Systems Immunology Approaches to Metabolism.

Over the last decade, immunometabolism has emerged as a novel interdisciplinary field of research and yielded significant fundamental insights into the regulation of immune responses. Multiple classical approaches to interrogate immunometabolism, including bulk metabolic profiling and analysis of metabolic regulator expression, paved the way to appreciating the physiological complexity of immunometabolic regulation in vivo. Studying immunometabolism at the systems level raised the need to transition towards the next-generation technology for metabolic profiling and analysis. Spatially resolved metabolic imaging and computational algorithms for multi-modal data integration are new approaches to connecting metabolism and immunity. In this review, we discuss recent studies that highlight the complex physiological interplay between immune responses and metabolism and give an overview of technological developments that bear the promise of capturing this complexity most directly and comprehensively.

Atlas of fetal metabolism during mid-to-late gestation and diabetic pregnancy.

Mounting evidence suggests metabolism instructs stem cell fate decisions. However, how fetal metabolism changes during development and how altered maternal metabolism shapes fetal metabolism remain unexplored. We present a descriptive atlas of in vivo fetal murine metabolism during mid-to-late gestation in normal and diabetic pregnancy. Using 13C-glucose and liquid chromatography-mass spectrometry (LC-MS), we profiled the metabolism of fetal brains, hearts, livers, and placentas harvested from pregnant dams between embryonic days (E)10.5 and 18.5. Our analysis revealed metabolic features specific to a hyperglycemic environment and signatures that may denote developmental transitions during euglycemic development. We observed sorbitol accumulation in fetal tissues and altered neurotransmitter levels in fetal brains isolated from hyperglycemic dams. Tracing 13C-glucose revealed disparate fetal nutrient sourcing depending on maternal glycemic states. Regardless of glycemic state, histidine-derived metabolites accumulated in late-stage fetal tissues. Our rich dataset presents a comprehensive overview of in vivo fetal tissue metabolism and alterations due to maternal hyperglycemia.

Evolution of diapause in the African turquoise killifish by remodeling the ancient gene regulatory landscape.

Suspended animation states allow organisms to survive extreme environments. The African turquoise killifish has evolved diapause as a form of suspended development to survive a complete drought. However, the mechanisms underlying the evolution of extreme survival states are unknown. To understand diapause evolution, we performed integrative multi-omics (gene expression, chromatin accessibility, and lipidomics) in the embryos of multiple killifish species. We find that diapause evolved by a recent remodeling of regulatory elements at very ancient gene duplicates (paralogs) present in all vertebrates. CRISPR-Cas9-based perturbations identify the transcription factors REST/NRSF and FOXOs as critical for the diapause gene expression program, including genes involved in lipid metabolism. Indeed, diapause shows a distinct lipid profile, with an increase in triglycerides with very-long-chain fatty acids. Our work suggests a mechanism for the evolution of complex adaptations and offers strategies to promote long-term survival by activating suspended animation programs in other species.

Emergence of community behaviors in the gut microbiota upon drug treatment.

Pharmaceuticals can directly inhibit the growth of gut bacteria, but the degree to which such interactions manifest in complex community settings is an open question. Here, we compared the effects of 30 drugs on a 32-species synthetic community with their effects on each community member in isolation. While most individual drug-species interactions remained the same in the community context, communal behaviors emerged in 26% of all tested cases. Cross-protection during which drug-sensitive species were protected in community was 6 times more frequent than cross-sensitization, the converse phenomenon. Cross-protection decreased and cross-sensitization increased at higher drug concentrations, suggesting that the resilience of microbial communities can collapse when perturbations get stronger. By metabolically profiling drug-treated communities, we showed that both drug biotransformation and bioaccumulation contribute mechanistically to communal protection. As a proof of principle, we molecularly dissected a prominent case: species expressing specific nitroreductases degraded niclosamide, thereby protecting both themselves and sensitive community members.

