RESUMEN
Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.
Asunto(s)
Proteínas Mitocondriales , Mitofagia , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte de ProteínasRESUMEN
TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex, SHARPIN, HOIP and HOIL-1, to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production, independently of tumor necrosis factor. Consistent with this, Traf1-/- mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling, providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP.
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Artritis Reumatoide/genética , Leucocitos Mononucleares/inmunología , Monocitos/inmunología , Transducción de Señal , Factor 1 Asociado a Receptor de TNF/metabolismo , Animales , Citocinas/metabolismo , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polimorfismo de Nucleótido Simple , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Factor 1 Asociado a Receptor de TNF/genética , Receptores Toll-Like/metabolismoRESUMEN
T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome, but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL, 4-1BBL, OX40L, and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40, CD25, and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation, revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion.
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Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Factores de Necrosis Tumoral/análisis , Animales , Ligando CD27/análisis , Receptor 1 de Quimiocinas CX3C/análisis , Células Dendríticas/inmunología , Femenino , Proteína Relacionada con TNFR Inducida por Glucocorticoide/análisis , Proteína Relacionada con TNFR Inducida por Glucocorticoide/fisiología , Glicoproteínas de Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Ligando OX40RESUMEN
Secretion of IL-1ß, a potent cytokine that plays a key role in gout pathogenesis, is regulated by inflammasomes. TRAF1 has been linked to heightened risk to inflammatory arthritis. In this article, we show that TRAF1 negatively regulates inflammasome activation to limit caspase-1 and IL-1ß secretion in human and mouse macrophages. TRAF1 reduces linear ubiquitination and subsequent oligomerization of the adapter protein, ASC. i.p. injection of monosodium urate crystals resulted in increased inflammatory cell infiltrates and IL-1ß production in Traf1 knockout mice compared with wild type littermates. In a model of monosodium urate crystal-induced gout, Traf1 knockout mice exhibited more swelling in the knee joints, increased infiltration of inflammatory cells, and higher expression of proinflammatory cytokines. In summary, this study identifies TRAF1 as a key regulator of IL-1ß production and a potential therapeutic target for inflammasome-driven diseases such as gout.
Asunto(s)
Gota , Inflamasomas , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales , Citocinas , Interleucina-1beta , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Factor 1 Asociado a Receptor de TNF/genética , Ácido ÚricoRESUMEN
Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.
Asunto(s)
Distrofia Muscular de Duchenne , Animales , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Adiponectina/genética , Modelos Animales de Enfermedad , Interleucina-6/metabolismo , Ratones Endogámicos C57BL , Peróxido de Hidrógeno/metabolismo , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Ratones Endogámicos DBA , Músculo Esquelético/metabolismo , Diafragma/metabolismo , Fibrosis , Inflamación/metabolismo , Progresión de la Enfermedad , Atrofia/metabolismo , Atrofia/patologíaRESUMEN
Inflammasomes are important in human health and disease, whereby they control the secretion of IL-1ß and IL-18, two potent proinflammatory cytokines that play a key role in inflammatory responses to pathogens and danger signals. Several inflammasomes have been discovered over the past two decades. NLRP3 inflammasome is the best characterized and can be activated by a wide variety of inducers. It is composed of a sensor, NLRP3, an adapter protein, ASC, and an effector enzyme, caspase-1. After activation, caspase-1 mediates the cleavage and secretion of bioactive IL-1ß and IL-18 via gasdermin-D pores in the plasma membrane. Aberrant activation of NLRP3 inflammasomes has been implicated in a multitude of human diseases, including inflammatory, autoimmune, and metabolic diseases. Therefore, several mechanisms have evolved to control their activity. In this review, we describe the posttranslational modifications that regulate NLRP3 inflammasome components, including ubiquitination, phosphorylation, and other forms of posttranslational modifications.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Procesamiento Proteico-Postraduccional/genética , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Caspasa 1/genética , Caspasa 1/metabolismo , Membrana Celular/metabolismo , Activación Enzimática/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas de Unión a Fosfato/metabolismo , Fosforilación/fisiología , Ubiquitinación/fisiologíaRESUMEN
