In pancreatic ductal adenocarcinoma (PDAC), metabolic rewiring and resistance to standard therapy are closely associated. PDAC cells show enormous requirements for glucose‐derived citrate, the first rate‐limiting metabolite in the synthesis of new lipids. Both the expression and activity of citrate synthase (CS) are extraordinarily upregulated in PDAC. However, no previous relationship between gemcitabine response and citrate metabolism has been documented in pancreatic cancer. Here, we report for the first time that pharmacological doses of vitamin C are capable of exerting an inhibitory action on the activity of CS, reducing glucose‐derived citrate levels. Moreover, ascorbate targets citrate metabolism towards the de novo lipogenesis pathway, impairing fatty acid synthase (FASN) and ATP citrate lyase (ACLY) expression. Lowered citrate availability was found to be directly associated with diminished proliferation and, remarkably, enhanced gemcitabine response. Moreover, the deregulated citrate‐derived lipogenic pathway correlated with a remarkable decrease in extracellular pH through inhibition of lactate dehydrogenase (LDH) and overall reduced glycolytic metabolism. Modulation of citric acid metabolism in highly chemoresistant pancreatic adenocarcinoma, through molecules such as vitamin C, could be considered as a future clinical option to improve patient response to standard chemotherapy regimens.

Chronic cholestatic damage is associated to both accumulation of cytotoxic levels of bile acids and expansion of adult hepatic progenitor cells (HPC) as part of the ductular reaction contributing to the regenerative response. Here, we report a bile acid-specific cytotoxic response in mouse HPC, which is partially impaired by EGF signaling. Additionally, we show that EGF synergizes with bile acids to trigger inflammatory signaling and NLRP3 inflammasome activation in HPC. Aiming at understanding the impact of this HPC specific response on the liver microenvironment we run a proteomic analysis of HPC secretome. Data show an enrichment in immune and TGF-β regulators, ECM components and remodeling proteins in HPC secretome. Consistently, HPC-derived conditioned medium promotes hepatic stellate cell (HSC) activation and macrophage M1-like polarization. Strikingly, EGF and bile acids co-treatment leads to profound changes in the secretome composition, illustrated by an abolishment of HSC activating effect and by promoting macrophage M2-like polarization. Collectively, we provide new specific mechanisms behind HPC regulatory action during cholestatic liver injury, with an active role in cellular interactome and inflammatory response regulation. Moreover, findings prove a key contribution for EGFR signaling jointly with bile acids in HPC-mediated actions.

Activated B cells experience metabolic changes that require mitochondrial remodeling, in a process incompletely defined. In this study, we report that mitochondrial antiviral signaling protein (MAVS) is involved in BCR-initiated cellular proliferation and prolonged survival. MAVS is well known as a mitochondrial-tethered signaling adaptor with a central role in viral RNA-sensing pathways that induce type I IFN. The role of MAVS downstream of BCR stimulation was recognized in absence of IFN, indicative of a path for MAVS activation that is independent of viral infection. Mitochondria of BCR-activated MAVS-deficient mouse B cells exhibited a damaged phenotype including disrupted mitochondrial morphology, excess mitophagy, and the temporal progressive blunting of mitochondrial oxidative capacity with mitochondrial hyperpolarization and cell death. Costimulation of MAVS-deficient B cells with anti-CD40, in addition to BCR stimulation, partially corrected the mitochondrial structural defects and functionality. Our data reveal a (to our knowledge) previously unrecognized role of MAVS in controlling the metabolic fitness of B cells, most noticeable in the absence of costimulatory help.

Although NAD +levels modulate metabolism, how this regulates immunometabolism/inflammation remains unclear. Employing Nicotinamide Riboside (NR) to boost NAD, we explored T H17-linked inflammatory in Psoriasis. Primary Psoriasis CD4 +T cells exposed to NR reduced IL-17 secretion and RNA-seq/pathway analysis implicated NR in modulating sequestosome 1 (SQSTM1/p62)-coupled oxidative stress. In parallel, NR reduced CD4 +T cell reactive oxygen species (ROS) levels and activated the NRF2 transcription factor. Depletion of NRF2 or SQSTM1 diminished NR effects on ROS and T H17 activation. Metabolomic analysis implicated NR in arginine biosynthesis and L-arginine replicated NR CD4 +T cell effects. Genetic disruption of the argininosuccinate lyase synthesis enzyme ameliorated NR antiinflammatory effects, supporting that NR, via arginine biosynthesis orchestrates NRF2 activation, CD4 +T cell antioxidant defenses and blunted T H17 responsiveness. A pilot in-vivo human study similarly showed that NR increased arginine, antioxidant enzyme gene expression and blunted T H17 regulation, implicating NAD metabolism in CD4 +T cell-linked inflammation. NHLBI Division of Intramural Research (MNS – ZIA-HL005102), NIH Bench to Bedside award (MNS and RT, HL-129510-04S1) and the NIH Office of Dietary Supplementation (JT) and the UK MRC (JLG – MR/P011705/2; UKDRI-5002; MAP UK).

