RESUMEN
The pathogenesis of acute lung injury is complex. Studies have demonstrated the role of neutrophil extracellular traps (NETs) in the process of lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the underlying mechanism remains unclear. In this study, the regulation of Nrf2 in the formation of NETs, which was pathogenic in LPS-induced ALI, was identified by analyzing the levels of Cit-H3, lung function, lung tissue pathology, lung wet/dry ratio, the inflammatory cells, cytokines and proteins in the bronchoalveolar lavage fluid (BALF) and in addition, the activity of lung myeloperoxidase (MPO) was also measured. Results showed that the levels of Cit-H3 measured by western blot in Nrf2-knockout (KO) mice were higher compared with the WT mice after LPS stimulation. To further investigate the NETs formation was pathogenic during LPS-induced ALI, the Nrf2-KO mice were treated with DNase I. Results showed that DNase I improved lung function and lung tissue pathology and significantly reduced lung wet/dry ratio and proteins in the BALF. Besides, DNase I also attenuated the infiltration of inflammatory cells and the cytokines (TNF-α, IL-1ß) production in the BALF and the activity of lung MPO. Therefore, these results together indicate that Nrf2 may intervene in the release of NETs during LPS-induced ALI in mice.
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Lesión Pulmonar Aguda , Líquido del Lavado Bronquioalveolar , Trampas Extracelulares , Lipopolisacáridos , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2 , Animales , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Factor 2 Relacionado con NF-E2/metabolismo , Ratones , Trampas Extracelulares/metabolismo , Líquido del Lavado Bronquioalveolar/química , Masculino , Peroxidasa/metabolismo , Neutrófilos/metabolismo , Pulmón/metabolismo , Pulmón/patología , Interleucina-1beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Desoxirribonucleasa I/metabolismo , Citocinas/metabolismo , Western BlottingRESUMEN
Neutrophils utilize a variety of metabolic sources to support their crucial functions as the first responders in innate immunity. Here, through in vivo and ex vivo isotopic tracing, we examined the contributions of different nutrients to neutrophil metabolism under specific conditions. Human peripheral blood neutrophils, in contrast to a neutrophil-like cell line, rely on glycogen storage as a major metabolic source under resting state but rapidly switch to primarily using extracellular glucose upon activation with various stimuli. This shift is driven by a substantial increase in glucose uptake, enabled by rapidly increased GLUT1 on cell membrane, that dominates the simultaneous increase in gross glycogen cycling capacity. Shifts in nutrient utilization impact neutrophil functions in a function-specific manner: oxidative burst depends on glucose utilization, whereas NETosis and phagocytosis can be flexibly supported by either glucose or glycogen, and neutrophil migration and fungal control are enhanced by the shift from glycogen utilization to glucose utilization. This work provides a quantitative and dynamic understanding of fundamental features in neutrophil metabolism and elucidates how metabolic remodeling shapes neutrophil functions, which has broad health relevance.
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Glucosa , Glucógeno , Neutrófilos , Fagocitosis , Humanos , Neutrófilos/metabolismo , Neutrófilos/inmunología , Glucosa/metabolismo , Glucógeno/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Nutrientes/metabolismo , Activación Neutrófila , Estallido Respiratorio , Trampas Extracelulares/metabolismoRESUMEN
Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.
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Toxinas Bacterianas , Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Escherichia coli Enterotoxigénica , Enterotoxinas , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Neutrófilos , Fagocitosis , Enterotoxinas/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Humanos , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Trampas Extracelulares/metabolismo , Trampas Extracelulares/inmunología , Transducción de SeñalRESUMEN
BACKGROUND: At present, hepatic ischemia-reperfusion injury (IRI) is an important complication of partial hepatectomy and liver transplantation, and it is an important cause of poor prognosis. Spleen tyrosine kinase(SYK) plays an important role in a variety of signaling pathways in the liver, but its role in hepatic IRI is still unclear. This study aims to investigate the role and mechanism of SYK in hepatic IRI and tumor recurrence. METHODS: We first observed the activation of SYK in the liver of mice in response to hepatic IRI. Subsequently, Pharmacological inhibitions of SYK were used to evaluated the effect of SYK on neutrophil recruitment and NETosis, and further explored the effect of SYK on IRI and tumor recurrence. RESULTS: Our study shows that SYK is activated in response to hepatic IRI and aggravates liver injury. On the one hand, neutrophils SYK during the early stage of liver reperfusion increases neutrophil extracellular traps (NETs) production by promoting Pyruvate kinase M2(PKM2) nuclear translocation leading to upregulation of phosphorylated STAT3, thereby exacerbating liver inflammation and tumor recurrence. On the other hand, macrophages SYK can promote the recruitment of neutrophils and increase the activation of NLRP3 inflammasome and IL1ß, which further promotes the formation of NETs. CONCLUSIONS: Our study demonstrates that neutrophil and macrophage SYK synergistically promote hepatic IRI and tumor recurrence, and SYK may be a potential target to improve postoperative hepatic IRI and tumor recurrence.