A metabolomics pipeline highlights microbial metabolism in bloodstream infections.

The growth of antimicrobial resistance (AMR) highlights an urgent need to identify bacterial pathogenic functions that may be targets for clinical intervention. Although severe infections profoundly alter host metabolism, prior studies have largely ignored microbial metabolism in this context. Here, we describe an iterative, comparative metabolomics pipeline to uncover microbial metabolic features in the complex setting of a host and apply it to investigate gram-negative bloodstream infection (BSI) in patients. We find elevated levels of bacterially derived acetylated polyamines during BSI and discover the enzyme responsible for their production (SpeG). Blocking SpeG activity reduces bacterial proliferation and slows pathogenesis. Reduction of SpeG activity also enhances bacterial membrane permeability and increases intracellular antibiotic accumulation, allowing us to overcome AMR in culture and in vivo. This study highlights how tools to study pathogen metabolism in the natural context of infection can reveal and prioritize therapeutic strategies for addressing challenging infections.

A multi-tissue metabolome atlas of primate pregnancy.

Pregnancy induces dramatic metabolic changes in females; yet, the intricacies of this metabolic reprogramming remain poorly understood, especially in primates. Using cynomolgus monkeys, we constructed a comprehensive multi-tissue metabolome atlas, analyzing 273 samples from 23 maternal tissues during pregnancy. We discovered a decline in metabolic coupling between tissues as pregnancy progressed. Core metabolic pathways that were rewired during primate pregnancy included steroidogenesis, fatty acid metabolism, and arachidonic acid metabolism. Our atlas revealed 91 pregnancy-adaptive metabolites changing consistently across 23 tissues, whose roles we verified in human cell models and patient samples. Corticosterone and palmitoyl-carnitine regulated placental maturation and maternal tissue progenitors, respectively, with implications for maternal preeclampsia, diabetes, cardiac hypertrophy, and muscle and liver regeneration. Moreover, we found that corticosterone deficiency induced preeclampsia-like inflammation, indicating the atlas's potential clinical value. Overall, our multi-tissue metabolome atlas serves as a framework for elucidating the role of metabolic regulation in female health during pregnancy.

Gut bacterial nutrient preferences quantified in vivo.

Great progress has been made in understanding gut microbiomes' products and their effects on health and disease. Less attention, however, has been given to the inputs that gut bacteria consume. Here, we quantitatively examine inputs and outputs of the mouse gut microbiome, using isotope tracing. The main input to microbial carbohydrate fermentation is dietary fiber and to branched-chain fatty acids and aromatic metabolites is dietary protein. In addition, circulating host lactate, 3-hydroxybutyrate, and urea (but not glucose or amino acids) feed the gut microbiome. To determine the nutrient preferences across bacteria, we traced into genus-specific bacterial protein sequences. We found systematic differences in nutrient use: most genera in the phylum Firmicutes prefer dietary protein, Bacteroides dietary fiber, and Akkermansia circulating host lactate. Such preferences correlate with microbiome composition changes in response to dietary modifications. Thus, diet shapes the microbiome by promoting the growth of bacteria that preferentially use the ingested nutrients.

Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels.

Some people are more attractive to mosquitoes than others, but the mechanistic basis of this phenomenon is poorly understood. We tested mosquito attraction to human skin odor and identified people who are exceptionally attractive or unattractive to mosquitoes. These differences were stable over several years. Chemical analysis revealed that highly attractive people produce significantly more carboxylic acids in their skin emanations. Mutant mosquitoes lacking the chemosensory co-receptors Ir8a, Ir25a, or Ir76b were severely impaired in attraction to human scent, but retained the ability to differentiate highly and weakly attractive people. The link between elevated carboxylic acids in "mosquito-magnet" human skin odor and phenotypes of genetic mutations in carboxylic acid receptors suggests that such compounds contribute to differential mosquito attraction. Understanding why some humans are more attractive than others provides insights into what skin odorants are most important to the mosquito and could inform the development of more effective repellents.

