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1.
Immunity ; 54(3): 454-467.e6, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33561388

RESUMEN

Heparin, a mammalian polysaccharide, is a widely used anticoagulant medicine to treat thrombotic disorders. It is also known to improve outcomes in sepsis, a leading cause of mortality resulted from infection-induced immune dysfunction. Whereas it is relatively clear how heparin exerts its anticoagulant effect, the immunomodulatory mechanisms enabled by heparin remain enigmatic. Here, we show that heparin prevented caspase-11-dependent immune responses and lethality in sepsis independent of its anticoagulant properties. Heparin or a chemically modified form of heparin without anticoagulant function inhibited the alarmin HMGB1-lipopolysaccharide (LPS) interaction and prevented the macrophage glycocalyx degradation by heparanase. These events blocked the cytosolic delivery of LPS in macrophages and the activation of caspase-11, a cytosolic LPS receptor that mediates lethality in sepsis. Survival was higher in septic patients treated with heparin than those without heparin treatment. The identification of this previously unrecognized heparin function establishes a link between innate immune responses and coagulation.


Asunto(s)
Anticoagulantes/uso terapéutico , Caspasas/metabolismo , Heparina/uso terapéutico , Macrófagos/inmunología , Sepsis/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Caspasas/genética , Línea Celular , Femenino , Glucuronidasa/genética , Glucuronidasa/metabolismo , Glicocálix/metabolismo , Proteína HMGB1/metabolismo , Humanos , Inmunomodulación , Lipopolisacáridos/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Sepsis/mortalidad , Análisis de Supervivencia , Adulto Joven
2.
Immunity ; 51(6): 983-996.e6, 2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31836429

RESUMEN

Excessive activation of the coagulation system leads to life-threatening disseminated intravascular coagulation (DIC). Here, we examined the mechanisms underlying the activation of coagulation by lipopolysaccharide (LPS), the major cell-wall component of Gram-negative bacteria. We found that caspase-11, a cytosolic LPS receptor, activated the coagulation cascade. Caspase-11 enhanced the activation of tissue factor (TF), an initiator of coagulation, through triggering the formation of gasdermin D (GSDMD) pores and subsequent phosphatidylserine exposure, in a manner independent of cell death. GSDMD pores mediated calcium influx, which induced phosphatidylserine exposure through transmembrane protein 16F, a calcium-dependent phospholipid scramblase. Deletion of Casp11, ablation of Gsdmd, or neutralization of phosphatidylserine or TF prevented LPS-induced DIC. In septic patients, plasma concentrations of interleukin (IL)-1α and IL-1ß, biomarkers of GSDMD activation, correlated with phosphatidylserine exposure in peripheral leukocytes and DIC scores. Our findings mechanistically link immune recognition of LPS to coagulation, with implications for the treatment of DIC.


Asunto(s)
Caspasas Iniciadoras/metabolismo , Coagulación Intravascular Diseminada/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Fosfatidilserinas/metabolismo , Tromboplastina/metabolismo , Animales , Coagulación Sanguínea/fisiología , Caspasas Iniciadoras/genética , Línea Celular Tumoral , Endotoxemia/patología , Activación Enzimática , Células HT29 , Células HeLa , Humanos , Interleucina-1alfa/sangre , Interleucina-1beta/sangre , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión a Fosfato/genética , Piroptosis/fisiología , Transducción de Señal/fisiología
3.
Immunity ; 49(4): 740-753.e7, 2018 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-30314759

RESUMEN

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.