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Trampas Extracelulares , Proteínas de la Membrana , Neutrófilos , Daño por Reperfusión , Factor de Transcripción STAT3 , Quinasa Syk , Quinasa Syk/metabolismo , Animales , Factor de Transcripción STAT3/metabolismo , Trampas Extracelulares/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Fosforilación , Ratones , Proteínas de la Membrana/metabolismo , Masculino , Neutrófilos/metabolismo , Proteínas Portadoras/metabolismo , Piruvato Quinasa/metabolismo , Hígado/metabolismo , Hígado/patología , Proteínas de Unión a Hormona Tiroide , Recurrencia Local de Neoplasia/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Humanos , Transducción de SeñalRESUMEN
BACKGROUND: Vascular endothelial damage is involved in the development and exacerbation of ventilator-induced lung injury (VILI). Pulmonary endothelial glycocalyx and neutrophil extracellular traps (NETs) are endothelial protective and damaging factors, respectively; however, their dynamics in VILI and the effects of recombinant thrombomodulin and antithrombin on these dynamics remain unclear. We hypothesized that glycocalyx degradation and NETs are induced by VILI and suppressed by recombinant thrombomodulin, recombinant antithrombin, or their combination. METHODS: VILI was induced in male C57BL/6J mice by intraperitoneal lipopolysaccharide injection (20 mg/kg) and high tidal volume ventilation (20 mL/kg). In the intervention groups, recombinant thrombomodulin, recombinant antithrombin, or their combination was administered at the start of mechanical ventilation. Glycocalyx degradation was quantified by measuring serum syndecan-1, fluorescence-labeled lectin intensity, and glycocalyx-occupied area in the pulmonary vascular lumen. Double-stranded DNA in the bronchoalveolar fluid and fluorescent areas of citrullinated histone H3 and myeloperoxidase were quantified as NET formation. RESULTS: Serum syndecan-1 increased, and lectin fluorescence intensity decreased in VILI. Electron microscopy revealed decreases in glycocalyx-occupied areas within pulmonary microvessels in VILI. Double-stranded DNA levels in the bronchoalveolar lavage fluid and the fluorescent area of citrullinated histone H3 and myeloperoxidase in lung tissues increased in VILI. Recombinant thrombomodulin, recombinant antithrombin, and their combination reduced glycocalyx injury and NET marker levels. There was little difference in glycocalyx injury and NET makers between the intervention groups. CONCLUSION: VILI induced glycocalyx degradation and NET formation. Recombinant thrombomodulin and recombinant antithrombin attenuated glycocalyx degradation and NETs in our VILI model. The effect of their combination did not differ from that of either drug alone. Recombinant thrombomodulin and antithrombin have the potential to be therapeutic agents for biotrauma in VILI.
