TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation.

The ligation of Toll-like receptors (TLRs) leads to rapid activation of dendritic cells (DCs). However, the metabolic requirements that support this process remain poorly defined. We found that DC glycolytic flux increased within minutes of exposure to TLR agonists and that this served an essential role in supporting the de novo synthesis of fatty acids for the expansion of the endoplasmic reticulum and Golgi required for the production and secretion of proteins that are integral to DC activation. Signaling via the kinases TBK1, IKKɛ and Akt was essential for the TLR-induced increase in glycolysis by promoting the association of the glycolytic enzyme HK-II with mitochondria. In summary, we identified the rapid induction of glycolysis as an integral component of TLR signaling that is essential for the anabolic demands of the activation and function of DCs.

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.

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.

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.

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.

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.

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.

Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development.

CD8(+) T cells undergo major metabolic changes upon activation, but how metabolism influences the establishment of long-lived memory T cells after infection remains a key question. We have shown here that CD8(+) memory T cells, but not CD8(+) T effector (Teff) cells, possessed substantial mitochondrial spare respiratory capacity (SRC). SRC is the extra capacity available in cells to produce energy in response to increased stress or work and as such is associated with cellular survival. We found that interleukin-15 (IL-15), a cytokine critical for CD8(+) memory T cells, regulated SRC and oxidative metabolism by promoting mitochondrial biogenesis and expression of carnitine palmitoyl transferase (CPT1a), a metabolic enzyme that controls the rate-limiting step to mitochondrial fatty acid oxidation (FAO). These results show how cytokines control the bioenergetic stability of memory T cells after infection by regulating mitochondrial metabolism.

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.

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.

Cognate interaction with iNKT cells expands IL-10-producing B regulatory cells.

Successful induction of B-cell activation and memory depends on help from CD4+ T cells. Invariant natural killer T (iNKT) cells (glycolipid-specific, CD1d-restricted innate lymphocytes) provide both cognate (direct) and noncognate (indirect) helper signals to enhance B-cell responses. Both forms of iNKT-cell help induce primary humoral immune responses, but only noncognate iNKT-cell help drives humoral memory and plasma cells. Here, we show that iNKT cognate help for B cells is fundamentally different from the help provided by conventional CD4+ T cells. Cognate iNKT-cell help drives an early, unsustained germinal center B-cell expansion, less reduction of T follicular regulatory cells, an expansion of marginal zone B cells, and early increases in regulatory IL-10-producing B-cell numbers compared with noncognate activation. These results are consistent with a mechanism whereby iNKT cells preferentially provide an innate form of help that does not generate humoral memory and has important implications for the application of glycolipid molecules as vaccine adjuvants.

IL-4-secreting secondary T follicular helper (Tfh) cells arise from memory T cells, not persisting Tfh cells, through a B cell-dependent mechanism.

Humoral immunity requires cross-talk between T follicular helper (Tfh) cells and B cells. Nevertheless, a detailed understanding of this intercellular interaction during secondary immune responses is lacking. We examined this by focusing on the response to a soluble, unadjuvanted, pathogen-derived Ag (soluble extract of Schistosoma mansoni egg [SEA]) that induces type 2 immunity. We found that activated Tfh cells persisted for long periods within germinal centers following primary immunization. However, the magnitude of the secondary response did not appear to depend on pre-existing Tfh cells. Instead, Tfh cell populations expanded through a process that was dependent on memory T cells recruited into the reactive LN, as well as the participation of B cells. We found that, during the secondary response, IL-4 was critical for the expansion of a population of plasmablasts that correlated with increased SEA-specific IgG1 titers. Additionally, following immunization with SEA (but not with an Ag that induced type 1 immunity), IL-4 and IL-21 were coproduced by individual Tfh cells, revealing a potential mechanism through which appropriate class-switching can be coupled to plasmablast proliferation to enforce type 2 immunity. Our findings demonstrate a pivotal role for IL-4 in the interplay between T and B cells during a secondary Th2 response and have significant implications for vaccine design.

Mechanistic target of rapamycin inhibition extends cellular lifespan in dendritic cells by preserving mitochondrial function.

TLR-mediated activation of dendritic cells (DCs) is associated with a metabolic transition in which mitochondrial oxidative phosphorylation is inhibited by endogenously synthesized NO and the cells become committed to glucose and aerobic glycolysis for survival. We show that inhibition of mechanistic target of rapamycin (mTOR) extends the lifespan of TLR-activated DCs by inhibiting the induction of NO production, thereby allowing the cells to continue to use their mitochondria to generate ATP, and allowing them the flexibility to use fatty acids or glucose as nutrients to fuel core metabolism. These data provide novel mechanistic insights into how mTOR modulates DC metabolism and cellular longevity following TLR activation and provide an explanation for previous findings that mTOR inhibition enhances the efficacy of DCs in autologous vaccination.