Asunto(s)
Caspasas/inmunología , Endotoxinas/inmunología , Proteína HMGB1/inmunología , Piroptosis/inmunología , Sepsis/inmunología , Animales , Caspasas/genética , Caspasas/metabolismo , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Endotoxinas/metabolismo , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada/inmunología , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Sepsis/genética , Sepsis/metabolismo , Células THP-1
4.
Semin Immunol ; 70: 101844, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37778179

RESUMEN

Sepsis remains one of the most common and lethal conditions globally. Currently, no proposed target specific to sepsis improves survival in clinical trials. Thus, an in-depth understanding of the pathogenesis of sepsis is needed to propel the discovery of effective treatment. Recently attention to sepsis has intensified because of a growing recognition of a non-canonical inflammasome-triggered lytic mode of cell death termed pyroptosis upon sensing cytosolic lipopolysaccharide (LPS). Although the consequences of activation of the canonical and non-canonical inflammasome are similar, the non-canonical inflammasome formation requires caspase-4/5/11, which enzymatically cleave the pore-forming protein gasdermin D (GSDMD) and thereby cause pyroptosis. The non-canonical inflammasome assembly triggers such inflammatory cell death by itself; or leverages a secondary activation of the canonical NLRP3 inflammasome pathway. Excessive cell death induced by oligomerization of GSDMD and NINJ1 leads to cytokine release and massive tissue damage, facilitating devastating consequences and death. This review summarized the updated mechanisms that initiate and regulate non-canonical inflammasome activation and pyroptosis and highlighted various endogenous or synthetic molecules as potential therapeutic targets for treating sepsis.


Asunto(s)
Sepsis , Choque Séptico , Humanos , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacología , Piroptosis , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Caspasas/metabolismo , Caspasas/farmacología , Factores de Crecimiento Nervioso/farmacología , Moléculas de Adhesión Celular Neuronal/farmacología
5.
Mol Med ; 30(1): 127, 2024 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-39179968

RESUMEN

BACKGROUND: Cognitive dysfunction caused by infection frequently emerges as a complication in sepsis survivor patients. However, a comprehensive understanding of its pathogenesis remains elusive. METHODS: In our in vivo experiments, an animal model of endotoxemia was employed, utilizing the Novel Object Recognition Test and Morris Water Maze Test to assess cognitive function. Various techniques, including immunofluorescent staining, Western blotting, blood‒brain barrier permeability assessment, Limulus Amebocyte Lysate (LAL) assay, and Proximity-ligation assay, were employed to identify brain pathological injury and neuroinflammation. To discern the role of Caspase-11 (Casp11) in hematopoietic or non-hematopoietic cells in endotoxemia-induced cognitive decline, bone marrow chimeras were generated through bone marrow transplantation (BMT) using wild-type (WT) and Casp11-deficient mice. In vitro studies involved treating BV2 cells with E. coli-derived outer membrane vesicles to mimic in vivo conditions. RESULTS: Our findings indicate that the deficiency of Casp11-GSDMD signaling pathways reverses infection-induced cognitive dysfunction. Moreover, cognitive dysfunction can be ameliorated by blocking the IL-1 effect. Mechanistically, the absence of Casp11 signaling significantly mitigated blood‒brain barrier leakage, microglial activation, and synaptic damage in the hippocampal CA3 region, ultimately leading to improved cognitive function. CONCLUSION: This study unveils the crucial contribution of Casp11 and GSDMD to cognitive impairments and spatial memory loss in a murine sepsis model. Targeting Casp11 signaling emerges as a promising strategy for preventing or treating cognitive dysfunction in patients with severe infections.


Asunto(s)
Caspasas Iniciadoras , Caspasas , Disfunción Cognitiva , Modelos Animales de Enfermedad , Transducción de Señal , Animales , Disfunción Cognitiva/etiología , Disfunción Cognitiva/metabolismo , Ratones , Caspasas/metabolismo , Caspasas Iniciadoras/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas de Unión a Fosfato/genética , Barrera Hematoencefálica/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Endotoxemia/complicaciones , Endotoxemia/metabolismo , Endotoxemia/etiología , Hipocampo/metabolismo , Hipocampo/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Sepsis/complicaciones , Sepsis/metabolismo , Gasderminas
6.
Blood ; 135(14): 1087-1100, 2020 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-32016282