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Antitrombinas , Endotoxemia , Trampas Extracelulares , Glicocálix , Ratones Endogámicos C57BL , Proteínas Recombinantes , Trombomodulina , Lesión Pulmonar Inducida por Ventilación Mecánica , Animales , Glicocálix/metabolismo , Glicocálix/efectos de los fármacos , Glicocálix/patología , Trombomodulina/metabolismo , Trombomodulina/administración & dosificación , Trampas Extracelulares/metabolismo , Trampas Extracelulares/efectos de los fármacos , Masculino , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Ratones , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/patología , Lesión Pulmonar Inducida por Ventilación Mecánica/tratamiento farmacológico , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & control , Endotoxemia/metabolismo , Endotoxemia/patología , Endotoxemia/tratamiento farmacológico , Endotoxemia/inducido químicamente , Antitrombinas/farmacología , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Pulmón/patología , Modelos Animales de Enfermedad , Sindecano-1/metabolismoRESUMEN
BACKGROUND: Exercise can promote sustainable protection against cold and warm liver ischemia-reperfusion injury (IRI) and tumor metastases. We have shown that this protection is by the induction of hepatic mitochondrial biogenesis pathway. In this study, we hypothesize that ZLN005, a PGC-1α activator, can be utilized as an alternative therapeutic strategy. METHODS: Eight-week-old mice were pretreated with ZLN005 and subjected to liver warm IRI. To establish a liver metastatic model, MC38 cancer cells (1 × 106) were injected into the spleen, followed by splenectomy and liver IRI. RESULTS: ZLN005-pretreated mice showed a significant decrease in IRI-induced tissue injury as measured by serum ALT/AST/LDH levels and tissue necrosis. ZLN005 pretreatment decreased ROS generation and cell apoptosis at the site of injury, with a significant decrease in serum pro-inflammatory cytokines, innate immune cells infiltration, and intrahepatic neutrophil extracellular trap (NET) formation. Moreover, mitochondrial mass was significantly upregulated in hepatocytes and maintained after IRI. This was confirmed in murine and human hepatocytes treated with ZLN005 in vitro under normoxic and hypoxic conditions. Additionally, ZLN005 preconditioning significantly attenuated tumor burden and increased the percentage of intratumoral cytotoxic T cells. CONCLUSIONS: Our study highlights the effective protection of ZLN005 pretreatment as a therapeutic alternative in terms of acute liver injury and tumor metastases.
Asunto(s)
Neoplasias Hepáticas , Hígado , Ratones Endogámicos C57BL , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Daño por Reperfusión , Animales , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Daño por Reperfusión/metabolismo , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/patología , Hígado/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Humanos , Masculino , Apoptosis/efectos de los fármacos , Progresión de la Enfermedad , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Línea Celular Tumoral , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Trampas Extracelulares/metabolismo , Trampas Extracelulares/efectos de los fármacosRESUMEN
Neutrophil extracellular traps (NETs) are three-dimensional reticular structures that release chromatin and cellular contents extracellularly upon neutrophil activation. As a novel effector mechanism of neutrophils, NETs possess the capacity to amplify localized inflammation and have been demonstrated to contribute to the exacerbation of various inflammatory diseases, including cardiovascular diseases and tumors. It is suggested that lysophosphatidylcholine (LPC), as the primary active component of oxidized low-density lipoprotein, represents a significant risk factor for various inflammatory diseases, such as cardiovascular diseases and neurodegenerative diseases. However, the specific mechanism of NETs formation induced by LPC remains unclear. Quercetin has garnered considerable attention due to its anti-inflammatory properties, serving as a prevalent flavonoid in daily diet. However, little is currently known about the underlying mechanisms by which quercetin inhibits NETs formation and alleviates associated diseases. In our study, we utilized LPC-treated primary rat neutrophils to establish an in vitro model of NETs formation, which was subsequently subjected to treatment with a combination of quercetin or relevant inhibitors/activators. Compared to the control group, the markers of NETs and the expression of P2X7R/P38MAPK/NOX2 pathway-associated proteins were significantly increased in cells treated with LPC alone. Quercetin intervention decreased the LPC-induced upregulation of the P2X7R/P38MAPK/NOX2 pathway and effectively reduced the expression of NETs markers. The results obtained using a P2X7R antagonist/activator and P38MAPK inhibitor/activator support these findings. In summary, quercetin reversed the upregulation of the LPC-induced P2X7R/P38MAPK/NOX2 pathway, further mitigating NETs formation. Our study investigated the potential mechanism of LPC-induced NETs formation, elucidated the inhibitory effect of quercetin on NETs formation, and offered new insights into the anti-inflammatory properties of quercetin.
