Detection of nitric oxide-mediated metabolic effects using real-time extracellular flux analysis.

Dendritic cell (DC) activation is marked by key events including: (I) rapid induction and shifting of metabolism favoring glycolysis for generation of biosynthetic metabolic intermediates and (II) large scale changes in gene expression including the upregulation of the antimicrobial enzyme inducible nitric oxide synthase (iNOS) which produces the toxic gas nitric oxide (NO). Historically, acute metabolic reprogramming and NO-mediated effects on cellular metabolism have been studied at specific timepoints during the DC activation process, namely at times before and after NO production. However, no formal method of real time detection of NO-mediated effects on DC metabolism have been fully described. Here, using Real-Time Extracellular Flux Analysis, we experimentally establish the phenomenon of an NO-dependent mitochondrial respiration threshold, which shows how titration of an activating stimulus is inextricably linked to suppression of mitochondrial respiration in an NO-dependent manner. As part of this work, we explore the efficacy of two different iNOS inhibitors in blocking the iNOS reaction kinetically in real time and explore/discuss parameters and considerations for application using Real Time Extracellular Flux Analysis technology. In addition, we show, the temporal relationship between acute metabolic reprogramming and NO-mediated sustained metabolic reprogramming kinetically in single real-time assay. These findings provide a method for detection of NO-mediated metabolic effects in DCs and offer novel insight into the timing of the DC activation process with its associated key metabolic events, revealing a better understanding of the nuances of immune cell biology.

Inhibition of Glycogen Metabolism Induces Reactive Oxygen Species-Dependent Cytotoxicity in Anaplastic Thyroid Cancer in Female Mice.

Anaplastic thyroid cancer (ATC) is one of the most lethal solid tumors, yet there are no effective, long-lasting treatments for ATC patients. Most tumors, including tumors of the endocrine system, exhibit an increased consumption of glucose to fuel cancer progression, and some cancers meet this high glucose requirement by metabolizing glycogen. Our goal was to determine whether ATC cells metabolize glycogen and if this could be exploited for treatment. We detected glycogen synthase and glycogen phosphorylase (PYG) isoforms in normal thyroid and thyroid cancer cell lines and patient-derived biopsy samples. Inhibition of PYG using CP-91,149 induced apoptosis in ATC cells but not normal thyroid cells. CP-91,149 decreased NADPH levels and induced reactive oxygen species accumulation. CP-91,149 severely blunted ATC tumor growth in vivo. Our work establishes glycogen metabolism as a novel metabolic process in thyroid cells, which presents a unique, oncogenic target that could offer an improved clinical outcome.

Divergent Genetic Regulation of Nitric Oxide Production between C57BL/6J and Wild-Derived PWD/PhJ Mice Controls Postactivation Mitochondrial Metabolism, Cell Survival, and Bacterial Resistance in Dendritic Cells.

Dendritic cell (DC) activation is characterized by sustained commitment to glycolysis that is a requirement for survival in DC subsets that express inducible NO synthase (Nos2) due to NO-mediated inhibition of mitochondrial respiration. This phenomenon primarily has been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6) mice, where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced postactivation survival due to attenuated NO production in LPS-stimulated PWD DCs phenocopying human monocyte-derived DCs. To genetically map this phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. B6.PWD-Chr11.2 DCs show preserved mitochondrial function and produce lower NO levels than B6 DCs. We demonstrate that activated B6.PWD-Chr11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production to investigate the contribution of NO in regulating the interplay between DC metabolism and immune function. These findings suggest that reported differences between human and murine DCs may be an artifact of the limited genetic diversity of the mouse models used, underscoring the need for mouse genetic diversity in immunology research.

Determination of cell volume as part of metabolomics experiments.

Cells regulate their cell volume, but cell volumes may change in response to metabolic and other perturbations. Many metabolomics experiments use cultured cells to measure changes in metabolites in response to physiological and other experimental perturbations, but the metabolomics workflow by mass spectrometry only determines total metabolite amounts in cell culture extracts. To convert metabolite amount to metabolite concentration requires knowledge of the number and volume of the cells. Measuring only metabolite amount can lead to incorrect or skewed results in cell culture experiments because cell size may change due to experimental conditions independent of change in metabolite concentration. We have developed a novel method to determine cell volume in cell culture experiments using a pair of stable isotopically labeled phenylalanine internal standards incorporated within the normal liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics workflow. This method relies on the flooding-dose technique where the intracellular concentration of a particular compound (in this case phenylalanine) is forced to equal its extracellular concentration. We illustrate the LC-MS/MS technique for two different mammalian cell lines. Although the method is applicable in general for determining cell volume, the major advantage of the method is its seamless incorporation within the normal metabolomics workflow.