RESUMEN

Bacterial infection not only stimulates innate immune responses but also activates coagulation cascades. Overactivation of the coagulation system in bacterial sepsis leads to disseminated intravascular coagulation (DIC), a life-threatening condition. However, the mechanisms by which bacterial infection activates the coagulation cascade are not fully understood. Here we show that type 1 interferons (IFNs), a widely expressed family of cytokines that orchestrate innate antiviral and antibacterial immunity, mediate bacterial infection-induced DIC by amplifying the release of high-mobility group box 1 (HMGB1) into the bloodstream. Inhibition of the expression of type 1 IFNs and disruption of their receptor IFN-α/ßR or downstream effector (eg, HMGB1) uniformly decreased gram-negative bacteria-induced DIC. Mechanistically, extracellular HMGB1 markedly increased the procoagulant activity of tissue factor by promoting the externalization of phosphatidylserine to the outer cell surface, where phosphatidylserine assembles a complex of cofactor-proteases of the coagulation cascades. These findings not only provide novel insights into the link between innate immune responses and coagulation, but they also open a new avenue for developing novel therapeutic strategies to prevent DIC in sepsis.


Asunto(s)
Coagulación Intravascular Diseminada/inmunología , Endotoxemia/inmunología , Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Interferón-alfa/inmunología , Interferón beta/inmunología , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Animales , Coagulación Sanguínea , Coagulación Intravascular Diseminada/sangre , Coagulación Intravascular Diseminada/etiología , Endotoxemia/sangre , Endotoxemia/complicaciones , Infecciones por Bacterias Gramnegativas/sangre , Infecciones por Bacterias Gramnegativas/complicaciones , Proteína HMGB1/sangre , Proteína HMGB1/inmunología , Humanos , Inmunidad Innata , Ratones Endogámicos C57BL
7.
J Biol Chem ; 294(22): 8872-8884, 2019 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-31000631

RESUMEN

Receptor-interacting protein kinase 3 (RIPK3) is a key regulator of programmed cell death and inflammation during viral infection or sterile tissue injury. Whether and how bacterial infection also activates RIPK3-dependent immune responses remains poorly understood. Here we show that bacterial lipids (lipid IVa or lipid A) form a complex with high mobility group box 1 (HMGB1), released by activated immune cells or damaged tissue during bacterial infection, and that this complex triggers RIPK3- and TIR domain-containing adapter-inducing IFN-ß (TRIF)-dependent immune responses. We found that these responses lead to macrophage death, interleukin (IL)-1α release, and IL-1ß maturation. In an air-pouch inflammatory infiltration model, genetic deletion of Ripk3, Trif, or IL-1 receptor (Il-1R), or monoclonal antibody-mediated HMGB1 neutralization uniformly attenuated inflammatory responses induced by Gram-negative bacteria that release lipid IVa and lipid A. These findings uncover a previously unrecognized mechanism by which host factors and bacterial components work in concert to orchestrate immune responses.


Asunto(s)
Apoptosis , Proteína HMGB1/metabolismo , Lípido A/metabolismo , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Glucolípidos/inmunología , Glucolípidos/metabolismo , Bacterias Gramnegativas/metabolismo , Proteína HMGB1/inmunología , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Lípido A/análogos & derivados , Lípido A/inmunología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Transducción de Señal , Receptor Toll-Like 4/metabolismo
8.
J Biol Chem ; 294(21): 8384-8394, 2019 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-30971430