Despite its importance in protecting the host from infections and injury, excessive inflammation may lead to serious human diseases including autoimmune disorders, cardiovascular diseases, diabetes, and cancer. Exercise is a known immunomodulator; however, whether exercise causes long-term changes in inflammatory responses and how these changes occur are lacking. Here, we show that chronic moderate-intensity training of mice leads to persistent metabolic rewiring and changes to chromatin accessibility in bone marrow-derived macrophages (BMDMs), which, in turn, tempers their inflammatory responses. We show that BMDMs from exercised mice exhibited a decrease in lipopolysaccharide (LPS)-induced NF-κB activation and proinflammatory gene expression along with an increase in M2-like-associated genes when compared with BMDMs from sedentary mice. This was associated with improved mitochondrial quality and increased reliance on oxidative phosphorylation accompanied with reduced mitochondrial reactive oxygen species (ROS) production. Mechanistically, assay for transposase-accessible chromatin (ATAC)-seq analysis showed changes in chromatin accessibility of genes associated with inflammatory and metabolic pathways. Overall, our data suggest that chronic moderate exercise can influence the inflammatory responses of macrophages by reprogramming their metabolic and epigenetic landscape.NEW & NOTEWORTHY In this study, we explain how long-term moderate exercise training can reduce inflammation in mouse macrophages by reprogramming the way they sense and respond to the presence of pathogens. We completed a thorough analysis and showed that these changes persist in macrophages because exercise improves the ability of cells to utilize oxygen without producing damaging compounds, and changes the way they access their DNA.
Asunto(s)
Macrófagos , Inmunidad Entrenada , Humanos , Animales , Ratones , Macrófagos/metabolismo , Inflamación/metabolismo , Transducción de Señal , Cromatina/metabolismo , Lipopolisacáridos/farmacologíaRESUMEN
NEW FINDINGS: What is the central question of this study? Can adiponectin receptor agonism improve recognition memory in a mouse model of Duchenne muscular dystrophy? What is the main finding and its importance? Short-term treatment with the new adiponectin receptor agonist ALY688 improves recognition memory in D2.mdx mice. This finding suggests that further investigation into adiponectin receptor agonism is warranted, given that there remains an unmet need for clinical approaches to treat this cognitive dysfunction in people with Duchenne muscular dystrophy. ABSTRACT: Memory impairments have been well documented in people with Duchenne muscular dystrophy (DMD). However, the underlying mechanisms are poorly understood, and there is an unmet need to develop new therapies to treat this condition. Using a novel object recognition test, we show that recognition memory impairments in D2.mdx mice are completely prevented by daily treatment with the new adiponectin receptor agonist ALY688 from day 7 to 28 of age. In comparison to age-matched wild-type mice, untreated D2.mdx mice demonstrated lower hippocampal mitochondrial respiration (carbohydrate substrate), greater serum interleukin-6 cytokine content and greater hippocampal total tau and Raptor protein contents. Each of these measures was partly or fully preserved after treatment with ALY688. Collectively, these results indicate that adiponectin receptor agonism improves recognition memory in young D2.mdx mice.
Asunto(s)
Distrofia Muscular de Duchenne , Animales , Ratones , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Ratones Endogámicos mdx , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/uso terapéutico , Adiponectina/metabolismo , Respiración , Modelos Animales de Enfermedad , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Músculo Esquelético/metabolismoRESUMEN
IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αß unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αß is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αß to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αß in the absence of Stat1. These studies reveal that IFN-αß is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.