Natural killer (NK) cells are cytotoxic cells that mediate anti-tumor and anti-viral immunity. The response of NK cells to different cytokines and stimuli may involve cell survival, proliferation, and changes in their cytotoxic function. These responses will be supported by changes in cellular metabolism. Therefore, changes in NK metabolic parameters could somehow predict changes in NK cell function and cytotoxicity. In this chapter, we describe a protocol to measure NK cell metabolism in primary human NK cells by using an extracellular flux analyzer. This machine measures pH and oxygen changes in the medium and allows the study of NK cell glycolysis and mitochondrial respiration in real time with a small number of cells.

The NLRP3 inflammasome, a key component of the innate immune system that mediates caspase-1 activation, which in turn induces cleavage of the pyroptosis executioner gasdermin D and the proinflammatory cytokines IL-1β and IL-18, requires two signals to be activated. First, inflammasome priming is achieved after activation of Toll-like receptors, which leads to NF-κB signaling and transcriptional activation of the genes for NLRP3 and IL-1β. Next, the inflammasome complex is activated by a second signal that induces extrusion of mitochondrial DNA to the cytosol of the cell, which leads to its oligomerization by a not fully understood mechanism. Here we describe a simple method that employs quantitative polymerase chain reaction (qPCR) using SYBR green to measure the presence of mitochondrial DNA (mtDNA) in the cytosol, which can be used to measure cytosolic mtDNA levels after inflammasome activation.

The mitochondria play an important role in the activation of the innate immune system. This organelle modulates the metabolic reprogramming of the immune cell into proinflammatory or antiinflammatory subtypes, which typically utilize very different metabolic pathways to fulfill their functions. It also acts as a signaling platform to activate immune routes in both immune and nonimmune cells, as it can generate agonists for inflammatory pathways, including toll-like receptors, inflammasomes, or the cyclic GMP-AMP synthase-stimulator of interferon genes pathway, which lead to the generation of proinflammatory cytokines and antiviral molecules such as type-I interferons. These novel functions of the mitochondria are important in the fight against pathogens, but also contribute to human disease when dysregulated. This review describes recent findings in this field and highlights the role of mitochondrial nucleic acids in the regulation of innate immune signaling pathways.

Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

In mice, time of day strongly influences lethality in response to LPS, with survival greatest at the beginning compared to the end of the light cycle. Here we show that feeding, rather than light, controls time-of-day dependent LPS sensitivity. Mortality following LPS administration is independent of cytokine production and the clock regulator BMAL1 expressed in myeloid cells. In contrast, deletion of BMAL1 in hepatocytes globally disrupts the transcriptional response to the feeding cycle in the liver and results in constitutively high LPS sensitivity. Using RNAseq and functional validation studies we identify hepatic farnesoid X receptor (FXR) signalling as a BMAL1 and feeding-dependent regulator of LPS susceptibility. These results show that hepatocyte-intrinsic BMAL1 and FXR signalling integrate nutritional cues to regulate survival in response to innate immune stimuli. Understanding hepatic molecular programmes operational in response to these cues could identify novel pathways for targeting to enhance endotoxemia resistance.

Constitutive activity of the immune surveillance system detects and kills cancerous cells, although many cancers have developed strategies to avoid detection and to resist their destruction. Cancer immunotherapy entails the manipulation of components of the endogenous immune system as targeted approaches to control and destroy cancer cells. Since one of the major limitations for the antitumor activity of immune cells is the immunosuppressive tumor microenvironment (TME), boosting the immune system to overcome the inhibition provided by the TME is a critical component of oncotherapeutics. In this article, we discuss the main effects of the TME on the metabolism and function of immune cells, and review emerging strategies to potentiate immune cell metabolism to promote antitumor effects either as monotherapeutics or in combination with conventional chemotherapy to optimize cancer management.

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFNγ) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the β and common γ (γc) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα-IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα-IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα-IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα-IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor-ligand interaction occurs can influence functional outcome.

Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for four days following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Despite elevated ROS production, it was successfully counterbalanced by antioxidant enzymes, hence the mitochondria did not display signs of oxidative stress as evidenced by the healthy cristae structure and overall morphology as well as the maintained mitochondrial reserve. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.

GCN5L1 regulates protein acetylation and mitochondrial energy metabolism in diverse cell types. In the heart, loss of GCN5L1 sensitizes the myocardium to injury from exposure to nutritional excess and ischemia/reperfusion injury. This phenotype is associated with the reduced acetylation of metabolic enzymes and elevated mitochondrial reactive oxygen species (ROS) generation, although the direct molecular targets of GCN5L1 remain largely unknown. In this study, we sought to determine the mechanism by which GCN5L1 impacts energy substrate utilization and mitochondrial health. We find that hypoxia and reoxygenation (H/R) leads to a reduction in cell viability and Akt phosphorylation in GCN5L1 knockdown AC16 cardiomyocytes, in parallel with elevated glucose utilization and impaired fatty acid use. We demonstrate that glycolysis is uncoupled from glucose oxidation under normoxic conditions in GCN5L1-depleted cells. We show that GCN5L1 directly binds to the Akt-activating mTORC2 component Rictor, and that loss of Rictor acetylation is evident in GCN5L1 knockdown cells. Finally, we show that restoring Rictor acetylation in GCN5L1-depleted cells reduces mitochondrial ROS generation and increases cell survival in response to H/R. These studies suggest that GCN5L1 may play a central role in energy substrate metabolism and cell survival via the regulation of Akt/mTORC2 signaling.