RESUMEN

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a multimeric protein complex that mediates maturation of the cytokines IL-1ß and IL-18 as well as release of the proinflammatory protein high-mobility group box 1 (HMGB1) and contributes to several inflammatory diseases, including sepsis, gout, and type 2 diabetes. In this context, the well-studied active complement fragment C5a and its receptor C5aR1 or C5aR2 orchestrate the inflammatory responses in many diseases. Although a C5a-C5aR interaction in NLRP3-associated diseases has been suggested, little is known about the details of C5a-C5aR cross-talk with the NLRP3 inflammasome in macrophages. In this study, using mice and murine macrophages and cytokines, immunoblotting, siRNA, and quantitative real-time PCR assays, we demonstrate that C5aR2 deficiency restricts activation of the NLRP3 inflammasome and release of HMGB1 both in vitro and in vivo Mechanistically, we found that C5aR2 promotes NLRP3 activation by amplifying dsRNA-dependent PKR expression, which is an important NLRP3-activating factor. We also observed that elevation of PKR expression because of the C5a-C5aR2 interaction depends on the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase pathway and type I IFN signaling. In conclusion, these findings reveal that C5aR2 contributes to NLRP3 inflammasome activation and HMGB1 release from macrophages.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Proteína HMGB1/metabolismo , Inflamasomas/metabolismo , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , eIF-2 Quinasa/biosíntesis , Animales , Complemento C5a/genética , Complemento C5a/metabolismo , Proteína HMGB1/genética , Inflamasomas/genética , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Receptor de Anafilatoxina C5a/genética , eIF-2 Quinasa/genética
9.
Biochem Biophys Res Commun ; 533(4): 1519-1526, 2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33158480

RESUMEN

Cognitive impairment, acute or long-term, is a common complication in patients with severe bacterial infection. However, the underlying mechanisms are not fully verified and effective medicine is not available in clinics. Interferon gamma (IFNγ) is a pivotal cytokine against infection and is believed to be a tune in homeostasis of cognitive function. Here, we collected blood and cerebrospinal fluid (CF) from human subjects and mice, and found that plasma and CF levels of IFNγ were significantly increased in septic patients and endotoxin-challenged mice when compared with healthy controls. IFNγ signaling was boosted in the hippocampus of mice after a challenge of lipopolysaccharide (LPS), which was accompanied with cognitive impairment and decline of neurogenesis. Deficiency of IFNγ or its receptor (IFNγR) dramatically attenuated microglia-induced A1 astrocytes and consequently restored neurogenesis and cognitive function in endotoxemia mice model. Using primary microglia, astrocytes and neurons, we found that IFNγ remarkably increased LPS-mediated release of TNFα and IL-1α in microglia and consequently induced the transformation of astrocyte to A1 subtype, which ultimately resulted in neuron damage. Thus, IFNγ promotes cognitive impairment in endotoxemia by enhancing microglia-induced A1 astrocytes. Targeting IFNγ would be a novel strategy for preventing or treating cognitive dysfunction in patients with Gram-negative infection.


Asunto(s)
Astrocitos/fisiología , Disfunción Cognitiva/fisiopatología , Endotoxemia/fisiopatología , Interferón gamma/antagonistas & inhibidores , Neurogénesis/fisiología , Animales , Astrocitos/patología , Estudios de Casos y Controles , Células Cultivadas , Disfunción Cognitiva/genética , Disfunción Cognitiva/terapia , Modelos Animales de Enfermedad , Endotoxemia/genética , Endotoxemia/psicología , Silenciador del Gen , Humanos , Interferón gamma/deficiencia , Interferón gamma/genética , Interferón gamma/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/patología , Microglía/fisiología , Neurogénesis/genética , Tratamiento con ARN de Interferencia , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptores de Interferón/fisiología , Receptor de Interferón gamma
10.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 45(2): 128-133, 2020 Feb 28.
Artículo en Inglés, Zh | MEDLINE | ID: mdl-32386036