Personalized microbiome-driven effects of non-nutritive sweeteners on human glucose tolerance.

Non-nutritive sweeteners (NNS) are commonly integrated into human diet and presumed to be inert; however, animal studies suggest that they may impact the microbiome and downstream glycemic responses. We causally assessed NNS impacts in humans and their microbiomes in a randomized-controlled trial encompassing 120 healthy adults, administered saccharin, sucralose, aspartame, and stevia sachets for 2 weeks in doses lower than the acceptable daily intake, compared with controls receiving sachet-contained vehicle glucose or no supplement. As groups, each administered NNS distinctly altered stool and oral microbiome and plasma metabolome, whereas saccharin and sucralose significantly impaired glycemic responses. Importantly, gnotobiotic mice conventionalized with microbiomes from multiple top and bottom responders of each of the four NNS-supplemented groups featured glycemic responses largely reflecting those noted in respective human donors, which were preempted by distinct microbial signals, as exemplified by sucralose. Collectively, human NNS consumption may induce person-specific, microbiome-dependent glycemic alterations, necessitating future assessment of clinical implications.

Metabolic analysis as a driver for discovery, diagnosis, and therapy.

Metabolic anomalies contribute to tissue dysfunction. Current metabolism research spans from organelles to populations, and new technologies can accommodate investigation across these scales. Here, we review recent advancements in metabolic analysis, including small-scale metabolomics techniques amenable to organelles and rare cell types, functional screening to explore how cells respond to metabolic stress, and imaging approaches to non-invasively assess metabolic perturbations in diseases. We discuss how metabolomics provides an informative phenotypic dimension that complements genomic analysis in Mendelian and non-Mendelian disorders. We also outline pressing challenges and how addressing them may further clarify the biochemical basis of human disease.

Multiple early factors anticipate post-acute COVID-19 sequelae.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Proteomic and Metabolomic Characterization of COVID-19 Patient Sera.

Early detection and effective treatment of severe COVID-19 patients remain major challenges. Here, we performed proteomic and metabolomic profiling of sera from 46 COVID-19 and 53 control individuals. We then trained a machine learning model using proteomic and metabolomic measurements from a training cohort of 18 non-severe and 13 severe patients. The model was validated using 10 independent patients, 7 of which were correctly classified. Targeted proteomics and metabolomics assays were employed to further validate this molecular classifier in a second test cohort of 19 COVID-19 patients, leading to 16 correct assignments. We identified molecular changes in the sera of COVID-19 patients compared to other groups implicating dysregulation of macrophage, platelet degranulation, complement system pathways, and massive metabolic suppression. This study revealed characteristic protein and metabolite changes in the sera of severe COVID-19 patients, which might be used in selection of potential blood biomarkers for severity evaluation.

A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.

Metabolic Dynamics and Prediction of Gestational Age and Time to Delivery in Pregnant Women.

Metabolism during pregnancy is a dynamic and precisely programmed process, the failure of which can bring devastating consequences to the mother and fetus. To define a high-resolution temporal profile of metabolites during healthy pregnancy, we analyzed the untargeted metabolome of 784 weekly blood samples from 30 pregnant women. Broad changes and a highly choreographed profile were revealed: 4,995 metabolic features (of 9,651 total), 460 annotated compounds (of 687 total), and 34 human metabolic pathways (of 48 total) were significantly changed during pregnancy. Using linear models, we built a metabolic clock with five metabolites that time gestational age in high accordance with ultrasound (R = 0.92). Furthermore, two to three metabolites can identify when labor occurs (time to delivery within two, four, and eight weeks, AUROC ≥ 0.85). Our study represents a weekly characterization of the human pregnancy metabolome, providing a high-resolution landscape for understanding pregnancy with potential clinical utilities.

A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans.

Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.

Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes.

Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.