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Trampas Extracelulares , Lisofosfatidilcolinas , NADPH Oxidasa 2 , Neutrófilos , Quercetina , Receptores Purinérgicos P2X7 , Proteínas Quinasas p38 Activadas por Mitógenos , Quercetina/farmacología , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacología , Trampas Extracelulares/metabolismo , Trampas Extracelulares/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Ratas , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Receptores Purinérgicos P2X7/metabolismo , NADPH Oxidasa 2/metabolismo , Transducción de Señal/efectos de los fármacos , MasculinoRESUMEN
Oxidative stress elicited by reactive oxygen species (ROS) and chronic inflammation are involved both in deterring and the generation/progression of human cancers. Exogenous ROS can injure mitochondria and induce them to generate more endogenous mitochondrial ROS to further perpetuate the deteriorating condition in the affected cells. Dysfunction of these cancer mitochondria may possibly be offset by the Warburg effect, which is characterized by amplified glycolysis and metabolic reprogramming. ROS from neutrophil extracellular traps (NETs) are an essential element for neutrophils to defend against invading pathogens or to kill cancer cells. A chronic inflammation typically includes consecutive NET activation and tissue damage, as well as tissue repair, and together with NETs, ROS would participate in both the destruction and progression of cancers. This review discusses human mitochondrial plasticity and the glucose metabolic reprogramming of cancer cells confronting oxidative stress by the means of chronic inflammation and neutrophil extracellular traps (NETs).
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Trampas Extracelulares , Glucosa , Inflamación , Mitocondrias , Neoplasias , Neutrófilos , Estrés Oxidativo , Especies Reactivas de Oxígeno , Humanos , Trampas Extracelulares/metabolismo , Inflamación/metabolismo , Inflamación/patología , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/inmunología , Mitocondrias/metabolismo , Glucosa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neutrófilos/metabolismoRESUMEN
The complement system and neutrophils play crucial roles in innate immunity. Neutrophils release neutrophil extracellular traps (NETs), which are composed of decondensed DNA entangled with granular contents, as part of their innate immune function. Mechanisms governing complement-mediated NET formation remain unclear. In this study, we tested a two-step NETosis mechanism, as follows: classical complement-mediated neutrophil activation in serum and subsequent NET formation in serum-free conditions, using neutrophils from healthy donors, endothelial cells, and various assays (Fluo-4AM, DHR123, and SYTOX), along with flow cytometry and confocal microscopy. Our findings reveal that classical complement activation on neutrophils upregulated the membrane-anchored complement regulators CD46, CD55, and CD59. Additionally, complement activation increased CD11b on neutrophils, signifying activation and promoting their attachment to endothelial cells. Complement activation induced calcium influx and citrullination of histone 3 (CitH3) in neutrophils. However, CitH3 formation alone was insufficient for NET generation. Importantly, NET formation occurred only when neutrophils were in serum-free conditions. In such environments, neutrophils induced NADPH oxidase-dependent reactive oxygen species (ROS) production, leading to NET formation. Hence, we propose that complement-mediated NET formation involves a two-step process, as follows: complement deposition, neutrophil priming, calcium influx, CitH3 formation, and attachment to endothelial cells in serum. This is followed by NADPH-dependent ROS production and NET completion in serum-free conditions. Understanding this process may unveil treatment targets for pathologies involving complement activation and NET formation.
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Calcio , Activación de Complemento , Trampas Extracelulares , NADPH Oxidasas , Activación Neutrófila , Neutrófilos , Especies Reactivas de Oxígeno , Trampas Extracelulares/metabolismo , Humanos , Neutrófilos/metabolismo , Neutrófilos/inmunología , NADPH Oxidasas/metabolismo , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Histonas/metabolismoRESUMEN
Non-alcoholic Fatty Liver Disease (NAFLD), noted for its widespread prevalence among adults, has become the leading chronic liver condition globally. Simultaneously, the annual disease burden, particularly liver cirrhosis caused by NAFLD, has increased significantly. Neutrophil Extracellular Traps (NETs) play a crucial role in the progression of this disease and are key to the pathogenesis of NAFLD. However, research into the specific roles of NETs-related genes in NAFLD is still a field requiring thorough investigation. Utilizing techniques like AddModuleScore, ssGSEA, and WGCNA, our team conducted gene screening to identify the genes linked to NETs in both single-cell and bulk transcriptomics. Using algorithms including Random Forest, Support Vector Machine, Least Absolute Shrinkage, and Selection Operator, we identified ZFP36L2 and PHLDA1 as key hub genes. The pivotal role of these genes in NAFLD diagnosis was confirmed using the training dataset GSE164760. This study identified 116 genes linked to NETs across single-cell and bulk transcriptomic analyses. These genes demonstrated enrichment in immune and metabolic pathways. Additionally, two NETs-related hub genes, PHLDA1 and ZFP36L2, were selected through machine learning for integration into a prognostic model. These hub genes play roles in inflammatory and metabolic processes. scRNA-seq results showed variations in cellular communication among cells with different expression patterns of these key genes. In conclusion, this study explored the molecular characteristics of NETs-associated genes in NAFLD. It identified two potential biomarkers and analyzed their roles in the hepatic microenvironment. These discoveries could aid in NAFLD diagnosis and management, with the ultimate goal of enhancing patient outcomes.