RESUMEN

OBJECTIVES: To predict renal biopsy with pathological acute and chronic index via comprehensive analysis of clinical indices from lupus nephritis patients. METHODS: We collected 107 inpatients' data from the Third Xiangya Hospital of Central South University from January 2011 to September 2018. These inpatients have already had renal biopsy results. These clinical indices were set as variables. Using multiple linear regression, we built the prediction models for renal pathological acute and chronic index. Simultaneously, we evaluated each vital variable's importance in models by standardized coefficient of regression equation, and model prediction accuracy by 5-fold cross validation. RESULTS: Acute index and chronic index prediction models were built with 19 and 23 clinical variables, respectively. To evaluate the two models' accuracy of prediction, we used 5-fold cross validation and found that the prediction accuracy level were satisfactory with the acute index model (Q2=0.649, R2=0.791) and chronic index model (Q2=0.563, R2=0.744). Standardized coefficient of regression equation showed serum total cholesterol, serum iron, NAG, ß2 microglobulin and BUN were important variables to predict acute index while total cholesterol, ß2 microglobulin, homocystein and serum low density lipoprotein-cholesterol were important for chronic index prediction. CONCLUSIONS: Clinical indices are able to effectively predict renal biopsy conditions, which are valuable to assess lupus nephritis severity.


Asunto(s)
Nefritis Lúpica , Biopsia , Humanos , Riñón , Análisis Multivariante
11.
Clin Immunol ; 205: 148-152, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30731209

RESUMEN

Ferritin is the major iron storage molecule of vertebrates, which can be detected in serum under numerous conditions, including inflammatory, neurodegenerative, and malignant diseases. Given this character, serum ferritin is frequently used as a biomarker in clinical settings. How the ferritin secreted to the serum has attracted much attention. Although some studies have found ferritin was mediated via the endoplasmic reticulum (ER)-Golgi secretion pathway or secretory lysosomes trafficking pathway under normal conditions, the secretion pathway of ferritin under pathological conditions, especially in sepsis is not very clear. In this report, we adopt a murine sepsis model to study the secretion pathway of ferritin in sepsis. We demonstrated caspase-11-GSDMD pathway and associated pyroptosis are required for secretion of ferritin in vitro and in vivo in sepsis. Moreover, our work connects pyroptosis to serum ferritin secretion and suggests a passive release process of ferritin, enhancing our understanding of the mechanism of ferritin secretion.


Asunto(s)
Caspasas Iniciadoras/genética , Ferritinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/metabolismo , Proteínas de Unión a Fosfato/genética , Piroptosis/genética , Sepsis/metabolismo , Adenosina Trifosfato/farmacología , Animales , Toxina del Cólera/farmacología , Ferritinas/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Piroptosis/efectos de los fármacos , Transducción de Señal
12.
Mol Med ; 24(1): 66, 2018 12 27.
Artículo en Inglés | MEDLINE | ID: mdl-30587103

RESUMEN

BACKGROUND: Caspase-11, a cytosolic receptor of bacterial endotoxin (lipopolysaccharide: LPS), mediates immune responses and lethality in endotoxemia and experimental sepsis. However, the upstream pathways that regulate caspase-11 activation in endotoxemia and sepsis are not fully understood. The aim of this study is to test whether TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling is critical for caspase-11-dependent immune responses and lethality in endotoxemia. METHODS: Mice of indicated genotypes were subjected to endotoxemia or cecum ligation and puncture (CLP) and monitored daily by signs of a moribund state for lethality. Serum interleukin (IL)-1α, IL-1ß, IL-6 and tumor necrosis factor (TNF) were measured by ELISA. Data were analyzed by using student's t-test or one-way ANOVA followed by post-hoc Bonferroni test. Survival data were analyzed by using the log-rank test. RESULTS: Blockade of type 1 interferon signaling or genetic deletion of TRIF or guanylate-binding proteins (GBPs) prevented caspase-11-dependent immune responses, organ injury and lethality in endotoxemia and experimental sepsis. In vitro, deletion of GBPs blocked cytosolic LPS-induced caspase-11 activation in mouse macrophages. CONCLUSIONS: These findings demonstrate that TRIF signaling is required for caspase-11-dependent immune responses and lethality in endotoxemia and sepsis, and provide novel mechanistic insights into how LPS induces caspase-11 activation during bacterial infection.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/inmunología , Caspasas/inmunología , Endotoxemia/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Caspasas Iniciadoras , Endotoxemia/inducido químicamente , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/inmunología , Interferón Tipo I/inmunología , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados
13.
Mol Med ; 24(1): 8, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-30134814

