RESUMO
BACKGROUND: L-Tryptophan (L-Trp), an essential amino acid, is the only amino acid whose level is regulated specifically by immune signals. Most proportions of Trp are catabolized via the kynurenine (Kyn) pathway (KP) which has evolved to align the food availability and environmental stimulation with the host pathophysiology and behavior. Especially, the KP plays an indispensable role in balancing the immune activation and tolerance in response to pathogens. SCOPE OF REVIEW: In this review, we elucidate the underlying immunological regulatory network of Trp and its KP-dependent catabolites in the pathophysiological conditions by participating in multiple signaling pathways. Furthermore, the KP-based regulatory roles, biomarkers, and therapeutic strategies in pathologically immune disorders are summarized covering from acute to chronic infection and inflammation. MAJOR CONCLUSIONS: The immunosuppressive effects dominate the functions of KP induced-Trp depletion and KP-produced metabolites during infection and inflammation. However, the extending minor branches from the KP are not confined to the immune tolerance, instead they go forward to various functions according to the specific condition. Nevertheless, persistent efforts should be made before the clinical use of KP-based strategies to monitor and cure infectious and inflammatory diseases.
Assuntos
Biomarcadores , Inflamação , Cinurenina , Triptofano , Triptofano/metabolismo , Cinurenina/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/imunologia , Animais , Biomarcadores/metabolismo , Infecções/imunologia , Infecções/metabolismoRESUMO
We reported that NAD(+)-dependent SIRT1, RELB, and SIRT6 nuclear proteins in monocytes regulate a switch from the glycolysis-dependent acute inflammatory response to fatty acid oxidation-dependent sepsis adaptation. We also found that disrupting SIRT1 activity during adaptation restores immunometabolic homeostasis and rescues septic mice from death. Here, we show that nuclear SIRT1 guides RELB to differentially induce SIRT3 expression and also increases mitochondrial biogenesis, which alters bioenergetics during sepsis adaptation. We constructed this concept using TLR4-stimulated THP1 human promonocytes, a model that mimics the initiation and adaptation stages of sepsis. Following increased expression, mitochondrial SIRT3 deacetylase activates the rate-limiting tricarboxylic acid cycle (TCA) isocitrate dehydrogenase 2 and superoxide dismutase 2, concomitant with increases in citrate synthase activity. Mitochondrial oxygen consumption rate increases early and decreases during adaptation, parallel with modifications to membrane depolarization, ATP generation, and production of mitochondrial superoxide and whole cell hydrogen peroxide. Evidence of SIRT1-RELB induction of mitochondrial biogenesis included increases in mitochondrial mass, mitochondrial-to-nuclear DNA ratios, and both nuclear and mitochondrial encoded proteins. We confirmed the SIRT-RELB-SIRT3 adaptation link to mitochondrial bioenergetics in both TLR4-stimulated normal and sepsis-adapted human blood monocytes and mouse splenocytes. We also found that SIRT1 inhibition ex vivo reversed the sepsis-induced changes in bioenergetics.
Assuntos
Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Monócitos/metabolismo , Sepse/metabolismo , Sirtuína 1/metabolismo , Sirtuína 3/metabolismo , Fator de Transcrição RelB/metabolismo , Animais , Linhagem Celular , Núcleo Celular/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Cultura Primária de Células , Sepse/genética , Sepse/imunologia , Sepse/patologia , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 3/genética , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Baço/patologia , Fator de Transcrição RelB/genéticaRESUMO
BACKGROUND: Transcriptomic studies hold great potential towards understanding the human aging process. Previous transcriptomic studies have identified many genes with age-associated expression levels; however, small samples sizes and mixed cell types often make these results difficult to interpret. RESULTS: Using transcriptomic profiles in CD14+ monocytes from 1,264 participants of the Multi-Ethnic Study of Atherosclerosis (aged 55-94 years), we identified 2,704 genes differentially expressed with chronological age (false discovery rate, FDR ≤ 0.001). We further identified six networks of co-expressed genes that included prominent genes from three pathways: protein synthesis (particularly mitochondrial ribosomal genes), oxidative phosphorylation, and autophagy, with expression patterns suggesting these pathways decline with age. Expression of several chromatin remodeler and transcriptional modifier genes strongly correlated with expression of oxidative phosphorylation and ribosomal protein synthesis genes. 17% of genes with age-associated expression harbored CpG sites whose degree of methylation significantly mediated the relationship between age and gene expression (p < 0.05). Lastly, 15 genes with age-associated expression were also associated (FDR ≤ 0.01) with pulse pressure independent of chronological age. Comparing transcriptomic profiles of CD14+ monocytes to CD4+ T cells from a subset (n = 423) of the population, we identified 30 age-associated (FDR < 0.01) genes in common, while larger sets of differentially expressed genes were unique to either T cells (188 genes) or monocytes (383 genes). At the pathway level, a decline in ribosomal protein synthesis machinery gene expression with age was detectable in both cell types. CONCLUSIONS: An overall decline in expression of ribosomal protein synthesis genes with age was detected in CD14+ monocytes and CD4+ T cells, demonstrating that some patterns of aging are likely shared between different cell types. Our findings also support cell-specific effects of age on gene expression, illustrating the importance of using purified cell samples for future transcriptomic studies. Longitudinal work is required to establish the relationship between identified age-associated genes/pathways and aging-related diseases.