American Association of Immunologists Recommendations for an Undergraduate Course in Immunology.

Identifying the "essential" components of an undergraduate immunology lecture course can be daunting because of the varying postgraduate pathways students take. The American Association of Immunologists Education Committee commissioned an Ad Hoc Committee, representing undergraduate, graduate, and medical institutions as well as the biotechnology community, to develop core curricular recommendations for teaching immunology to undergraduates. In a reiterative process involving the American Association of Immunologists teaching community, 14 key topics were identified and expanded to include foundational concepts, subtopics and examples, and advanced subtopics, providing a flexible list for curriculum development and avenues for higher-level learning. Recommendations for inclusive and antiracist teaching that outline opportunities to meet the needs of diverse student populations were also developed. The consensus recommendations can be used to accommodate various course settings and will bridge undergraduate and graduate teaching and prepare diverse students for subsequent careers in the biomedical field.

Probiotic and commensal gut microbial therapies in multiple sclerosis and its animal models: a comprehensive review.

The need for alternative treatments for multiple sclerosis (MS) has triggered copious amounts of research into microbial therapies focused on manipulating the microbiota-gut-brain axis. This comprehensive review was intended to present and systematically evaluate the current clinical and preclinical evidence for various probiotic and commensal gut microbial therapies as treatments for MS, using the Bradford Hill criteria (BHC) as a multi-parameter assessment rubric. Literature searches were performed to identify a total of 37 relevant studies (6 human, 31 animal), including 28 probiotic therapy and 9 commensal therapy studies. In addition to presenting qualitative summaries of these findings, therapeutic evidence for each bacterial formulation was assessed using the BHC to generate summative scores. These scores, which encompassed study quality, replication, and other considerations, were used to rank the most promising therapies and highlight deficiencies. Several therapeutic formulations, including VSL#3, Lactobacillus paracasei, Bifidobacterium animalis, E. coli Nissle 1917, and Prevotella histicola, emerged as the most promising. In contrast, a number of other therapies were hindered by limited evidence of replicable findings and other criteria, which need to be addressed by future studies in order to harness gut microbial therapies to ultimately provide cheaper, safer, and more durable treatments for MS.

A guided-inquiry investigation of genetic variants using Oxford nanopore sequencing for an undergraduate molecular biology laboratory course.

Next Generation Sequencing (NGS) has become an important tool in the biological sciences and has a growing number of applications across medical fields. Currently, few undergraduate programs provide training in the design and implementation of NGS applications. Here, we describe an inquiry-based laboratory exercise for a college-level molecular biology laboratory course that uses real-time MinION deep sequencing and bioinformatics to investigate characteristic genetic variants found in cancer cell-lines. The overall goal for students was to identify non-small cell lung cancer (NSCLC) cell-lines based on their unique genomic profiles. The units described in this laboratory highlight core principles in multiplex PCR primer design, real-time deep sequencing, and bioinformatics analysis for genetic variants. We found that the MinION device is an appropriate, feasible tool that provides a comprehensive, hands-on NGS experience for undergraduates. Student evaluations demonstrated increased confidence in using molecular techniques and enhanced understanding of NGS concepts. Overall, this exercise provides a pedagogical tool for incorporating NGS approaches in the teaching laboratory as way of enhancing students' comprehension of genomic sequence analysis. Further, this NGS lab module can easily be added to a variety of lab-based courses to help undergraduate students learn current DNA sequencing methods with limited effort and cost.

Glycolipid-Containing Nanoparticle Vaccine Engages Invariant NKT Cells to Enhance Humoral Protection against Systemic Bacterial Infection but Abrogates T-Independent Vaccine Responses.