RESUMEN

BACKGROUND: The NLRP3 inflammasome, a cytosolic complex that mediates the maturation of IL-1ß and IL-18 as well as the release of high mobility group box 1 (HMGB1), contributes to the lethality of endotoxic shock. Ethyl pyruvate (EP) was previously shown to inhibit HMGB1 release and promote survival during endotoxemia and experimental sepsis. However, the underlying protective mechanism remains elusive. RESULT: EP dose-dependently inhibited the ATP-, nigericin-, alum-, and silica-induced caspase-1 activation and HMGB1 release in mouse macrophages. EP failed to inhibit DNA transfection- or Salmonella Typhimurium-induced caspase-1 activation and HMGB1 release. Mechanistically, EP significantly attenuated mitochondrial damage and cytoplasmic translocation of mitochondrial DNA, a known NLRP3 ligand, without influencing the potassium efflux, the lysosomal rupture or the production of mitochondrial reactive oxygen species (mtROS). CONCLUSION: Ethyl pyruvate acts as a novel NLRP3 inflammasome inhibitor that preserves the integrity of mitochondria during inflammation.


Asunto(s)
Inflamasomas/antagonistas & inhibidores , Mitocondrias/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Piruvatos/farmacología , Animales , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismo
14.
Nature ; 488(7413): 670-4, 2012 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-22801494

RESUMEN

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1ß, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1ß, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.


Asunto(s)
Proteína HMGB1/metabolismo , Inflamasomas/metabolismo , eIF-2 Quinasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfato/farmacología , Animales , Antígenos Bacterianos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Toxinas Bacterianas/farmacología , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Células Cultivadas , Cristalinas/metabolismo , Escherichia coli/inmunología , Escherichia coli/fisiología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Femenino , Proteína HMGB1/sangre , Humanos , Inflamasomas/agonistas , Interleucina-18/sangre , Interleucina-1beta/sangre , Interleucina-6/análisis , Interleucina-6/sangre , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas NLR , Peritonitis/metabolismo , Fosforilación , ARN Bicatenario/inmunología , ARN Bicatenario/farmacología , Rotenona/farmacología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/inmunología , Salmonella typhimurium/fisiología , Transfección , Ácido Úrico/farmacología , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genética
15.
J Biol Chem ; 291(29): 15093-107, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27226571

RESUMEN

The sensing of double-stranded RNA (dsRNA) in the liver is important for antiviral defenses but can also contribute to sterile inflammation during liver injury. Hepatocytes are often the target of viral infection and are easily injured by inflammatory insults. Here we sought to establish the pathways involved in the production of type I interferons (IFN-I) in response to extracellular poly(I:C), a dsRNA mimetic, in hepatocytes. This was of interest because hepatocytes are long-lived and, unlike most immune cells that readily die after activation with dsRNA, are not viewed as cells with robust antimicrobial capacity. We found that poly(I:C) leads to rapid up-regulation of inducible nitric oxide synthase (iNOS), double-stranded RNA-dependent protein kinase (PKR), and Src. The production of IFN-ß was dependent on iNOS, PKR, and Src and partially dependent on TLR3/Trif. iNOS and Src up-regulation was partially dependent on TLR3/Trif but entirely dependent on PKR. The phosphorylation of TLR3 on tyrosine 759 was shown to increase in parallel to IFN-ß production in an iNOS- and Src-dependent manner, and Src was found to directly interact with TLR3 in the endosomal compartment of poly(I:C)-treated cells. Furthermore, we identified a robust NO/cGMP/PKG-dependent feedforward pathway for the amplification of iNOS expression. These data identify iNOS/NO as an integral component of IFN-ß production in response to dsRNA in hepatocytes in a pathway that involves the coordinated activities of TLR3/Trif and PKR.