Assuntos
Envelhecimento/genética , Monócitos/metabolismo , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Autofagia/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Feminino , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Ribossomos/genética , Ribossomos/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Transcription factor (TF) and microRNA (miRNA) have been discovered playing crucial roles in cancer development. However, the effect of TFs and miRNAs in pancreatic cancer pathogenesis remains vague. We attempted to reveal the possible mechanism of pancreatic cancer based on transcription level. Using GSE16515 datasets downloaded from gene expression omnibus database, we first identified the differentially expressed genes (DEGs) in pancreatic cancer by the limma package in R. Then the DEGs were mapped into DAVID to conduct the kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. TFs and miRNAs that DEGs significantly enriched were identified by Fisher's test, and then the pancreatic cancer double-factor regulatory network was constructed. In our study, total 1117 DEGs were identified and they significantly enriched in 4 KEGG pathways. A double-factor regulatory network was established, including 29 DEGs, 24 TFs, 25 miRNAs. In the network, LAMC2, BRIP1 and miR155 were identified which may be involved in pancreatic cancer development. In conclusion, the double-factor regulatory network was found to play an important role in pancreatic cancer progression and our results shed new light on the molecular mechanism of pancreatic cancer.
Assuntos
Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias Pancreáticas/genética , Transcrição Gênica , Estudos de Casos e Controles , Análise por Conglomerados , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pancreáticas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and ß to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.
Assuntos
Metabolismo Energético , Ácidos Graxos/metabolismo , Glucose/metabolismo , Sepse/metabolismo , Sirtuína 1/metabolismo , Sirtuínas/metabolismo , Adaptação Fisiológica/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Citocinas/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glicólise , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Células Precursoras de Monócitos e Macrófagos/imunologia , Células Precursoras de Monócitos e Macrófagos/metabolismo , Células Precursoras de Monócitos e Macrófagos/fisiologia , NAD/biossíntese , Nicotinamida Fosforribosiltransferase/metabolismo , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA , Sepse/imunologia , Receptor 4 Toll-Like/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Gene-selective epigenetic reprogramming and shifts in cellular bioenergetics develop when Toll-like receptors (TLR) recognize and respond to systemic life-threatening infections. Using a human monocyte cell model of endotoxin tolerance and human leukocytes from acute systemic inflammation with sepsis, we report that energy sensor sirtuin 1 (SIRT1) coordinates the epigenetic and bioenergy shifts. After TLR4 signaling, SIRT1 rapidly accumulated at the promoters of TNF-α and IL-1ß, but not IκBα; SIRT1 promoter binding was dependent on its co-factor, NAD(+). During this initial process, SIRT1 deacetylated RelA/p65 lysine 310 and nucleosomal histone H4 lysine 16 to promote termination of NFκB-dependent transcription. SIRT1 then remained promoter bound and recruited de novo induced RelB, which directed assembly of the mature transcription repressor complex that generates endotoxin tolerance. SIRT1 also promoted de novo expression of RelB. During sustained endotoxin tolerance, nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for endogenous production of NAD(+), and SIRT1 expression increased. The elevation of SIRT1 required protein stabilization and enhanced translation. To support the coordination of bioenergetics in human sepsis, we observed elevated NAD(+) levels concomitant with SIRT1 and RelB accumulation at the TNF-α promoter of endotoxin tolerant sepsis blood leukocytes. We conclude that TLR4 stimulation and human sepsis activate pathways that couple NAD(+) and its sensor SIRT1 with epigenetic reprogramming.
Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Endotoxinas/farmacologia , Epigênese Genética , Regiões Promotoras Genéticas , Sepse/metabolismo , Sirtuína 1/metabolismo , Linhagem Celular , Resistência a Medicamentos/genética , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , NAD/genética , NAD/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Estabilidade Proteica/efeitos dos fármacos , Sepse/genética , Sirtuína 1/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Cryptococcus neoformans infection in the central nervous system is a severe infectious disease with poor outcomes and high mortality. It has been estimated that there are 220,000 new cases each year. Over 90% of C. neoformans meningitis cases were diagnosed in AIDS patients with CD4+ T cell count <100 cells/µl; however, the mechanism of cryptococcal meningitis in patients with normal immune functions remains unclear. IL-17 is a pro-inflammatory cytokine and plays an important role in anti-fungal immunity. Here we report that significantly high levels of IL-17 were predominantly detected in the cerebrospinal fluid of patients with either AIDS- or non-AIDS-associated C. neoformans meningitis but not in patients with tuberculous meningitis or non-neurosyphilis. Antifungal therapy minimized the IL-17 level in the cerebrospinal fluid. An in vitro mechanistic study showed that C. neoformans stimulation of healthy peripheral blood mononuclear cells prompted IL-17 production, and CD4+ T cells were the predominant IL-17-producing cells. IL-17 production by C. neoformans stimulation was STAT3 signaling dependent. Inhibition of STAT3 phosphorylation attenuated the C. neoformans-mediated IL-17 expression. Our data highlighted the significance of CD4+ T cells in antifungal immunity and suggested IL-17 as a diagnostic biomarker of C. neoformans infection and STAT3 as a checkpoint for antifungal targeted therapies.
Assuntos
Síndrome da Imunodeficiência Adquirida , Criptococose , Cryptococcus neoformans , Meningite Criptocócica , Antifúngicos/farmacologia , Linfócitos T CD4-Positivos , Humanos , Interleucina-17 , Leucócitos Mononucleares , Fosforilação , Fator de Transcrição STAT3 , Linfócitos TRESUMO
Tryptophan (Trp) as an essential amino acid plays critical roles in regulating multiple cell activities, and the changes of its circulating level usually indicate disease status such as the severity of acute inflammation sepsis. However, the current technology for Trp detection mostly relies on chromatography that cannot meet the rapid and simple detection requirement in monitoring sepsis. Herein, a label-free fluorescent nanosensor was constructed to detect Trp and its carrier protein - human serum albumin (HSA) in plasma. The nanosensor consists of a cytosine (C) - rich signal probe of DNA-templated silver nanoclusters (AgNCs/DNA) and a Trp (Trp) aptamer - based capture probe with a guanine (G) - rich overhang. Trp was found to trigger G-rich sequence-mediated fluorescence enhancement effect for AgNCs/DNA under UV irradiation which offers energy to induce the redox process between limited Trp and silver ions bound on the DNA probes. Based on this photochemical property, the nanosensor exhibited a linear response in the range of 0.05-60 µM with the limit of detection of 0.43 µM for Trp, superior to the current fluorescence-based detection method, and it gave specific response to indole group. When applied in plasma detection, the nanosensor resisted physiological level of NaCl in plasma, but was quenched by trace volume of HSA, which facilitates the combined HSA detection using only 1 µL plasma sample. The simple procedure of "mix, exposure and detection" together with its ultralow sampling volume, time-saving, cost-effective, sensitive and selective properties endow the nanosensor great potentials for future Trp detection-based clinical use.