Asunto(s)
Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/inmunología , Legionelosis/inmunología , Macrófagos/inmunología , Receptor de Interferón alfa y beta/inmunología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT2/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/microbiología , Células de la Médula Ósea/patología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Interferón gamma/genética , Interferón gamma/inmunología , Legionella pneumophila/inmunología , Legionelosis/genética , Legionelosis/microbiología , Legionelosis/patología , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de Células , Multimerización de Proteína , Receptor de Interferón alfa y beta/genética , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/deficiencia , Factor de Transcripción STAT2/genética , Transducción de Señal , Factores de TiempoRESUMEN
Statins have been shown to exert anti-inflammatory and anti-fibrogenic properties in the liver. In the present study, we explored the mechanisms underlying anti-fibrogenic effects of statins in isolated hepatic myofibroblasts and focused on cyclooxyegnase-2, a major anti-proliferative pathway in these cells. We show that simvastatin and fluvastatin inhibit thymidine incorporation in hMF in a dose-dependent manner. Pretreatment of cells with NS398, a COX-2 inhibitor, partially blunted this effect. cAMP levels, essential to the inhibition of hMF proliferation, were increased by statins and inhibited by non-steroidal anti-inflammatory drugs. Since statins modify prenylation of some important proteins in gene expression, we investigated the targets involved using selective inhibitors of prenyltransferases. Inhibition of geranylgeranylation resulted in the induction of COX-2 and mPGES-1. Using gel retardation assays, we further demonstrated that statins potentially activated the NFκB and CRE/E-box binding for COX-2 promoter and the binding of GC-rich regions and GATA for mPGES-1. Together these data demonstrate that statin limit hepatic myofibroblasts proliferation via a COX-2 and mPGES-1 dependent pathway. These data suggest that statin-dependent increase of prostaglandin in hMF contributes to its anti-fibrogenic effect.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Miofibroblastos/efectos de los fármacos , Prostaglandina-E Sintasas/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Fluvastatina , Factores de Transcripción GATA/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Hígado/citología , Miofibroblastos/citología , Miofibroblastos/metabolismo , FN-kappa B/metabolismo , Nitrobencenos/farmacología , Regiones Promotoras Genéticas/genética , Prostaglandina-E Sintasas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simvastatina/farmacología , Sulfonamidas/farmacologíaRESUMEN
The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1ß through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.
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Proteínas Portadoras/metabolismo , GMP Cíclico/análogos & derivados , Fosfatos de Dinucleósidos/farmacología , Inflamasomas/metabolismo , Animales , Calcio/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , GMP Cíclico/farmacología , Humanos , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The tumor necrosis factor receptor-associated factor 1 (TRAF1) plays a key role in promoting lymphocyte survival, proliferation, and cytokine production. Recent evidence showed that TRAF1 plays opposing roles in monocytes and macrophages where it controls NF-κB activation and limits pro-inflammatory cytokine production as well as inflammasome-dependent IL-1ß secretion. Importantly, TRAF1 polymorphisms have been strongly linked to an increased risk of rheumatoid arthritis (RA). However, whether and how TRAF1 contributes to RA pathogenesis is not fully understood. Moreover, investigating the role of TRAF1 in driving RA pathogenesis is complicated by its multifaceted and opposing roles in various immune cells. In this study, we subjected wildtype (WT) mice to the collagen antibody-induced arthritis (CAIA) model of RA and injected them intra-articularly with WT- or TRAF1-deficient macrophages. We show that mice injected with TRAF1-deficient macrophages exhibited significantly exacerbated joint inflammation, immune cell infiltration, and tissue damage compared to mice injected with WT macrophages. This study may lay the groundwork for novel therapies for RA that target TRAF1 in macrophages.
Asunto(s)
Artritis Reumatoide , Macrófagos , Factor 1 Asociado a Receptor de TNF , Animales , Factor 1 Asociado a Receptor de TNF/genética , Factor 1 Asociado a Receptor de TNF/metabolismo , Factor 1 Asociado a Receptor de TNF/deficiencia , Artritis Reumatoide/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Macrófagos/metabolismo , Ratones , Artritis Experimental/patología , Artritis Experimental/genética , Artritis Experimental/metabolismo , Artritis Experimental/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Nod-like receptors (NLRs) are innate immune receptors that play a key role in sensing components from pathogens and from damaged cells or organelles. NLRs form signaling complexes that can lead to activation of transcription factors or effector caspases - by means of inflammasome activation -Inflammatory arthritis (IA) culminating in promoting inflammation. An increasing body of research supports the role of NLRs in driving pathogenesis of IA, a collection of diseases that include rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis, and pediatric arthritis. In this review, we briefly discuss the main drivers of IA diseases and dive into the evidence for - and against - various NLRs in driving these diseases. We also review the studies examining the use of NLR and inflammasome inhibitors as potential therapies for IA.
Asunto(s)
Artritis Psoriásica , Artritis Reumatoide , Espondilitis Anquilosante , Humanos , Niño , Artritis Psoriásica/patología , Artritis Psoriásica/terapia , Inflamasomas , Proteínas NLRRESUMEN
The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-ß release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.