Asunto(s)
Hepatocitos/inmunología , Hepatocitos/metabolismo , Interferón beta/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Bicatenario/inmunología , ARN Bicatenario/farmacología , Receptor Toll-Like 3/metabolismo , eIF-2 Quinasa/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Hepatocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Fosforilación/efectos de los fármacos , Poli I-C/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/deficiencia , Receptor Toll-Like 3/genética , Tirosina/química , Regulación hacia Arriba/efectos de los fármacos , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genética , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética
16.
Kidney Blood Press Res ; 42(6): 1045-1052, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29197864

RESUMEN

BACKGROUND/AIMS: Renal biopsy is the gold standard to determine the pathologic type of primary nephrotic syndrome, which is critical for diagnosis, choice of treatment and evaluation of prognosis. However, in some cases, renal biopsy cannot be performed. METHODS: To explore the possibility of predicting the histology type of primary nephrotic syndrome without the need for biopsy, we trained and validated a machine learning algorithm using data from 222 patients with biopsy-confirmed primary nephrotic syndrome treated at our hospital between May 2008 and January 2016. The model was then tested prospectively on another sample of 63 patients with biopsy-confirmed primary nephrotic syndrome. RESULTS: Overall accuracy of prediction from the retrospective set of 222 patients was 62.2% across all types of nephrotic syndrome. The accuracy of model prediction for the prospectively collected dataset of 63 patients was 61.9%. The algorithm identified 17 of 33 variables as contributing strongly to type of renal pathology. CONCLUSION: To our knowledge, this is the first such application of machine learning to predict the pathologic type of primary nephrotic syndrome, which may be clinically useful by itself as well as helpful for guiding future efforts at machine learning-based prediction in other disease contexts.


Asunto(s)
Aprendizaje Automático , Síndrome Nefrótico/diagnóstico , Adulto , Algoritmos , Biopsia , Femenino , Humanos , Masculino , Síndrome Nefrótico/patología , Valor Predictivo de las Pruebas , Pronóstico
17.
Proc Natl Acad Sci U S A ; 111(8): 3068-73, 2014 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-24469805

RESUMEN

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.


Asunto(s)
Núcleo Celular/metabolismo , Proteína HMGB1/metabolismo , Janus Quinasa 1/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/fisiología , Acetilación , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Análisis de Varianza , Animales , Bencimidazoles/farmacología , Western Blotting , Cromatografía Liquida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Inmunohistoquímica , Interferón Tipo I/farmacología , Lipopolisacáridos , Ratones , Piridonas/farmacología , Transducción de Señal/efectos de los fármacos , Espectrometría de Masas en Tándem
18.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 42(9): 1111-1115, 2017 Sep 28.
Artículo en Zh | MEDLINE | ID: mdl-28989160

RESUMEN

High mobility group box-1 (HMGB1) is an evolutionarily conserved protein, which widely exists in mammals. HMGB1 contains the nucleus localization sequences. Intracellular and extracellular HMGB1 shows different biological functions. Extracellular HMGB1 is closely related to sepsis, cancer, rheumatoid immune, atherosclerosis, ischemia-reperfusion injury and so on. The mobilization of HMGB1 from the nucleus to the cytoplasm and subsequent release involves the processes of post-translation modification, active secretion and nuclear localization.