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Biomarcadores , Trampas Extracelulares , Aprendizaje Automático , Enfermedad del Hígado Graso no Alcohólico , Análisis de la Célula Individual , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Humanos , Análisis de la Célula Individual/métodos , Trampas Extracelulares/metabolismo , Biomarcadores/metabolismo , Neutrófilos/metabolismo , Transcriptoma , Perfilación de la Expresión GénicaRESUMEN
In end-stage heart failure, which is characterized by persistent or progressive ventricular dysfunction despite optimal medical therapy, a left ventricular assist device (LVAD) can be beneficial. Congestive heart failure provokes inflammatory and prothrombotic activation. The aim of this study was to evaluate the serum concentration of citrullinated histone 3 (CH3) representing neutrophil extracellular trap (NET) formation in patients referred for LVAD implantation. There were 10 patients with a median age of 61 (57-65) years enrolled in a prospective single-center analysis who underwent LVAD implantation. The CH3 plasma concentration was measured preoperatively and on the 1st and 7th postoperative days, followed by control measurements on the median (Q1-3) 88th (49-143) day. The preoperative CH3 concentration strongly correlated with brain natriuretic peptide (r = 0.879, p < 0.001). Significant differences in CH3 serum concentration were observed between pre- and postoperative measurements, including an increase on the first postoperative day (p < 0.001), as well as a decrease on the seventh day (p = 0.016) and in follow-up (p < 0.001). CH3 concentration, as a marker of NET formation, decreases after LVAD implantation.
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Trampas Extracelulares , Insuficiencia Cardíaca , Corazón Auxiliar , Histonas , Humanos , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/terapia , Trampas Extracelulares/metabolismo , Persona de Mediana Edad , Masculino , Femenino , Anciano , Histonas/sangre , Histonas/metabolismo , Estudios Prospectivos , Biomarcadores/sangre , Neutrófilos/metabolismo , CitrulinaciónRESUMEN
Neutrophilia occurs in patients infected with SARS-CoV-2 (COVID-19) and is predictive of poor outcomes. Here, we link heterogenous neutrophil populations to disease severity in COVID-19. We identified neutrophils with features of cellular aging and immunosuppressive capacity in mild COVID-19 and features of neutrophil immaturity and activation in severe disease. The low-density neutrophil (LDN) number in circulating blood correlated with COVID-19 severity. Many of the divergent neutrophil phenotypes in COVID-19 were overrepresented in the LDN fraction and were less detectable in normal-density neutrophils. Functionally, neutrophils from patients with severe COVID-19 displayed defects in neutrophil extracellular trap formation and reactive oxygen species production. Soluble factors secreted by neutrophils from these patients inhibited T cell proliferation. Neutrophils from patients with severe COVID-19 had increased expression of arginase-1 protein, a feature that was retained in convalescent patients. Despite this increase in intracellular expression, there was a reduction in arginase-1 release by neutrophils into serum and culture supernatants. Furthermore, neutrophil-mediated T cell suppression was independent of arginase-1. Our results indicate the presence of dysfunctional, activated, and immature neutrophils in severe COVID-19.
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Arginasa , COVID-19 , Neutrófilos , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Humanos , COVID-19/inmunología , COVID-19/sangre , Arginasa/metabolismo , Neutrófilos/metabolismo , Neutrófilos/inmunología , SARS-CoV-2/inmunología , Masculino , Persona de Mediana Edad , Femenino , Linfocitos T/inmunología , Linfocitos T/metabolismo , Anciano , Adulto , Trampas Extracelulares/metabolismo , Trampas Extracelulares/inmunología , Especies Reactivas de Oxígeno/metabolismo , Activación NeutrófilaRESUMEN
The inducers of neutrophil extracellular trap (NET) formation are heterogeneous and consequently, there is no specific pathway or signature molecule indispensable for NET formation. But certain events such as histone modification, chromatin decondensation, nuclear envelope breakdown, and NET release are ubiquitous. During NET formation, neutrophils drastically rearrange their cytoplasmic, granular and nuclear content. Yet, the exact mechanism for decoding each step during NET formation still remains elusive. Here, we investigated the mechanism of nuclear envelope breakdown during NET formation. Immunofluorescence microscopic evaluation revealed a gradual disintegration of outer nuclear membrane protein nesprin-1 and alterations in nuclear morphology during NET formation. MALDI-TOF analysis of NETs that had been generated by various inducers detected the accumulation of nesprin-1 fragments. This suggests that nesprin-1 degradation occurs before NET release. In the presence of a calpain-1, inhibitor nesprin-1 degradation was decreased in calcium driven NET formation. Microscopic evaluation confirmed that the disintegration of the lamin B receptor (LBR) and the collapse of the actin cytoskeleton occurs in early and later phases of NET release, respectively. We conclude that the calpain-1 degrades nesprin-1, orchestrates the weakening of the nuclear membrane, contributes to LBR disintegration, and promoting DNA release and finally, NETs formation.