Asunto(s)
Adiponectina , Citocinas , Lipopolisacáridos , Ratones Endogámicos C57BL , Transducción de Señal , Animales , Adiponectina/sangre , Adiponectina/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Masculino , Citocinas/metabolismo , Citocinas/sangre , Bioensayo/métodos , Endotoxemia/inmunología , Endotoxemia/metabolismo , Receptores de Adiponectina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Modelos Animales de Enfermedad , FemeninoRESUMEN
Strategies to enhance autophagy flux have been suggested to improve outcomes in cardiac ischemic models. We explored the role of adiponectin in mediating cardiac autophagy under ischemic conditions induced by permanent coronary artery ligation. We studied the molecular mechanisms underlying adiponectin's cardio-protective effects in adiponectin knockout (Ad-KO) compared with wild-type (WT) mice subjected to ischemia by coronary artery ligation and H9c2 cardiomyocyte cell line exposed to hypoxia. Systemic infusion of a cathepsin-B activatable near-infrared probe as a biomarker for autophagy and detection via noninvasive three-dimensional fluorescence molecular tomography combined with computerized tomography to quantitate temporal changes, indicated increased activity in the myocardium of WT mice after myocardial infarction which was attenuated in Ad-KO. Seven days of ischemia increased myocardial adiponectin accumulation and elevated ULK1/AMPK phosphorylation and autophagy assessed by Western blotting for LC3 and p62, an outcome not observed in Ad-KO mice. Cell death, assessed by TUNEL analysis and the ratio of Bcl-2:Bax, plus cardiac dysfunction, measured using echocardiography with strain analysis, were exacerbated in Ad-KO mice. Using cellular models, we observed that adiponectin stimulated autophagy flux in isolated primary adult cardiomyocytes and increased basal and hypoxia-induced autophagy in H9c2 cells. Real-time temporal analysis of caspase-3/7 activation and caspase-3 Western blot indicated that adiponectin suppressed activation by hypoxia. Hypoxia-induced mitochondrial reactive oxygen species production and cell death were also attenuated by adiponectin. Importantly, the ability of adiponectin to reduce caspase-3/7 activation and cell death was not observed in autophagy-deficient cells generated by CRISPR-mediated deletion of Atg7. Collectively, our data indicate that adiponectin acts in an autophagy-dependent manner to attenuate cardiomyocyte caspase-3/7 activation and cell death in response to hypoxia in vitro and ischemia in mice.
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Adiponectina , Cardiopatías , Ratones , Animales , Adiponectina/genética , Adiponectina/metabolismo , Adiponectina/farmacología , Caspasa 3/metabolismo , Ratones Noqueados , Miocitos Cardíacos , Autofagia , Isquemia/metabolismo , Hipoxia , Cardiopatías/metabolismo , ApoptosisRESUMEN
Iron overload is associated with various pathological changes which contribute to metabolic syndrome, many of which have been proposed to occur via damaging tissue through an excessive amount of reactive oxygen species (ROS) production. In this study, we established a model of iron overload in L6 skeletal muscle cells and observed that iron enhanced cytochrome c release from depolarized mitochondria, assayed by immunofluorescent colocalization of cytochrome c with Tom20 and the use of JC-1, respectively. This subsequently elevated apoptosis, determined via use of a caspase-3/7 activatable fluorescent probe and western blotting for cleaved caspase-3. Using CellROX deep red and mBBr, we observed that iron increased generation of reactive oxygen species (ROS), and that pretreatment with the superoxide dismutase mimetic MnTBAP reduced ROS production and attenuated iron-induced intrinsic apoptosis and cell death. Furthermore, using MitoSox Red we observed that iron enhanced mROS and the mitochondria-targeted anti-oxidant SKQ1 reduced iron-induced ROS generation and cell death. Western blotting for LC3-II and P62 levels as well as immunofluorescent detection of autophagy flux with LC3B and P62 co-localization indicated that iron acutely (2-8 h) activated and later (12-24 h) attenuated autophagic flux. We used autophagy-deficient cell models generated by overexpressing a dominant-negative Atg5 mutant or CRISPR-mediated ATG7 knock out to test the functional significance of autophagy and observed that autophagy-deficiency exacerbated iron-induced ROS production and apoptosis. In conclusion, our study showed that high iron levels promoted ROS production, blunted the self-protective autophagy response and led to cell death in L6 skeletal muscle cells.
