Javier Traba

SCaMC-1 promotes cancer cell survival by desensitizing mitochondrial permeability transition via ATP/ADP-mediated matrix Ca(2+) buffering.

Ca2+-mediated mitochondrial permeability transition (mPT) is the final common pathway of stress-induced cell death in many major pathologies, but its regulation in intact cells is poorly understood. Here we report that the mitochondrial carrier SCaMC-1/SLC25A24 mediates ATP-Mg2−/Pi2− and/or HADP2−/Pi2− uptake into the mitochondria after an increase in cytosolic [Ca2+]. ATP and ADP contribute to Ca2+ buffering in the mitochondrial matrix, resulting in desensitization of the mPT. Comprehensive gene expression analysis showed that SCaMC-1 overexpression is a general feature of transformed and cancer cells. Knockdown of the transporter led to vast reduction of mitochondrial Ca2+ buffering capacity and sensitized cells to mPT-mediated necrotic death triggered by oxidative stress and Ca2+ overload. These findings revealed that SCaMC-1 exerts a negative feedback control between cellular Ca2+ overload and mPT-dependent cell death, suggesting that the carrier might represent a novel target for cancer therapy.

Fasting and refeeding differentially regulate NLRP3 inflammasome activation in human subjects

BACKGROUND Activation of the NLRP3 inflammasome is associated with metabolic dysfunction, and intermittent fasting has been shown to improve clinical presentation of NLRP3 inflammasome-linked diseases. As mitochondrial perturbations, which function as a damage-associated molecular pattern, exacerbate NLRP3 inflammasome activation, we investigated whether fasting blunts inflammasome activation via sirtuin-mediated augmentation of mitochondrial integrity. METHODS We performed a clinical study of 19 healthy volunteers. Each subject underwent a 24-hour fast and then was fed a fixed-calorie meal. Blood was drawn during the fasted and fed states and analyzed for NRLP3 inflammasome activation. We enrolled an additional group of 8 healthy volunteers to assess the effects of the sirtuin activator, nicotinamide riboside, on NLRP3 inflammasome activation. RESULTS In the fasting/refeeding study, individuals showed less NLRP3 inflammasome activation in the fasted state compared with that in refed conditions. In a human macrophage line, depletion of the mitochondrial-enriched sirtuin deacetylase SIRT3 increased NLRP3 inflammasome activation in association with excessive mitochondrial ROS production. Furthermore, genetic and pharmacologic SIRT3 activation blunted NLRP3 activity in parallel with enhanced mitochondrial function in cultured cells and in leukocytes extracted from healthy volunteers and from refed individuals but not in those collected during fasting. CONCLUSIONS Together, our data indicate that nutrient levels regulate the NLRP3 inflammasome, in part through SIRT3-mediated mitochondrial homeostatic control. Moreover, these results suggest that deacetylase-dependent inflammasome attenuation may be amenable to targeting in human disease. TRIAL REGISTRATION ClinicalTrials.gov NCT02122575 and NCT00442195. FUNDING Division of Intramural Research, NHLBI of the NIH.

Prolonged fasting suppresses mitochondrial NLRP3 inflammasome assembly and activation via SIRT3 mediated activation of Superoxide Dismutase 2

Twenty-four hours of fasting is known to blunt activation of the human NLRP3 inflammasome. This effect might be mediated by SIRT3 activation, controlling mitochondrial reactive oxygen species. To characterize the molecular underpinnings of this fasting effect, we comparatively analyzed the NLRP3 inflammasome response to nutrient deprivation in wild-type and SIRT3 knock-out mice. Consistent with previous findings for human NLRP3, prolonged fasting blunted the inflammasome in wild-type mice but not in SIRT3 knock-out mice. In SIRT3 knock-out bone marrow–derived macrophages, NLRP3 activation promoted excess cytosolic extrusion of mitochondrial DNA along with increased reactive oxygen species and reduced superoxide dismutase 2 (SOD2) activity. Interestingly, the negative regulatory effect of SIRT3 on NLRP3 was not due to transcriptional control or priming of canonical inflammasome components but, rather, occurred via SIRT3-mediated deacetylation of mitochondrial SOD2, leading to SOD2 activation. We also found that siRNA knockdown of SIRT3 or SOD2 increased NLRP3 supercomplex formation and activation. Moreover, overexpression of wild-type and constitutively active SOD2 similarly blunted inflammasome assembly and activation, effects that were abrogated by acetylation mimic–modified SOD2. Finally, in vivo administration of lipopolysaccharide increased liver injury and the levels of peritoneal macrophage cytokines, including IL-1β, in SIRT3 KO mice. These results support the emerging concept that enhancing mitochondrial resilience against damage-associated molecular patterns may play a pivotal role in preventing inflammation and that the anti-inflammatory effect of fasting-mimetic diets may be mediated, in part, through SIRT3-directed blunting of NLRP3 inflammasome assembly and activation.

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

Second signals rescue B cells from activation-induced mitochondrial dysfunction and death

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’.

ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1−/−) to determine how the enzyme affects PC biology. Although Enpp1−/− mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1−/− PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

A Pilot Study To Investigate the Immune-Modulatory Effects of Fasting in Steroid-Naive Mild Asthmatics

A fasting mimetic diet blunts inflammation, and intermittent fasting has shown ameliorative effects in obese asthmatics. To examine whether canonical inflammatory pathways linked with asthma are modulated by fasting, we designed a pilot study in mild asthmatic subjects to assess the effect of fasting on the NLRP3 inflammasome, Th2 cell activation, and airway epithelial cell cytokine production. Subjects with documented reversible airway obstruction and stable mild asthma were recruited into this study in which pulmonary function testing (PFT) and PBMCextraction was performed 24 h after fasting, with repeated PFT testing and blood draw 2.5 h after refeeding. PFTs were not changed by a prolonged fast. However, steroid-naive mild asthmatics showed fasting-dependent blunting of the NLRP3 inflammasome. Furthermore, PBMCs from these fasted asthmatics cocultured with human epithelial cells resulted in blunting of house dust mite–induced epithelial cell cytokine production and reduced CD4+ T cell Th2 activation compared with refed samples. This pilot study shows that prolonged fasting blunts the NLRP3 inflammasome and Th2 cell activation in steroid-naive asthmatics as well as diminishes airway epithelial cell cytokine production. This identifies a potential role for nutrient level–dependent regulation of inflammation in asthma. Our findings support the evaluation of this concept in a larger study as well as the potential development of caloric restriction interventions for the treatment of asthma.