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Calpaína , Trampas Extracelulares , Receptor de Lamina B , Neutrófilos , Membrana Nuclear , Membrana Nuclear/metabolismo , Calpaína/metabolismo , Humanos , Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Calcio/metabolismo , Proteínas del CitoesqueletoRESUMEN
Sickle cell disease (SCD) is the most common inherited monogenetic disorder. Chronic and acute pain are hallmark features of SCD involving neural and vascular injury and inflammation. Mast cells reside in the vicinity of nerve fibers and vasculature, but how they influence these structures remains unknown. We therefore examined the mechanism of mast cell activation in a sickle microenvironment replete with cell-free heme and inflammation. Mast cells exposed to this environment showed an explosion of nuclear contents with the release of citrullinated histones, suggestive of mast cell extracellular trap (MCET) release. MCETs interacted directly with the vasculature and nerve fibers, a cause of vascular and neural injury in sickle cell mice. MCET formation was dependent upon peptidylarginine deiminase 4 (PAD4). Inhibition of PAD4 ameliorated vasoocclusion, chronic and acute hyperalgesia, and inflammation in sickle mice. PAD4 activation may also underlie neutrophil trap formation in SCD, thus providing a novel target to treat the sequelae of vascular and neural injury in SCD.
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Anemia de Células Falciformes , Trampas Extracelulares , Hiperalgesia , Mastocitos , Arginina Deiminasa Proteína-Tipo 4 , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/metabolismo , Animales , Hiperalgesia/metabolismo , Hiperalgesia/etiología , Trampas Extracelulares/metabolismo , Ratones , Mastocitos/metabolismo , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Humanos , Masculino , Modelos Animales de Enfermedad , Inflamación/metabolismo , Ratones Endogámicos C57BL , Neutrófilos/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a chronic autoimmune disease with a complex etiology. Neutrophil extracellular traps (NETs are NETwork protein structures activated by neutrophils to induce the cleavage and release of DNA-protein complexes). Current studies have shown the critical involvement of NETs in the progression of autoimmune diseases, Neutrophils mostly gather in the inflammatory sites of patients and participate in the pathogenesis of autoimmune diseases in various ways. NETs, as the activated state of neutrophils, have attracted much attention in immune diseases. Many molecules released in NETs are targeted autoantigens in autoimmune diseases, such as histones, citrulline peptides, and myeloperoxidase. All of these suggest that NETs have a direct causal relationship between the production of autoantigens and autoimmune diseases. For RA in particular, as a disorder of the innate and adaptive immune response, the pathogenesis of RA is inseparable from the generation of RA. In this article, we investigate the emerging role of NETs in the pathogenesis of RA and suggest that NETs may be an important target for the treatment of inflammatory autoimmune diseases.
