RESUMO
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-ß 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.
Assuntos
Ácidos e Sais Biliares , Receptores ErbB , Inflamação , Fígado , Transdução de Sinais , Animais , Masculino , Camundongos , Ácidos e Sais Biliares/metabolismo , Receptores ErbB/metabolismo , Células Estreladas do Fígado/metabolismo , Inflamação/metabolismo , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Proteômica , Células-Tronco/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Linfócitos B , Transdução de Sinais , Animais , Camundongos , Antígenos CD40 , Proliferação de Células , MitocôndriasRESUMO
To evaluate whether nicotinamide adenine dinucleotide-positive (NAD+) boosting modulates adaptive immunity, primary CD4+ T cells from healthy control and psoriasis subjects were exposed to vehicle or nicotinamide riboside (NR) supplementation. NR blunts interferon γ (IFNγ) and interleukin (IL)-17 secretion with greater effects on T helper (Th) 17 polarization. RNA sequencing (RNA-seq) analysis implicates NR blunting of sequestosome 1 (sqstm1/p62)-coupled oxidative stress. NR administration increases sqstm1 and reduces reactive oxygen species (ROS) levels. Furthermore, NR activates nuclear factor erythroid 2-related factor 2 (Nrf2), and genetic knockdown of nrf2 and the Nrf2-dependent gene, sqstm1, diminishes NR amelioratory effects. Metabolomics analysis identifies that NAD+ boosting increases arginine and fumarate biosynthesis, and genetic knockdown of argininosuccinate lyase ameliorates NR effects on IL-17 production. Hence NR via amino acid metabolites orchestrates Nrf2 activation, augments CD4+ T cell antioxidant defenses, and attenuates Th17 responsiveness. Oral NR supplementation in healthy volunteers similarly increases serum arginine, sqstm1, and antioxidant enzyme gene expression and blunts Th17 immune responsiveness, supporting evaluation of NAD+ boosting in CD4+ T cell-linked inflammation.
Assuntos
Antioxidantes , NAD , Humanos , NAD/metabolismo , Proteína Sequestossoma-1/metabolismo , Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Inflamação/tratamento farmacológicoRESUMO
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.
Assuntos
Smegmamorpha , Animais , Metabolismo Energético , Glicólise , Humanos , Células Matadoras Naturais , Fosforilação OxidativaRESUMO
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-1ß 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-1ß. 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.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Caspase 1 , Citosol , DNA Mitocondrial/genética , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , PiroptoseRESUMO
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.
Assuntos
Hipóxia Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Receptores Notch/metabolismo , Antígenos CD34/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Biologia Computacional/métodos , Humanos , Anotação de Sequência Molecular , Receptores Notch/genética , Transdução de Sinais , TranscriptomaRESUMO
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.
Assuntos
Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/metabolismo , Metabolismo Energético , Neoplasias/etiologia , Neoplasias/metabolismo , Imunidade Adaptativa , Animais , Comunicação Celular/imunologia , Citocinas/metabolismo , Gerenciamento Clínico , Humanos , Imunidade Inata , Imunomodulação , Imunoterapia , Terapia de Alvo Molecular , Neoplasias/patologia , Neoplasias/terapia , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologiaRESUMO
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.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Comportamento Alimentar/fisiologia , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Sepse/mortalidade , Fatores de Transcrição ARNTL/genética , Animais , Ritmo Circadiano/genética , Modelos Animais de Doenças , Resistência à Doença , Hepatócitos/metabolismo , Hipoglicemia/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/genética , Sepse/induzido quimicamente , Sepse/genética , Sepse/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Proliferação de Células , Interferon gama/biossíntese , Interleucina-15/metabolismo , Células Matadoras Naturais/fisiologia , Ativação Linfocitária/imunologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologiaRESUMO
Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the ß 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.
Assuntos
Proliferação de Células , Células Dendríticas/metabolismo , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular , Endocitose/fisiologia , Voluntários Saudáveis , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação/fisiologia , Cultura Primária de Células , Proteína S6 Ribossômica/metabolismoRESUMO
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.
Assuntos
Glucose/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Morte Celular/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Glucose/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos , Proteínas Mitocondriais , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Oxirredução , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismoRESUMO
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.
Assuntos
Deleção de Genes , Proteínas Mitocondriais/deficiência , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/deficiência , Especificidade de Órgãos , Recuperação de Função Fisiológica , Animais , Regulação para Baixo/genética , Feminino , Sequestradores de Radicais Livres/metabolismo , Humanos , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
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 4 d 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. 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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Animais , Células Cultivadas , Metabolismo Energético , Feminino , Glicólise , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de SinaisRESUMO
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.
Assuntos
Linfócitos B/fisiologia , Mitocôndrias/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Receptor Toll-Like 9/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Citocinas/metabolismo , Glicólise , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células NIH 3T3 , Fosforilação Oxidativa , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais , Receptor Toll-Like 9/genéticaRESUMO
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.
Assuntos
Asma/imunologia , Jejum , Imunomodulação , Ativação Linfocitária , Células Th2/imunologia , Adulto , Asma/patologia , Células Cultivadas , Citocinas/imunologia , Feminino , Humanos , Inflamassomos/imunologia , Masculino , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Projetos Piloto , Esteroides , Células Th2/patologiaRESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Plasmócitos/metabolismo , Pirofosfatases/metabolismo , Animais , Formação de Anticorpos/imunologia , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Centro Germinativo/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Baço/metabolismo , Regulação para Cima/fisiologiaRESUMO
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-1ß, 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.
Assuntos
Jejum , Inflamassomos/metabolismo , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismo , Acetilação/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Ativação Enzimática , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/agonistas , Multimerização Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/químicaRESUMO
Sterile inflammation is a cornerstone of immune activation in obesity and type 2 Diabetes Mellitus. The molecular underpinnings of this inflammation include nutrient excess-mediated activation of the innate immune NLRP3 inflammasome. At the same time, disruption of mitochondrial integrity is emerging as an integral control node in NLRP3 inflammasome activation and is also associated with caloric overload conditions including obesity and diabetes. Conversely, caloric restriction and fasting mimetic interventions alleviate these caloric excess-linked diseases and reduce inflammation and the NLRP3 inflammasome. The objective of this review is to integrate the findings linking mitochondrial integrity to the activation of the NLRP3 inflammasome and to evaluate how caloric restriction or caloric restriction mimetic compounds may play a role in attenuating the NLRP3 inflammasome and sterile inflammation.
Assuntos
Diabetes Mellitus Tipo 2/imunologia , Inflamassomos/imunologia , Inflamação/imunologia , Mitocôndrias/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Obesidade/imunologia , Animais , Diabetes Mellitus Tipo 2/patologia , Ingestão de Energia , Humanos , Inflamação/patologia , Mitocôndrias/patologia , Sirtuínas/imunologiaRESUMO
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.