CD4+ T cells enable the critical B cell humoral immune protection afforded by most effective vaccines. We and others have recently identified an alternative source of help for B cells in mice, invariant NK T (iNKT) cells. iNKT cells are innate glycolipid-specific T cells restricted to the nonpolymorphic Ag-presenting molecule CD1d. As such, iNKT cells respond to glycolipids equally well in all people, making them an appealing adjuvant for universal vaccines. We tested the potential for the iNKT glycolipid agonist, α-galactosylceramide (αGC), to serve as an adjuvant for a known human protective epitope by creating a nanoparticle that delivers αGC plus antigenic polysaccharides from Streptococcus pneumoniae αGC-embedded nanoparticles activate murine iNKT cells and B cells in vitro and in vivo, facilitate significant dose sparing, and avoid iNKT anergy. Nanoparticles containing αGC plus S. pneumoniae polysaccharides elicits robust IgM and IgG in vivo and protect mice against lethal systemic S. pneumoniae However, codelivery of αGC via nanoparticles actually eliminated Ab protection elicited by a T-independent S. pneumoniae vaccine. This is consistent with previous studies demonstrating iNKT cell help for B cells following acute activation, but negative regulation of B cells during chronic inflammation. αGC-containing nanoparticles represent a viable platform for broadly efficacious vaccines against deadly human pathogens, but their potential for eliminating B cells under certain conditions suggests further clarity on iNKT cell interactions with B cells is warranted.

Healthy versus inflamed lung environments differentially affect mesenchymal stromal cells.

BACKGROUND: Despite increased interest in mesenchymal stromal cell (MSC)-based cell therapies for acute respiratory distress syndrome (ARDS), clinical investigations have not yet been successful and our understanding of the potential in vivo mechanisms of MSC actions in ARDS remains limited. ARDS is driven by an acute severe innate immune dysregulation, often characterised by inflammation, coagulation and cell injury. How this inflammatory microenvironment influences MSC functions remains to be determined. AIM: The aim of this study was to comparatively assess how the inflammatory environment present in ARDS lungs versus the lung environment present in healthy volunteers alters MSC behaviour. METHODS: Clinical-grade human bone marrow-derived MSCs (hMSCs) were exposed to bronchoalveolar lavage fluid (BALF) samples obtained from ARDS patients or from healthy volunteers. Following exposure, hMSCs and their conditioned media were evaluated for a broad panel of relevant properties, including viability, levels of expression of inflammatory cytokines, gene expression, cell surface human leukocyte antigen expression, and activation of coagulation and complement pathways. RESULTS: Pro-inflammatory, pro-coagulant and major histocompatibility complex (self-recognition) related gene expression was markedly upregulated in hMSCs exposed ex vivo to BALF obtained from healthy volunteers. These changes were less apparent and often opposite in hMSCs exposed to ARDS BALF samples. CONCLUSION: These data provide new insights into how hMSCs behave in healthy versus inflamed lung environments, and strongly suggest that the inflamed environment in ARDS induces hMSC responses that are potentially beneficial for cell survival and actions. This further highlights the need to understand how different disease environments affect hMSC functions.

Sweet talk: Metabolic conversations between host and microbe during infection.

In this issue, we introduce the second part of a series of reviews focusing on how immunometabolism influences host and pathogen interactions during infection. This part of the collection addresses the interface between metabolism and specific types of infection, including immunometabolism in macrophages during helminth infection, the role of metabolism in T-cell exhaustion during chronic viral infections and host immunometabolism in the defence against Mycobacterium tuberculosis infection. These reviews, together with the four articles published in part 1 of the series in November 2020, offer new insights into the complex interactions between mammalian hosts and microbial pathogens through the lens of cellular metabolic regulation.

Metabolic reprogramming of the myeloid lineage by Schistosoma mansoni infection persists independently of antigen exposure.

Macrophages have a defined role in the pathogenesis of metabolic disease and cholesterol metabolism where alternative activation of macrophages is thought to be beneficial to both glucose and cholesterol metabolism during high fat diet induced disease. It is well established that helminth infection protects from metabolic disease, but the mechanisms underlying protection are not well understood. Here, we investigated the effects of Schistosoma mansoni infection and cytokine activation in the metabolic signatures of bone marrow derived macrophages using an approach that integrated transcriptomics, metabolomics, and lipidomics in a metabolic disease prone mouse model. We demonstrate that bone marrow derived macrophages (BMDM) from S. mansoni infected male ApoE-/- mice have dramatically increased mitochondrial respiration compared to those from uninfected mice. This change is associated with increased glucose and palmitate shuttling into TCA cycle intermediates, increased accumulation of free fatty acids, and decreased accumulation of cellular cholesterol esters, tri and diglycerides, and is dependent on mgll activity. Systemic injection of IL-4 complexes is unable to recapitulate either reductions in systemic glucose AUC or the re-programing of BMDM mitochondrial respiration seen in infected males. Importantly, the metabolic reprogramming of male myeloid cells is transferrable via bone marrow transplantation to an uninfected host, indicating maintenance of reprogramming in the absence of sustained antigen exposure. Finally, schistosome induced metabolic and bone marrow modulation is sex-dependent, with infection protecting male, but not female mice from glucose intolerance and obesity. Our findings identify a transferable, long-lasting sex-dependent reprograming of the metabolic signature of macrophages by helminth infection, providing key mechanistic insight into the factors regulating the beneficial roles of helminth infection in metabolic disease.