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Artritis Reumatoide , Progresión de la Enfermedad , Trampas Extracelulares , Neutrófilos , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Humanos , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/etiología , Artritis Reumatoide/patología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Animales , Autoantígenos/inmunologíaRESUMEN
Recurring lung injury, chronic inflammation, aberrant tissue repair and impaired tissue remodelling contribute to the pathogenesis of pulmonary fibrosis (PF). Neutrophil extracellular traps (NETs) are released by activated neutrophils to trap, immobilise and kill invading pathogen and is facilitated by peptidyl arginine deiminase-4 (PAD-4). Dysregulated NETs release and abnormal PAD-4 activation plays a crucial role in activating pro-fibrotic events in PF. Developmental endothelial locus-1 (Del-1), expressed by the endothelial cells of lungs and brain acts as an endogenous inhibitor of inflammation and fibrosis. We have hypothesised that PAD-4 inhibitor exerts anti-inflammatory and anti-fibrotic effects in mice model of PF. We have also hypothesised by PAD-4 regulated the transcription of Del-1 through co-repression and its inhibition potentiates anti-fibrotic effects of Del-1. In our study, the PAD-4 inhibitor chloro-amidine (CLA) demonstrated anti-NETotic and anti-inflammatory effects in vitro in differentiated HL-60 cells. In a bleomycin-induced PF mice model, CLA administration in two doses (3 mg/kg, I.P and 10 mg/kg, I.P) improved lung function, normalized bronchoalveolar lavage fluid parameters, and attenuated fibrotic events, including markers of extracellular matrix and epithelial-mesenchymal transition. Histological analyses confirmed the restoration of lung architecture and collagen deposition with CLA treatment. ELISA, IHC, IF, RT-PCR, and immunoblot analysis supported the anti-NETotic effects of CLA. Furthermore, BLM-induced PF reduced Del-1 and p53 expression, which was normalized by CLA treatment. These findings suggest that inhibition of PAD-4 results in amelioration of PF in animal model and may involve modulation of Del-1 and p53 pathways, warranting further investigation.
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Bleomicina , Arginina Deiminasa Proteína-Tipo 4 , Fibrosis Pulmonar , Animales , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Humanos , Arginina Deiminasa Proteína-Tipo 4/antagonistas & inhibidores , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Ratones , Trampas Extracelulares/efectos de los fármacos , Trampas Extracelulares/metabolismo , Células HL-60 , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Pulmón/patología , Pulmón/efectos de los fármacos , Masculino , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Ornitina/análogos & derivadosRESUMEN
Sepsis is a life-threatening condition caused by an excessive inflammatory response to an infection. However, the precise regulatory mechanism of sepsis remains unclear. Using a strand-specific RNA-sequencing, we identified 115 hub differentially expressed long noncoding RNAs (lncRNAs) and 443 mRNAs in septic patients, primarily participated in crucial pathways including neutrophil extracellular trap (NET) formation and toll-like receptor signaling. Notably, NETs related gene aquaporin-9 (AQP9) and its associated lncRNAs exhibited significant upregulation in septic neutrophils. Functional experiments revealed AQP9 interacts with its lncRNAs to augment the formation of neutrophil NETs. In murine sepsis models, AQP9 inhibition with phloretin reduced proinflammatory cytokine production and lung damage. These findings provide crucial insights into the regulatory role of AQP9 in sepsis, unraveling its interaction with associated lncRNAs in transmitting downstream signals, holding promise in informing the development of novel therapeutic strategies aimed at ameliorating the debilitating effects of sepsis.
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Acuaporinas , Trampas Extracelulares , Neutrófilos , ARN Largo no Codificante , ARN Mensajero , Sepsis , Sepsis/inmunología , Sepsis/genética , Sepsis/metabolismo , Trampas Extracelulares/metabolismo , Trampas Extracelulares/inmunología , Humanos , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Neutrófilos/inmunología , Ratones , Masculino , Acuaporinas/genética , Acuaporinas/metabolismo , Ratones Endogámicos C57BL , Citocinas/metabolismo , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Transducción de SeñalRESUMEN
Crohn's disease (CD) presents with diverse clinical phenotypes due to persistent inflammation of the gastrointestinal tract. Its global incidence is on the rise. Neutrophil extracellular traps (NETs) are networks released by neutrophils that capture microbicidal proteins and oxidases targeting pathogens. Research has shown that NETs are implicated in the pathogenesis of several immune-mediated diseases such as rheumatoid arthritis, systemic lupus erythematosus and inflammatory bowel disease. The goal of this study was to identify a panel of NET-related genes to construct a diagnostic and therapeutic model for CD. Through analysis of the GEO database, we identified 1950 differentially expressed genes (DEGs) associated with CD. Gene enrichment and immune cell infiltration analyses indicate that neutrophil infiltrates and chemokine-related pathways are predominantly involved in CD, with other immune cells such as CD4 and M1 macrophages also playing a role in disease progression. Utilizing weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) networks, we identified six hub genes (SPP1, SOCS3, TIMP1, IRF1, CXCL2 and CD274). To validate the accuracy of our model, we performed external validation with statistical differences(p < 0.05). Additionally, immunohistochemical experiments demonstrated higher protein expression of the hub genes in colonic tissues from CD patients compared to healthy subjects (p < 0.05). In summary, we identified six effective hub genes associated with NETs as potential diagnostic markers for CD. These markers not only offer targets for future research but also hold promise for the development of novel therapeutic interventions for CD.