Assuntos
Nanopartículas Metálicas , Sepse , Albuminas , DNA/química , Corantes Fluorescentes/química , Humanos , Nanopartículas Metálicas/química , Sepse/diagnóstico , Prata/química , TriptofanoRESUMO
By virtue of the presence of multiple protein-protein interaction and signaling domains, PDZ proteins play important roles in assembling protein complexes that participate in diverse cell biological processes. GIPC is a versatile PDZ protein that binds a variety of target proteins in different cell types. In previous studies we showed that, in epidermal melanocytes, GIPC interacts with newly synthesized melanosomal protein TRP1 in the Golgi region and proposed that this interaction may facilitate intracellular trafficking of TRP1. However, since GIPC contains a single PDZ domain and no other known protein interaction motifs, it is not known how GIPC-TRP1 interaction affects melanosome biogenesis and/or melanin pigmentation. Here, we show that in human primary melanocytes GIPC interacts with AKT-binding protein APPL (adaptor protein containing pleckstrin homology, leucine zipper and phosphotyrosine binding domains), which readily co-precipitates with newly synthesized TRP1. Knockdown of either GIPC or APPL inhibits melanogenesis by decreasing tyrosinase protein levels and enzyme activity. In melanocytes, APPL exists in a complex with GIPC and phospho-AKT. Inhibition of AKT phosphorylation using a PI3-kinase inhibitor abolishes this interaction and results in retardation TRP1 in the Golgi. These data suggest that interactions between TRP1-GIPC and GIPC-APPL-AKT provide a potential link between melanogenesis and PI3 kinase signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Melaninas/biossíntese , Melanossomas/metabolismo , Tripsina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Melanossomas/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Transporte Proteico/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genéticaRESUMO
Objectives: Both coronavirus disease 2019 (COVID-19) pneumonia and influenza A (H1N1) pneumonia are highly contagious diseases. We aimed to characterize initial computed tomography (CT) and clinical features and to develop a model for differentiating COVID-19 pneumonia from H1N1 pneumonia. Methods: In total, we enrolled 291 patients with COVID-19 pneumonia from January 20 to February 13, 2020, and 97 patients with H1N1 pneumonia from May 24, 2009, to January 29, 2010 from two hospitals. Patients were randomly grouped into a primary cohort and a validation cohort using a seven-to-three ratio, and their clinicoradiologic data on admission were compared. The clinicoradiologic features were optimized by the least absolute shrinkage and selection operator (LASSO) logistic regression analysis to generate a model for differential diagnosis. Receiver operating characteristic (ROC) curves were plotted for assessing the performance of the model in the primary and validation cohorts. Results: The COVID-19 pneumonia mainly presented a peripheral distribution pattern (262/291, 90.0%); in contrast, H1N1 pneumonia most commonly presented a peribronchovascular distribution pattern (52/97, 53.6%). In LASSO logistic regression, peripheral distribution patterns, older age, low-grade fever, and slightly elevated aspartate aminotransferase (AST) were associated with COVID-19 pneumonia, whereas, a peribronchovascular distribution pattern, centrilobular nodule or tree-in-bud sign, consolidation, bronchial wall thickening or bronchiectasis, younger age, hyperpyrexia, and a higher level of AST were associated with H1N1 pneumonia. For the primary and validation cohorts, the LASSO model containing above eight clinicoradiologic features yielded an area under curve (AUC) of 0.963 and 0.943, with sensitivity of 89.7 and 86.2%, specificity of 89.7 and 89.7%, and accuracy of 89.7 and 87.1%, respectively. Conclusions: Combination of distribution pattern and category of pulmonary opacity on chest CT with clinical features facilitates the differentiation of COVID-19 pneumonia from H1N1 pneumonia.
RESUMO
PURPOSE: Hypoxia is a cause for resistance to cancer therapies. Molecularly targeted recombinant cytotoxins have shown clinical efficacy in the treatment of patients with primary brain tumors, glioblastoma multiforme, but it is not known whether hypoxia influences their antitumor effect. EXPERIMENTAL DESIGN: We have exposed glioblastoma multiforme cells, such as U-251 MG, U-373 MG, SNB-19, and A-172 MG, to either anoxia or hypoxia and then reoxygenated them while treating with an interleukin (IL)-13-based diphtheria toxin (DT)-containing cytotoxin, DT-IL13QM. We measured the levels of immunoreactive IL-13Ralpha2, a receptor that mediates IL-13-cytotoxin cell killing, and the levels of active form of furin, a protease that activates the bacterial toxin portion in a cytotoxin. RESULTS: We found that anoxia/hypoxia significantly alters the responsiveness of glioblastoma multiforme cells to DT-IL13QM. Interestingly, bringing these cells back to normoxia caused them to become even more susceptible to the cytotoxin than the cells maintained under normoxia. Anoxia/hypoxia caused a highly prominent decrease in the immunoreactive levels of both IL-13R and active forms of furin, and reoxygenation not only restored their levels but also became higher than that in normoxic glioblastoma multiforme cells. CONCLUSIONS: Our results show that a recombinant cytotoxin directed against glioblastoma multiforme cells kills these cells much less efficiently under anoxic/hypoxic conditions. The reoxygenation brings unexpected additional benefit of making glioblastoma multiforme cells even more responsive to the killing effect of a cytotoxin.