Metabolic mediators: How immunometabolism directs the immune response to infection.

Here we announce the first part of an exciting new series of reviews exploring the impact of immunometabolism in the interaction between host and pathogen, and in the outcome of infection. This collection discusses the links between metabolism and epigenetic control of cell function, post-translation modifications of host proteins that determine protein fate and host cell function, the metabolic determinants of cell migration and immune cell activity, and the tussle for iron as a metabolic mediator of host-pathogen domination. Together these reviews provide engaging new insight into the metabolic signals that guide the dynamic conversation between microbial pathogens and the mammalian hosts they aim to occupy.

Differential effects of the cystic fibrosis lung inflammatory environment on mesenchymal stromal cells.

Growing evidence demonstrates that human mesenchymal stromal cells (MSCs) modify their in vivo anti-inflammatory actions depending on the specific inflammatory environment encountered. Understanding this better is crucial to refine MSC-based cell therapies for lung and other diseases. Using acute exacerbations of cystic fibrosis (CF) lung disease as a model, the effects of ex vivo MSC exposure to clinical bronchoalveolar lavage fluid (BALF) samples, as a surrogate for the in vivo clinical lung environment, on MSC viability, gene expression, secreted cytokines, and mitochondrial function were compared with effects of BALF collected from healthy volunteers. CF BALF samples that cultured positive for Aspergillus sp. (Asp) induced rapid MSC death, usually within several hours of exposure. Further analyses suggested the fungal toxin gliotoxin as a potential mediator contributing to CF BALF-induced MSC death. RNA sequencing analyses of MSCs exposed to either Asp+ or Asp- CF BALF samples identified a number of differentially expressed transcripts, including those involved in interferon signaling, antimicrobial gene expression, and cell death. Toxicity did not correlate with bacterial lung infections. These results suggest that the potential use of MSC-based cell therapies for CF or other lung diseases may not be warranted in the presence of Aspergillus.

Glycogen Metabolism Supports Early Glycolytic Reprogramming and Activation in Dendritic Cells in Response to Both TLR and Syk-Dependent CLR Agonists.

Dendritic cells (DCs) increase their metabolic dependence on glucose and glycolysis to support their maturation, activation-associated cytokine production, and T-cell stimulatory capacity. We have previously shown that this increase in glucose metabolism can be initiated by both Toll-like receptor (TLR) and C-type lectin receptor (CLR) agonists. In addition, we have shown that the TLR-dependent demand for glucose is partially satisfied by intracellular glycogen stores. However, the role of glycogen metabolism in supporting CLR-dependent DC glycolytic demand has not been formally demonstrated. In this work, we have shown that DCs activated with fungal-associated ß-glucan ligands exhibit acute glycolysis induction that is dependent on glycogen metabolism. Furthermore, glycogen metabolism supports DC maturation, inflammatory cytokine production, and priming of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in response to both TLR- and CLR-mediated activation. These data support a model in which different classes of innate immune receptors functionally converge in their requirement for glycogen-dependent glycolysis to metabolically support early DC activation. These studies provide new insight into how DC immune effector function is metabolically regulated in response to diverse inflammatory stimuli.

Syk-dependent glycolytic reprogramming in dendritic cells regulates IL-1ß production to ß-glucan ligands in a TLR-independent manner.