Asunto(s)
Proteína HMGB1 , Transporte de Proteínas , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína HMGB1/metabolismo , Humanos
19.
Arterioscler Thromb Vasc Biol ; 35(12): 2579-93, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26515416

RESUMEN

OBJECTIVE: Endoluminal vascular interventions such as angioplasty initiate a sterile inflammatory response resulting from local tissue damage. This response drives the development of intimal hyperplasia (IH) that, in turn, can lead to arterial occlusion. We hypothesized that the ubiquitous nuclear protein and damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1), is one of the endogenous mediators that activates processes leading to IH after endoluminal injury to the arterial wall. The aim of this study is to investigate whether approaches that reduce the levels of HMGB1 or inhibit its activity suppresses IH after arterial injury. APPROACH AND RESULTS: Here, we show that HMGB1 regulates IH in a mouse carotid wire injury model. Induced genetic deletion or neutralization of HMGB1 prevents IH, monocyte recruitment, and smooth muscle cell growth factor production after endoluminal carotid artery injury. A specific inhibitor of HMGB1 myeloid differentiation factor 2-toll-like receptor 4 (TLR4) interaction, P5779, also significantly inhibits IH. HMGB1 deletion is mimicked in this model by global deletion of TLR4 and partially replicated by myeloid-specific deletion of TLR4 but not TLR2 or receptor for advanced glycation endproducts deletion. The specific HMGB1 isoform known to activate TLR4 signaling (disulfide HMGB1) stimulates smooth muscle cell to migrate and produce monocyte chemotactic protein 1/CCL2) via TLR4. Macrophages produce smooth muscle cell mitogens in response to disulfide HMGB1 also in a TLR4/myeloid differentiation primary response gene (88)/Trif-dependent manner. CONCLUSIONS: These findings place HMGB1 and its receptor, TLR4 as critical regulators of the events that drive the inflammation leading to IH after endoluminal arterial injury and identify this pathway as a possible therapeutic target to limit IH to attenuate damage-associated molecular pattern molecule-mediated vascular inflammatory responses.


Asunto(s)
Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Proteína HMGB1/metabolismo , Neointima , Receptor Toll-Like 4/metabolismo , Lesiones del Sistema Vascular/metabolismo , Vasculitis/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Quimiotaxis de Leucocito , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteína HMGB1/deficiencia , Proteína HMGB1/genética , Humanos , Hiperplasia , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Transducción de Señal , Factores de Tiempo , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patología , Vasculitis/genética , Vasculitis/patología
20.
Clin Chem Lab Med ; 54(9): 1451-9, 2016 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-26872314

RESUMEN

BACKGROUND: A pilot study showing a decrease in androstenedione concentration in serum collected into gel-containing serum tubes (STs) triggered an investigation of the effect of serum collection tube on steroid hormone stability. METHODS: In the main study, two tube types were examined: BD Vacutainer® SST™II Advance and BD Vacutainer® Serum Tube. Forty-seven serum samples from apparently healthy volunteers were collected and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP) (n=20); and oestradiol (n=27). Primary specimens were centrifuged once, maintained at room temperature and extracted within 2 h for day zero (d0) results. To assess stability following refrigeration (2-8 °C), aliquots were taken from the primary tube on day one (d1) and day five (d5) and analysed immediately. Differences in measurand concentration between tubes at d0 and following storage (d1 and d5) were evaluated for statistical significance. RESULTS: There was a progressive and statistically significant decrease in androstenedione concentration from d0 to d5 (p<0.001) in the SST™II tubes. In addition, there was a statistically significant reduction in testosterone, 17-OHP and oestradiol concentrations at d5 (p<0.01). Interestingly, oestradiol and testosterone concentrations increased with time in plain STs (p<0.01). The only change likely to have a clinical impact was that of androstenedione in serum gel tubes. CONCLUSIONS: To optimise conditions and to reduce pre-analytical error we recommend the use of plain serum collection tubes for androstenedione and rapid separation of serum from cells when oestradiol and testosterone are requested.


Asunto(s)
17-alfa-Hidroxiprogesterona/sangre , Androstenodiona/sangre , Recolección de Muestras de Sangre/instrumentación , Testosterona/sangre , Adulto , Anciano , Cromatografía Liquida , Femenino , Geles/química , Humanos , Masculino , Persona de Mediana Edad , Espectrometría de Masas en Tándem , Adulto Joven
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