Assuntos
Neoplasias Encefálicas/metabolismo , Hipóxia Celular , Citotoxinas/farmacologia , Toxina Diftérica/farmacologia , Glioblastoma/metabolismo , Interleucina-13 , Oxigênio/farmacologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Furina/análise , Glioblastoma/terapia , Humanos , Imunotoxinas/farmacologia , Subunidade alfa2 de Receptor de Interleucina-13/metabolismoRESUMO
BACKGROUND: It has been pointed out that only low-dose arsenic trioxide (ATO) presents therapeutic benefits outweighing the toxic side effects. Low-dose ATO can effectively alleviate acute promyelocytic leukemia (APL). However, it is quite challenging in treating solid tumors. The purpose of this study was to investigate the effect of ATO at low concentrations on the metastatic potential of mouse hepatoma H(22) cells and the anti-metastatic mechanism of ATO. METHODS: The metastatic potential of H(22) cells was evaluated by adhesion, migration and invasion assays after exposure to a low dose of ATO in vitro. The mouse lung metastatic model induced by injection of H(22) cells via the tail vein was adopted for the evaluation of metastatic potential. Different proteins in the lysate of H(22) cells exposed to ATO at different concentrations were investigated by surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Finally, Western blotting analyses were made to detect the expression pattern of MMP-2 and nm23-M1 proteins. RESULTS: Significant cell death started at ATO concentrations above 2 micromol/L. The growth and adhesion potential of H(22) cells was inhibited in a time- and dose-dependent manner, and the migration and invasion potential of H(22) cells was inhibited in a dose-dependent manner while ATO concentration was below 2 micromol/L. Mice injected with ATO at a dose of 0.5 mg/kg had fewer lung metastases. However, mice injected with ATO at a dose of 2 mg/kg or 4 mg/kg had a high mortality rate and more liver injuries. A total of 15 different protein peaks were identified between the lysate of H(22) cells treated with ATO and controls. Two proteins that peaked at m/z 5302 and 17207 coincided with MMP-2 (fragment) and nm23-M1, respectively. Western blotting analyses demonstrated that MMP-2 and MMP-2 fragments were down-regulated and nm23-M1 was up-regulated in H(22) cells treated with 2 micromol/L ATO for 48 hours. CONCLUSIONS: ATO at a low dose inhibits the metastatic potential of mouse hepatoma H(22) cells in vitro and in vivo, and involves down-regulation of MMP-2 and up-regulation of nm23-M1.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Carcinoma Hepatocelular/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Óxidos/farmacologia , Animais , Antineoplásicos/efeitos adversos , Trióxido de Arsênio , Arsenicais/efeitos adversos , Carcinoma Hepatocelular/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Óxidos/efeitos adversos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A typical inflammatory response sequentially progresses from pro-inflammatory, immune suppressive to inflammatory repairing phases. Although the physiological inflammatory response resolves in time, severe acute inflammation usually sustains immune tolerance and leads to high mortality, yet the underlying mechanism is not completely understood. Here, using the leukemia-derived THP-1 human monocytes, healthy and septic human peripheral blood mononuclear cells (PBMC), we report that endotoxin dose-dependent switch of nicotinamide adenine dinucleotide (NAD) biosynthesis pathways sustain immune tolerant status. Low dose endotoxin triggered nicotinamide phosphoribosyltransferase (NAMPT)-dependent NAD salvage activity to adapt pro-inflammation. In contrast, high dose endotoxin drove a shift of NAD synthesis pathway from early NAMPT-dependent NAD salvage to late indoleamine 2,3-dioxygenase-1 (IDO1)-dependent NAD de novo biosynthesis, leading to persistent immune suppression. This is resulted from the IDO1-dependent expansion of nuclear NAD pool and nuclear NAD-dependent prolongation of sirtuin1 (SIRT1)-directed epigenetics of immune tolerance. Inhibition of IDO1 activity predominantly decreased nuclear NAD level, which promoted sequential dissociations of immunosuppressive SIRT1 and RelB from the promoter of pro-inflammatory TNF-α gene and broke endotoxin tolerance. Thus, NAMPT-NAD-SIRT1 axis adapts pro-inflammation, but IDO1-NAD-SIRT1-RelB axis sustains endotoxin tolerance during acute inflammatory response. Remarkably, in contrast to the prevention of sepsis death of animal model by IDO1 inhibition before sepsis initiation, we demonstrated that the combination therapy of IDO1 inhibition by 1-methyl-D-tryptophan (1-MT) and tryptophan supplementation rather than 1-MT administration alone after sepsis onset rescued sepsis animals, highlighting the translational significance of tryptophan restoration in IDO1 targeting therapy of severe inflammatory diseases like sepsis.
Assuntos
Tolerância Imunológica , NAD/imunologia , Sirtuína 1/imunologia , Fator de Transcrição RelB/imunologia , Animais , Citocinas/imunologia , Endotoxinas/toxicidade , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Nicotinamida Fosforribosiltransferase/imunologia , Sepse/induzido quimicamente , Sepse/tratamento farmacológico , Sepse/imunologia , Células THP-1 , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: The pathogenesis of acute pancreatitis is complex and largely unclear. The aim of this study was to explore the relationship between modes of cell death in pancreatic acinar cells, the release of cell contents and the inflammatory response of macrophages. METHODS: Our experiment included four groups: group A (the control group), group B (AR42J cells overstimulated by caerulein), group C (AR42J cells treated with lipopolysaccharide and caerulein), and group D (AR42J cells treated with octreotide and caerulein). Apoptosis and oncosis, and the release of amylase and lactate dehydrogenase (LDH) from AR42J cells were detected. Rat macrophages were stimulated by 1 ml supernatant of culture medium of AR42J cells. Finally, NF-kappaB activation and TNF-alpha and IL-1beta secretion by macrophages were detected. RESULTS: Oncotic cells in group C increased while apoptotic cells decreased (P < 0.05); cells in group D had the inverse reaction. The release of amylase and LDH changed directly with the occurrence of oncosis. The transcription factor NF-kappaB was activated and secretion of TNF-alpha and IL-1beta were significantly higher in group C than in group B (P < 0.05); in group D, these actions were significantly lower than in group B (P < 0.05). This trend was in line with changes in amylase and LDH production. CONCLUSION: There is a close relationship between modes of pancreatic acinar cell death, the release of cell contents and the inflammatory reaction of macrophages.
Assuntos
Apoptose , Ativação de Macrófagos , Pâncreas/patologia , Amilases/metabolismo , Animais , Interleucina-1beta/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The goal of this investigation was to define the molecular mechanism underlying physiologic conversion of immune tolerance to resolution of the acute inflammatory response, which is unknown. An example of this knowledge gap and its clinical importance is the broad-based energy deficit and immunometabolic paralysis in blood monocytes from non-survivors of human and mouse sepsis that precludes sepsis resolution. This immunometabolic dysregulation is biomarked by ex vivo endotoxin tolerance to increased glycolysis and TNF-α expression. To investigate how tolerance switches to resolution, we adapted our previously documented models associated with acute inflammatory, immune, and metabolic reprogramming that induces endotoxin tolerance as a model of sepsis in human monocytes. We report here that mitochondrial sirtuin 4 (SIRT4) physiologically breaks tolerance and resolves acute inflammation in human monocytes by coordinately reprogramming of metabolism and bioenergetics. We find that increased SIRT4 mRNA and protein expression during immune tolerance counters the increase in pyruvate dehydrogenase kinase 1 (PDK1) and SIRT1 that promote tolerance by switching glucose-dependent support of immune resistance to fatty acid oxidation support of immune tolerance. By decreasing PDK1, pyruvate dehydrogenase complex reactivation rebalances mitochondrial respiration, and by decreasing SIRT1, SIRT4 represses fatty acid oxidation. The precise mechanism for the mitochondrial SIRT4 nuclear feedback is unclear. Our findings are consistent with a new concept in which mitochondrial SIRT4 directs the axis that controls anabolic and catabolic energy sources.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Monócitos/fisiologia , Sepse/imunologia , Sirtuínas/metabolismo , Reprogramação Celular , Metabolismo Energético , Glucose/metabolismo , Glicólise , Homeostase , Humanos , Tolerância Imunológica , Oxirredução , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dysregulation of microRNAs (miRNAs) is correlated with cancer progression. In vitro detection methods using extracts from cell lysis cannot provide information about the spatial distribution of miRNAs. Due to the development of miRNA fluorescence in situ hybridization (FISH), increasing amounts of intracellular expression information are being obtained. However, miRNA FISH suffers from weak signals and complex steps and thus remains very challenging. Herein, a strategy based on DNA-templated silver nanoclusters (AgNCs/DNAs) and their G-rich fluorescence enhancement effect was developed for FISH detection of miRNAs in gastric cancer cells. The method combines hybridization and signal amplification into one step, which allows imaging of intracellular miRNAs immediately after hybridization. Most importantly, using the method based on our design, miR-101-3p, miR-16-5p and miR-19b-3p were found to be located in the nuclei of MGC803 cells with granulated shapes, indicating an unanticipated distribution pattern. In addition, before the final miRNA FISH, we performed an optimization of AgNCs/DNAs and their G-rich fluorescence enhancement effect; we found that the effect occurred at shorter wavelengths emitting green fluorescence, with weakened red fluorescence at longer wavelengths. However, the components involved in the FISH process impacted the fluorescence properties so greatly that the probes finally exhibited slightly strengthened red fluorescence signals. Our method enables facile visualization of miRNAs at the subcellular level, which may benefit the precise localization of miRNAs in single cells in the future.