The mechanism of splenic invariant NKT cell activation dictates localization in vivo.

Invariant NKT (iNKT) cells are glycolipid-specific innate lymphocytes emerging as critical players in the immune response to diverse infections and disease. iNKT cells are activated through cognate interactions with lipid-loaded APCs, by Ag-independent cytokine-mediated signaling pathways, or a combination of both. Although each of these modes of iNKT cell activation plays an important role in directing the humoral and cell-mediated immune response, the spatio-temporal nature of these interactions and the cellular requirements for activation are largely undefined. Combining novel in situ confocal imaging of αGalactosylceramide-loaded CD1d tetramer labeling to localize the endogenous iNKT cell population with cytokine reporter mice, we reveal the choreography of early murine splenic iNKT cell activation across diverse settings of glycolipid immunization and systemic infection with Streptococcus pneumoniae. We find that iNKT cells consolidate in the marginal zone and require dendritic cells lining the splenic marginal zone for activation following administration of cognate glycolipids and during systemic infection but not following exogenous cytokine administration. Although further establishing the importance of cognate iNKT cell interactions with APCs, we also show that noncognate iNKT-dependent mechanisms are sufficient to mediate effector outcomes, such as STAT signaling and dendritic cell licensing throughout the splenic parenchyma. Collectively, these data provide new insight into how iNKT cells may serve as a natural adjuvant in facilitating adaptive immune responses, irrespective of their tissue localization.

IL-10R blockade during chronic schistosomiasis mansoni results in the loss of B cells from the liver and the development of severe pulmonary disease.

In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4+ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.

Fatty acid oxidation is essential for egg production by the parasitic flatworm Schistosoma mansoni.

Schistosomes, parasitic flatworms that cause the neglected tropical disease schistosomiasis, have been considered to have an entirely carbohydrate based metabolism, with glycolysis playing a dominant role in the adult parasites. However, we have discovered a close link between mitochondrial oxygen consumption by female schistosomes and their ability to produce eggs. We show that oxygen consumption rates (OCR) and egg production are significantly diminished by pharmacologic inhibition of carnitine palmitoyl transferase 1 (CPT1), which catalyzes a rate limiting step in fatty acid ß-oxidation (FAO) and by genetic loss of function of acyl CoA synthetase, which complexes with CPT1 and activates long chain FA for use in FAO, and of acyl CoA dehydrogenase, which catalyzes the first step in FAO within mitochondria. Declines in OCR and egg production correlate with changes in a network of lipid droplets within cells in a specialized reproductive organ, the vitellarium. Our data point to the importance of regulated lipid stores and FAO for the compartmentalized process of egg production in schistosomes.

Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells.

TLR agonists initiate a rapid activation program in dendritic cells (DCs) that requires support from metabolic and bioenergetic resources. We found previously that TLR signaling promotes aerobic glycolysis and a decline in oxidative phosphorylation (OXHPOS) and that glucose restriction prevents activation and leads to premature cell death. However, it remained unclear why the decrease in OXPHOS occurs under these circumstances. Using real-time metabolic flux analysis, in the present study, we show that mitochondrial activity is lost progressively after activation by TLR agonists in inflammatory blood monocyte-derived DCs that express inducible NO synthase. We found that this is because of inhibition of OXPHOS by NO and that the switch to glycolysis is a survival response that serves to maintain ATP levels when OXPHOS is inhibited. Our data identify NO as a profound metabolic regulator in inflammatory monocyte-derived DCs.

Inhibition of mechanistic target of rapamycin promotes dendritic cell activation and enhances therapeutic autologous vaccination in mice.

Dendritic cells (DCs) are potent inducers of T cell immunity, and autologous DC vaccination holds promise for the treatment of cancers and chronic infectious diseases. In practice, however, therapeutic vaccines of this type have had mixed success. In this article, we show that brief exposure to inhibitors of mechanistic target of rapamycin (mTOR) in DCs during the period that they are responding to TLR agonists makes them particularly potent activators of naive CD8+ T cells and able to enhance control of B16 melanoma in a therapeutic autologous vaccination model in the mouse. The improved performance of DCs in which mTOR has been inhibited is correlated with an extended life span after activation and prolonged, increased expression of costimulatory molecules. Therapeutic autologous vaccination with DCs treated with TLR agonists plus the mTOR inhibitor rapamycin results in improved generation of Ag-specific CD8+ T cells in vivo and improved antitumor immunity compared with that observed with DCs treated with TLR agonists alone. These findings define mTOR as a molecular target for augmenting DC survival and activation, and document a novel pharmacologic approach for enhancing the efficacy of therapeutic autologous DC vaccination.

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.