Dendritic cells (DCs) activated via TLR ligation experience metabolic reprogramming, in which the cells are heavily dependent on glucose and glycolysis for the synthesis of molecular building blocks essential for maturation, cytokine production, and the ability to stimulate T cells. Although the TLR-driven metabolic reprogramming events are well documented, fungal-mediated metabolic regulation via C-type lectin receptors such as Dectin-1 and Dectin-2 is not clearly understood. Here, we show that activation of DCs with fungal-associated ß-glucan ligands induces acute glycolytic reprogramming that supports the production of IL-1ß and its secretion subsequent to NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. This acute glycolytic induction in response to ß-glucan ligands requires spleen tyrosine kinase signaling in a TLR-independent manner, suggesting now that different classes of innate immune receptors functionally induce conserved metabolic responses to support immune cell activation. These studies provide new insight into the complexities of metabolic regulation of DCs immune effector function regarding cellular activation associated with protection against fungal microbes.

Serum Amyloid A3 is required for normal lung development and survival following influenza infection.

Serum amyloid A (SAA) proteins are a family of acute phase apolipoproteins implicated to directly modulate innate and adaptive immune responses. However, new studies comparing endogenous SAAs and recombinant forms of these proteins have questioned the function of SAA in inflammation and immunity. We generated SAA3 knockout mice to evaluate the contribution of SAA3 to lung development and immune-mediated lung disease. While SAA3 deficiency does not affect the generation of house dust mite-induced allergic asthma, mice lacking SAA3 develop adult-onset obesity, intrinsic airway hyperresponsiveness, increased inflammatory and fibrotic gene expression in the lung, and elevated levels of lung citrullinated proteins. Polyclonally stimulated CD4+ T cells from SAA3-/- mice exhibit impaired glycolytic activity, decreased TH2 and TH1 cytokine secretion, and elevated IL-17A production compared to wild type cells. Polyclonally stimulated CD8+ T cells from SAA3-/- mice also exhibit impaired glycolytic activity as well as a diminished capacity to produce IL-2 and IFNγ. Finally, SAA3-/- mice demonstrate increased mortality in response to H1N1 influenza infection, along with higher copy number of viral RNAs in the lung, a lack of CD8+ T cell IFNγ secretion, and decreased flu-specific antibodies. Our findings indicate that endogenous SAA3 regulates lung development and homeostasis, and is required for protection against H1N1 influenza infection.

Analysis of glycogen metabolic pathway utilization by dendritic cells and T cells using custom phenotype metabolic assays.

In the field of immunology, there is an increasing interest in cellular energy metabolism and its outcome on immune cell effector function. Activation of immune cells leads to rapid metabolic changes that are central to cellular biology in order to support the effector responses. Therefore, the need for user-friendly and dependable assay technologies to address metabolic regulation and nutrient utilization in immune cells is an important need in this field. Redox-dye reduction-based Phenotype MicroArray (PM) assays, which measure NADH reduction as a readout, developed by Biolog Inc., provide a wide screening of metabolites both in bacteria and mammalian cells. In this study, we delineate a detailed protocol of a customized Biolog assay for investigation of a specific metabolic pathway of interest. The option to be able to easily customize this technology offers researchers with a convenient assay platform to methodically examine specific nutrient substrates or metabolic pathways of interest in a rapid and cost effective manner.

Regulation of Dendritic Cell Immune Function and Metabolism by Cellular Nutrient Sensor Mammalian Target of Rapamycin (mTOR).

Dendritic cell (DC) activation is characterized by an acute increase in glucose metabolic flux that is required to fuel the high anabolic rates associated with DC activation. Inhibition of glycolysis significantly attenuates most aspects of DC immune effector function including antigen presentation, inflammatory cytokine production, and T cell stimulatory capacity. The cellular nutrient sensor mammalian/mechanistic Target of Rapamycin (mTOR) is an important upstream regulator of glycolytic metabolism and plays a central role in coordinating DC metabolic changes and immune responses. Because mTOR signaling can be activated by a variety of immunological stimuli, including signaling through the Toll-like Receptor (TLR) family of receptors, mTOR is involved in orchestrating many aspects of the DC metabolic response to microbial stimuli. It has become increasingly clear that mTOR's role in promoting or attenuating inflammatory processes in DCs is highly context-dependent and varies according to specific cellular subsets and the immunological conditions being studied. This review will address key aspects of the complex role of mTOR in regulating DC metabolism and effector function.

IL-1/inhibitory κB kinase ε-induced glycolysis augment epithelial effector function and promote allergic airways disease.

BACKGROUND: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma. OBJECTIVES: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma. METHODS: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis. RESULTS: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1ß into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1ß or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1ß- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1ß on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1ß and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1ß levels, and lactate content correlated negatively with lung function. CONCLUSIONS: Collectively, these findings demonstrate that IL-1ß/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease.