RESUMEN
Inflammation plays a crucial role in the development and progression of many diseases, and is often caused by dysregulation of signalling from pattern recognition receptors, such as TLRs. Inhibition of key protein-protein interactions is an attractive target for treating inflammation. Recently, we demonstrated that the signalling lymphocyte activation molecule family 1 (SLAMF1) positively regulates signalling downstream of TLR4 and identified the interaction interface between SLAMF1 and the TLR4 adaptor protein TRIF-related adapter molecule (TRAM). Based on these findings, we developed a SLAMF1-derived peptide, P7, which is linked to a cell-penetrating peptide for intracellular delivery. We found that P7 peptide inhibits the expression and secretion of IFNß and pro-inflammatory cytokines (TNF, IL-1ß, IL-6) induced by TLR4, and prevents death in mice subjected to LPS shock. The mechanism of action of P7 peptide is based on interference with several intracellular protein-protein interactions, including TRAM-SLAMF1, TRAM-Rab11FIP2, and TIRAP-MyD88 interactions. Overall, P7 peptide has a unique mode of action and demonstrates high efficacy in inhibiting TLR4-mediated signalling in vitro and in vivo.
Asunto(s)
Transducción de Señal , Receptor Toll-Like 4 , Animales , Ratones , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Péptidos/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , InflamaciónRESUMEN
Cytokine-mediated activation of host immunity is central to the control of pathogens. Interferon-gamma (IFNγ) is a key cytokine in protective immunity that induces major histocompatibility complex class II molecules (MHCII) to amplify CD4+ T cell activation and effector function. Despite its central role, the dynamic regulation of IFNγ-induced MHCII is not well understood. Using a genome-wide CRISPR-Cas9 screen in murine macrophages, we identified genes that control MHCII surface expression. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase three beta (GSK3ß) or the mediator complex subunit 16 (MED16). Both pathways control distinct aspects of the IFNγ response and are necessary for IFNγ-mediated induction of the MHCII transactivator Ciita, MHCII expression, and CD4+ T cell activation. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/metabolismo , Interferón gamma/metabolismo , Activación de Linfocitos/genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Antígenos de Histocompatibilidad Clase II/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Linfocitos T , Transactivadores/metabolismoRESUMEN
Receptor interacting protein kinase 1 (RIPK1) mediates cell death and inflammatory signaling and is increased in multiple sclerosis (MS) brain samples. Here, we investigate the role of glial RIPK1 kinase activity in mediating MS pathogenesis. We demonstrate RIPK1 levels correlate with MS disease progression. We find microglia are susceptible to RIPK1-mediated cell death and identify an inflammatory gene signature that may contribute to the neuroinflammatory milieu in MS patients. We uncover a distinct role for RIPK1 in astrocytes in regulating inflammatory signaling in the absence of cell death and confirm RIPK1-kinase-dependent regulation in human glia. Using a murine MS model, we show RIPK1 inhibition attenuates disease progression and suppresses deleterious signaling in astrocytes and microglia. Our results suggest RIPK1 kinase activation in microglia and astrocytes induces a detrimental neuroinflammatory program that contributes to the neurodegenerative environment in progressive MS.
Asunto(s)
Microglía/metabolismo , Esclerosis Múltiple/genética , Enfermedades Neuroinflamatorias/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Esclerosis Múltiple/patología , Transducción de SeñalRESUMEN
Caspase-8 is an apical caspase involved in the programmed form of cell death called apoptosis that is critically important for mammalian development and immunity. Apoptosis was historically described as immunologically silent in contrast to other types of programmed cell death such as necroptosis or pyroptosis. Recent reports suggest considerable crosstalk between these different forms of cell death. It is becoming increasingly clear that caspase-8 has many non-apoptotic roles, participating in multiple processes including regulation of necroptosis (mediated by receptor-interacting serine/threonine kinases, RIPK1-RIPK3), inflammatory cytokine expression, inflammasome activation, and cleavage of IL-1ß and gasdermin D, and protection against shock and microbial infection. In this review, we discuss the involvement of caspase-8 in cell death and inflammation and highlight its role in innate immune responses and in the relationship between different forms of cell death. Caspase-8 is one of the central components in this type of crosstalk.
Asunto(s)
Caspasa 8/inmunología , Muerte Celular/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Animales , HumanosRESUMEN
Hepatocyte cell death and liver inflammation have been well recognized as central characteristics of nonalcoholic steatohepatitis (NASH), however, the underlying molecular basis remains elusive. The kinase receptor-interacting protein 1 (RIP1) is a multitasking molecule with distinct functions in regulating apoptosis, necroptosis, and inflammation. Dissecting the role of RIP1 distinct functions in different pathophysiology has absorbed huge research enthusiasm. Wild-type and RIP1 kinase-dead (Rip1K45A/K45A) mice were fed with high-fat diet (HFD) to investigate the role of RIP1 kinase activity in the pathogenesis of NASH. Rip1K45A/K45A mice exhibited significantly alleviated NASH phenotype of hepatic steatosis, liver damage, fibrosis as well as reduced hepatic cell death and inflammation compared to WT mice. Our results also indicated that both in vivo lipotoxicity and in vitro saturated fatty acids (palmitic acid) treatment were able to induce the kinase activation of RIP1 in liver macrophages. RIP1 kinase was required for mediating inflammasome activation, apoptotic and necrotic cell death induced by palmitic acid in both bone marrow-derived macrophage and mouse primary Kupffer cells. Results from chimeric mice established through lethal irradiation and bone marrow transplantation further confirmed that the RIP1 kinase in hematopoietic-derived macrophages contributed mostly to the disease progression in NASH. Consistent with murine models, we also found that RIP1 kinase was markedly activated in human NASH, and the kinase activation mainly occurred in liver macrophages as indicated by immunofluorescence double staining. In summary, our study indicated that RIP1 kinase was phosphorylated and activated mainly in liver macrophages in both experimental and clinical NASH. We provided direct genetic evidence that the kinase activity of RIP1 especially in hematopoietic-derived macrophages contributes to the pathogenesis of NASH, through mediating inflammasome activation and cell death induction. Macrophage RIP1 kinase represents a specific and potential therapeutic target for NASH.
Asunto(s)
Muerte Celular/fisiología , Inflamación/metabolismo , Macrófagos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1ß (IL-1ß) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1ß conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.
Asunto(s)
Histona Desacetilasa 6/metabolismo , Vigilancia Inmunológica , Inflamasomas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Pirina/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Histona Desacetilasa 6/genética , Humanos , Inflamasomas/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Transporte de ProteínasRESUMEN
BACKGROUND: Activation of NLRP3 in liver macrophages contributes to alcohol-associated liver disease (ALD). Molecular chaperone heat shock protein (HSP) 90 facilitates NLRP3 inflammasome activity during infections and inflammatory diseases. We previously reported that HSP90 is induced in ALD and regulates proinflammatory cytokines, tumor necrosis factor alpha, and IL-6. Whether HSP90 affects IL-1ß and IL-18 regulated by NLRP3 inflammasome in ALD is unknown. Here, we hypothesize that HSP90 modulated NLRP3 inflammasome activity and affects IL-1ß and IL-18 secretion in ALD. METHODS: The expression of HSP90AA1 and NLRP3 inflammasome genes was evaluated in human alcoholic livers and in mouse model of ALD. The importance of HSP90 on NLRP3 inflammasome activation in ALD was evaluated by administering HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) to mice subjected to ALD, and in vitro to bone marrow-derived macrophages (BMDM) stimulated with LPS and ATP. The effect of activation of HSF1/HSPA1A axis during HSP90 inhibition or direct activation during heat shock of BMDMs on NLRP3 activity and secretion of downstream cytokines was evaluated. RESULTS: We found positive correlation between induction of HSP90 and NLRP3 inflammasome genes in human alcoholic cirrhotic livers. Administration of 17-DMAG in mouse model of ALD significantly down-regulated NLRP3 inflammasome-mediated caspase-1 (CASP-1) activity and cytokine secretion, with reduction in ALD. 17-DMAG-mediated decrease in NLRP3 was restricted to liver macrophages. Using BMDMs, we show that inhibition of HSP90 prevented CASP-1 activity, and Gasdermin D (GSDMD) cleavage, important in release of active IL-1ß and IL-18. Interestingly, activation of the heat shock factor 1 (HSF1)/HSPA1A axis, either during HSP90 inhibition or by heat shock, decreased NLRP3 inflammasome activity and reduced secretion of cytokines. CONCLUSION: Our studies indicate that inhibition of HSP90 and activation of HSF1/HSPA1A reduce IL-1ß and IL-18 via decrease in NLRP3/CASP-1 and GSDMD activity in ALD.