GCN5L1 regulates mitochondrial protein acetylation, cellular bioenergetics, reactive oxygen species (ROS) generation, and organelle positioning in a number of diverse cell types. However, the functional role of GCN5L1 in the heart is currently unknown. As many of the factors regulated by GCN5L1 play a major role in ischemia-reperfusion (I/R) injury, we sought to determine if GCN5L1 is an important nexus in the response to cardiac ischemic stress. Deletion of GCN5L1 in cardiomyocytes resulted in impaired myocardial post-ischemic function and increased infarct development in isolated work-performing hearts. GCN5L1 knockout hearts displayed hallmarks of ROS damage, and scavenging of ROS restored cardiac function and reduced infarct volume in vivo. GCN5L1 knockdown in cardiac-derived AC16 cells was associated with reduced activation of the pro-survival MAP kinase ERK1/2, which was also reversed by ROS scavenging, leading to restored cell viability. We therefore conclude that GCN5L1 activity provides an important protection against I/R induced, ROS-mediated damage in the ischemic heart.

The signaling cascades initiated by binding of the antigen to B cell receptor (BCR) leads to rapid increases in cellular respiration, nutrient uptake and expression of various activation markers. However, unless a second, independent stimulus is received soon after antigen binding, BCR signaling does not lead to B cell proliferation and differentiation. Instead, BCR signaling alone leads to a dysregulation of cellular calcium homeostasis which in turn causes mitochondrial dysfunction and a phenomenon called activation induced cell death. The second signal can be provided by Toll like receptor (TLR) signaling, indicating the presence of an acute infection or through B cell-T cell interactions that rules out that the initial antigen was a prohibited self-antigen. Additionally, there is an interplay between TLR versus T cell mediated second signals, namely that TLR signaling trumped the need for the antigen-stimulated B cell to interact with T cells to receive a ‘go’ signal. TLR stimulation decreased B cells’ ability to capture, process and present antigens to solicit help from T cells. Rather TLRs directly boosted B cell antibody and cytokine secretion and proliferation. Thus, TLR signals drive B cells toward rapid T cell-independent differentiation to plasma cells providing rapid neutralizing antibody production to fight off infections quickly. In contrast B cells that receive their survival signals from T cells are induced to undergo a time-consuming process that is necessary for antibody affinity maturation and long-lasting immune memory. Therefore, the TLR driven shortcut to quick antibody comes with a price which is the absence of “crème de la crème” antibodies that would protect the body from subsequent infections for years to come.

Significance Interleukin 15 (IL-15) is essential for natural killer (NK) cell survival and proliferation. The IL-15 receptor consists of 3 subunits: the α chain and the β and common γ (γ c ) chains, which are shared with the IL-2 receptor. NK cells express the β-γ c chains and are activated in trans by cells presenting IL-15 bound to IL-15Rα, such as dendritic cells. We show here that the release of soluble IL-15Rα–IL-15 complex from presenting cells contributes to efficient signaling for NK cell survival, whereas uptake of the entire membrane-associated-IL-15Rα–IL-15 complex from presenting cells into NK cells contributes to proliferation. Thus, outcomes are dictated not only by signal strength, but also by location where IL-15 trans -presentation occurs in NK cells.

Activation of B cells to proliferate and differentiate into antibody secreting cells is regulated to avoid unwanted antibody responses by two temporally distinct signals, the first provided by antigen binding to the B cell receptor (BCR) and the second by T helper cells. Despite the central role of these two signals in driving antibody responses, our understanding of the consequences of each signal is incomplete. Here using in vitro and in vivo models, we investigated the metabolic changes that accompany B cell activation and show that antigen binding to the BCR signals for rapid increases in both oxidative phosphorylation and glycolysis, however early events following antigen engagement including spreading and contraction of antigen bound BCR and internalization of the bound antigen depend solely on the energy produced by oxidative phosphorylation. Changes in gene expression, mitochondrial performance and glucose uptake follow the initial activation, preparing the cells to respond to a second signal. However, in the absence of a second signal B cells undergo progressive loss of mitochondria function and glycolytic capacity ultimately leading to apoptosis. Mitochondria dysfunction is a result of the gradual accumulation of intracellular calcium which leads to inefficient oxidative phosphorylation and increased ROS production and eventually swollen patches of mitochondria. This process can be avoided for approximately nine hours after B cell antigen binding by either T helper cells or by signaling through Toll-like receptor 9. Thus, antigen binding to B cells appears to activate a metabolic program that imposes a short time window in which B cells either receive a second signal and survive or alternatively face elimination.

B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response–activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated. B cells need at least two signals to terminally differentiate into antibody-secreting cells. Pierce and colleagues show that persistent exposure to antigen in the absence of T cell help or ‘pathogen pattern motifs’ leads to B cell death via a calcium-dependent ‘metabolic timer’.