducible results. A metabolomics analysis is divided into 4 main steps: 1) Sample collection, 2) Metabolite extraction, 3) Data acquisition and 4) Data analysis. Here, we describe a protocol for gas chromatography coupled to mass spectrometry (GC–MS) based metabolic analysis for biological matrices, especially body fluids. This protocol can be applied on blood serum/plasma, saliva and cerebrospinal fluid (CSF) samples of humans and other vertebrates. It covers sample collection, sample pre-processing, metabolite extraction, GC–MS measurement and guidelines for the subsequent data analysis. Advantages of this protocol include: • Robust and reproducible metabolomics results, taking into account pre-analytical variations that may occur during the sampling process • Small sample volume required • Rapid and cost-effective processing of biological samples • Logistic regression based determination of biomarker signatures for in-depth data analysis

Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here, we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using 13C/2H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth, chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration, altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development.

Food supplementation with a fiber mix of guar gum and chickpea flour represents a promising approach to reduce the risk of type 2 diabetes mellitus (T2DM) by attenuating postprandial glycemia. To investigate the effects on postprandial metabolic fluxes of glucose-derived metabolites in response to this fiber mix, a randomized, cross-over study was designed. Twelve healthy, male subjects consumed three different flatbreads either supplemented with 2% guar gum or 4% guar gum and 15% chickpea flour or without supplementation (control). The flatbreads were enriched with ~2% of 13C-labeled wheat flour. Blood was collected at 16 intervals over a period of 360 min after bread intake and plasma samples were analyzed by GC-MS based metabolite profiling combined with stable isotope-assisted metabolomics. Although metabolite levels of the downstream metabolites of glucose, specifically lactate and alanine, were not altered in response to the fiber mix, supplementation of 4% guar gum was shown to significantly delay and reduce the exogenous formation of these metabolites. Metabolic modeling and computation of appearance rates revealed that the effects induced by the fiber mix were strongest for glucose and attenuated downstream of glucose. Further investigations to explore the potential of fiber mix supplementation to counteract the development of metabolic diseases are warranted.

Continuous cell culture monitoring as a way of investigating growth, proliferation, and kinetics of biological experiments is in high demand. However, commercially available solutions are typically expensive and large in size. Digital inline-holographic microscopes (DIHM) can provide a cost-effective alternative to conventional microscopes, bridging the gap towards live-cell culture imaging. In this work, a DIHM is built from inexpensive components and applied to different cell cultures. The images are reconstructed by computational methods and the data are analyzed with particle detection and tracking methods. Counting of cells as well as movement tracking of living cells is demonstrated, showing the feasibility of using a field-portable DIHM for basic cell culture investigation and bringing about the potential to deeply understand cell motility.