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Biopelículas , Sobrecarga de Hierro , Humanos , Especies Reactivas de Oxígeno/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Citocromos c , Reactores Biológicos , Autofagia , Apoptosis , Hierro , Músculo Esquelético/metabolismoRESUMEN
While inflammation is an important immune response for protection from infections, excessive or prolonged inflammation can lead to a variety of debilitating diseases including skin disease, diabetes, heart disease, stroke, autoimmune diseases and cancer. Inflammation is a graded response that is typically initiated when resident macrophages sense the presence of pathogens or damage in the tissue and produce inflammatory cytokines and chemokines to kill the pathogen, clear debris and dead tissue, and initiate tissue repair. Here we show that copper-infused fabrics can prevent inflammation by blocking the production of inflammatory cytokines from macrophages after being exposed to LPS, a component of bacterial cell wall. Mechanistically, we show that copper-infused fabrics can significantly reduce the NF-κB and IRF3 activation in LPS-stimulated macrophages. Given the importance of excessive inflammation in diabetes, we show that copper can reduce insulin resistance mediated by inflammatory cytokines in muscle cells. Our data show that copper infused fabrics may be useful to reduce excessive inflammation in macrophages and improve insulin sensitivity in skeletal muscles.
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Cobre , Resistencia a la Insulina , Humanos , Cobre/farmacología , Lipopolisacáridos , Citocinas , Inflamación , MacrófagosRESUMEN
Bacterial infections of the mucosal epithelium are a major cause of human disease. The prolonged presence of microbial pathogens stimulates inflammation of the local tissues, which leads to changes in the molecular composition of the extracellular milieu. A well-characterized molecule that is released to the extracellular milieu by stressed or infected cells is extracellular ATP and its ecto-enzymatic degradation products, which function as signaling molecules through ligation of purinergic receptors. There has been little information, however, on the effects of the extracellular metabolites on bacterial growth in inflamed tissues. Millimolar concentrations of ATP have been previously shown to inhibit irreversibly bacterial infection through ligation of P2X(7) receptors. We show here that the proinflammatory mediator, ATP, is released from Chlamydia trachomatis-infected epithelial cells. Moreover, further stimulation of the infected cells with micromolar extracellular ADP or ATP significantly impairs the growth of the bacteria, with a profile characteristic of the involvement of P2X(4) receptors. A specific role for P2X(4) was confirmed using cells overexpressing P2X(4). The chlamydiae remain viable and return to normal growth kinetics after removal of the extracellular stimulus, similar to responses previously described for persistence of chlamydial infection.
Asunto(s)
Adenosina Trifosfato/farmacología , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/patogenicidad , Células Epiteliales/microbiología , Receptores Purinérgicos P2X4/efectos de los fármacos , Receptores Purinérgicos P2X4/metabolismo , Adenosina Trifosfato/metabolismo , Chlamydia trachomatis/efectos de los fármacos , Células HEK293 , Células HeLa , HumanosRESUMEN
A growing body of work has recently highlighted the pivotal role of mitochondria in the initiation and modulation of inflammasome activation. Specifically, mitochondrial dysfunction can induce NLRP3 inflammasome activation, where loss of mitochondrial potential leads to production of reactive oxygen species (ROS) and release of Ca2+, which in turn trigger inflammasome assembly. Therefore, several measures of mitochondrial parameters and components are routinely utilized in studies assessing mechanisms of inflammasome activation. In this chapter, we show detailed protocols on how to employ flow cytometry using three distinct mitochondria-specific dyes to measure mitochondrial ROS (MitoSOX), mitochondrial respiration (Mitotracker deep red), and total mitochondria (Mitotracker green), as well as a dye that measures reduced glutathione (mBBr ).
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Biopelículas , Reactores Biológicos , Citometría de Flujo , Mitocondrias , Especies Reactivas de OxígenoRESUMEN
Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is an adaptor protein that is essential for the activation of several inflammasome complexes. Activation of inflammasomes leads to pathogenic clearance and inflammatory cell death called pyroptosis. Upon inflammasome activation, ASC oligomerization leads to the recruitment and activation of caspase-1, which in turn converts pro-inflammatory cytokines (e.g., pro-IL-1ß, pro-IL-18) to their mature active form. Given its central role in inflammasome activation, ASC oligomerization is used as an indicator of inflammasome activation. Here we describe how ASC oligomerization can be detected by Western blotting.