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Biología Computacional , Enfermedad de Crohn , Trampas Extracelulares , Redes Reguladoras de Genes , Mapas de Interacción de Proteínas , Humanos , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Biología Computacional/métodos , Trampas Extracelulares/metabolismo , Trampas Extracelulares/genética , Mapas de Interacción de Proteínas/genética , Perfilación de la Expresión Génica , Neutrófilos/metabolismo , Neutrófilos/inmunología , Regulación de la Expresión Génica , Biomarcadores , Bases de Datos GenéticasRESUMEN
BACKGROUND: Hyperlipidemia is one of the main causes of aggravated hepatic ischemia-reperfusion injury (IRI). Simvastatin (SIM), a lipid-lowering drug, has been shown to effectively alleviate IRI caused by hyperlipidemia. However, the regulatory mechanism by which SIM alleviates hyperlipidemia-induced hepatic IRI is still not clear. This study aims to explore the potential mechanisms of SIM in inhibiting hyperlipidemia-induced hepatic IRI, providing new therapeutic strategies for the alleviation of hepatic IRI. METHODS: An animal model of hyperlipidemia was induced by feeding mice a high-fat diet for 8 weeks. Subsequently, a hepatic IRI animal model of hyperlipidemia was established by occluding the hepatic artery and portal vein for one hour, followed by reperfusion for 6 or 12 h. Enzyme linked immunosorbent assay, Western blotting, hematoxylin-eosin (H&E) staining, immunohistochemistry, immunofluorescence, and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling assay, were used to evaluate liver injury, neutrophil extracellular traps (NETs) formation, and related molecular mechanisms. RESULTS: Hepatic IRI was accelerated by hyperlipidemia, which enhanced the expression of oxidized low-density lipoprotein (oxLDL) and Macrophage-1antigen (Mac-1), leading to the promotion of NETs formation and apoptosis of liver cells. The administration of simvastatin reduced the levels of oxLDL and Mac-1, decreased the formation of NETs, and alleviated hepatic IRI induced by hyperlipidemia. CONCLUSIONS: Simvastatin reduced hyperlipidemia-induced hepatic IRI by inhibiting the formation of NETs through the regulation of the oxLDL/Mac-1 pathway.
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Dieta Alta en Grasa , Trampas Extracelulares , Hiperlipidemias , Hígado , Daño por Reperfusión , Simvastatina , Animales , Simvastatina/farmacología , Simvastatina/uso terapéutico , Daño por Reperfusión/prevención & control , Daño por Reperfusión/patología , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Ratones , Trampas Extracelulares/efectos de los fármacos , Trampas Extracelulares/metabolismo , Masculino , Dieta Alta en Grasa/efectos adversos , Hígado/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Hiperlipidemias/complicaciones , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Lipoproteínas LDL/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
Neutrophil extracellular trap formation has been identified as a new cell death mediator, termed NETosis, which is distinct from apoptosis and necrosis. NETs capture foreign substances, such as bacteria, by releasing DNA into the extracellular environment, and have been associated with inflammatory diseases and altered immune responses. Short-chain fatty acids, such as acetate, are produced by the gut microbiota and reportedly enhance innate immune responses; however, the underlying molecular mechanisms remain unclear. Here, we investigated the effects of sodium acetate, which has the highest SCFA concentration in the blood and gastrointestinal tract, on NETosis by focusing on the mechanisms associated with histone acetylation in neutrophil-like HL-60 cells. Sodium acetate enhanced NETosis, as shown by fluorescence staining with SYTOX green, and the effect was directly proportional to the treatment duration (16-24 h). Moreover, the addition of sodium acetate significantly enhanced the acetylation of Ace-H3, H3K9ace, and H3K14ace. Sodium acetate-induced histone acetylation rapidly decreased upon stimulation with the calcium ionophore A23187, whereas histone citrullination markedly increased. These results demonstrate that sodium acetate induces NETosis via histone acetylation in neutrophil-like HL-60 cells, providing new insights into the therapeutic effects based on the innate immunity-enhancing effect of dietary fiber.