Assuntos
Sondas de DNA/química , Nanopartículas Metálicas , MicroRNAs/análise , Prata , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Humanos , Hibridização in Situ FluorescenteRESUMO
PURPOSE: The novel fusion protein, DAB389EGF, composed of the catalytic and translocation domains of diphtheria toxin (DAB389) fused with a His-Ala linker to human epidermal growth factor (EGF) was tested for antiglioma efficacy in an in vivo model of human glioma. EXPERIMENTAL DESIGN: Female athymic nude mice (ages 4-6 weeks) were inoculated s.c. with 10 million U87MG human glioma cells in the right flank. When tumor volumes reached approximately 100 mm3 (approximately 6-8 days), i.t. injections of saline, DAB389IL2, or DAB389EGF 1, 3, 5 or 10 microg in 50 microL were given every other day for three to six doses. Animals were monitored twice daily and tumor measurements were made by calipers. RESULTS: The maximal tolerated dose (MTD) of DAB389EGF was 3 microg every other day. Above the MTD, animals experienced loss of activity, reduced oral intake, and dehydration. Blood chemistries confirmed elevated blood urea nitrogen, creatinine, aspartate transaminase, and alanine transaminase. Histopathology revealed renal tubular necrosis. At the MTD, tumor regression was seen in all animals. Relapses occurred in 4 of 16 (25%) of animals after 1 month. These tumors contained EGF receptor, were sensitive in vitro to DAB389EGF, and responded to a second course of i.t. DAB389EGF. CONCLUSIONS: DAB389EGF fusion protein shows in vivo antiglioma efficacy in a s.c. tumor model and warrants further preclinical testing in an i.c. tumor model for eventual treatment of patients with recurrent or refractory EGF receptor-positive glioblastoma multiforme.
Assuntos
Toxina Diftérica/uso terapêutico , Fator de Crescimento Epidérmico/uso terapêutico , Glioblastoma/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Nitrogênio da Ureia Sanguínea , Linhagem Celular Tumoral , Feminino , Glioma/tratamento farmacológico , Humanos , Concentração Inibidora 50 , Rim/metabolismo , Fígado/metabolismo , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Necrose , Transplante de Neoplasias , Nitrogênio/metabolismo , Estrutura Terciária de Proteína , Fatores de Tempo , Resultado do TratamentoRESUMO
The cytotoxicity of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of DAB(389)EGF, the percentage reduction in thymidine incorporation was determined. For 13 of 14 cell lines, potent cytotoxicity was observed, with IC(50)s of 0.4-50 pM. The epidermal growth factor receptor (EGFR) density of these cell lines was determined by immunofluorescence microscopy, flow cytometry, and radioligand binding. These assays correlated well with each other and demonstrated EGFR levels of 15,000-230,000/cell for 13 of 14 cell lines. The cell line U138MG, which lacked EGFR, was the only cell line insensitive to DAB(389)EGF. Linear regression analysis showed a good correlation between EGFR density and DAB(389)EGF sensitivity (P < 0.001) and between results of flow cytometry and radiolabeled binding assays of EGFR density (P = 0.01). DAB(389)EGF may have potential for intracranial therapy of EGFR-positive glioblastomas.