Asunto(s)
Hepatopatías Alcohólicas/genética , Adulto , Anciano , Animales , Benzoquinonas/farmacología , Caspasa 1/efectos de los fármacos , Caspasa 1/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Técnicas In Vitro , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lactamas Macrocíclicas/farmacología , Cirrosis Hepática Alcohólica/genética , Cirrosis Hepática Alcohólica/metabolismo , Hepatopatías Alcohólicas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas de Neoplasias , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
The gasdermins are a family of pore-forming proteins recently implicated in the immune response. One of these proteins, gasdermin D (GSDMD), has been identified as the executioner of pyroptosis, an inflammatory form of lytic cell death that is induced upon formation of caspase-1-activating inflammasomes. The related proteins GSDME and GSDMA have also been implicated in autoimmune diseases and certain cancers. Most gasdermin proteins are believed to have pore-forming capabilities. The best-studied member, GSDMD, controls the release of the proinflammatory cytokines IL-1ß and IL-18 and pyroptotic cell death. Because of its potential as a driver of inflammation in septic shock and autoimmune diseases, GSDMD represents an attractive drug target. In this review, we discuss the gasdermin proteins with particular emphasis on GSDMD and its mechanism of action and biological significance.
Asunto(s)
Inmunidad/inmunología , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de Unión a Fosfato/inmunología , Piroptosis/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Caspasa 1/inmunología , Caspasa 1/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismoRESUMEN
Group A Streptococcus (GAS) is a common and versatile human pathogen causing a variety of diseases. One of the many virulence factors of GAS is the secreted pore-forming cytotoxin streptolysin O (SLO), which has been ascribed multiple properties, including inflammasome activation leading to release of the potent inflammatory cytokine IL-1ß from infected macrophages. IL-1ß is synthesized as an inactive pro-form, which is activated intracellularly through proteolytic cleavage. Here, we use a macrophage infection model to show that SLO specifically induces ubiquitination and degradation of pro-IL-1ß. Ubiquitination was dependent on SLO being released from the infecting bacterium, and pore formation by SLO was required but not sufficient for the induction of ubiquitination. Our data provide evidence for a novel SLO-mediated mechanism of immune regulation, emphasizing the importance of this pore-forming toxin in bacterial virulence and pathogenesis.
Asunto(s)
Interleucina-1beta/metabolismo , Macrófagos/inmunología , Infecciones Estreptocócicas/metabolismo , Streptococcus pyogenes/fisiología , Estreptolisinas/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Células Cultivadas , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/genética , Macrófagos/microbiología , Ratones , Ratones Noqueados , Proteolisis , UbiquitinaciónRESUMEN
Early detection of microbial patterns is a hallmark of innate immunity and essential for clearance of invading pathogens. A recent Nature publication by Zhou et al. (2018) has uncovered ALPK1 as a pattern recognition receptor for Gram-negative bacteria triggering NF-κB activation and identified the bacterial sugar ADP-Hep as its ligand.
Asunto(s)
Inmunidad Innata/inmunología , Proteínas Quinasas/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Azúcares/inmunología , Animales , Proteínas Portadoras , Bacterias Gramnegativas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Inflamación , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Proteínas Quinasas/genética , Transducción de Señal/fisiología , Sistemas de Secreción Tipo IIIRESUMEN
Limited proteolysis of gasdermin D (GSDMD) generates an N-terminal pore-forming fragment that controls pyroptosis in macrophages. GSDMD is processed via inflammasome-activated caspase-1 or -11. It is currently unknown whether macrophage GSDMD can be processed by other mechanisms. Here, we describe an additional pathway controlling GSDMD processing. The inhibition of TAK1 or IκB kinase (IKK) by the Yersinia effector protein YopJ elicits RIPK1- and caspase-8-dependent cleavage of GSDMD, which subsequently results in cell death. GSDMD processing also contributes to the NLRP3 inflammasome-dependent release of interleukin-1ß (IL-1ß). Thus, caspase-8 acts as a regulator of GSDMD-driven cell death. Furthermore, this study establishes the importance of TAK1 and IKK activity in the control of GSDMD cleavage and cytotoxicity.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 8/metabolismo , Interacciones Huésped-Patógeno , Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Peste/inmunología , Animales , Proteínas Bacterianas/metabolismo , Caspasa 8/genética , Muerte Celular , Humanos , Inflamasomas/inmunología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas de Unión a Fosfato , Peste/enzimología , Peste/patología , ProteolisisRESUMEN
The inflammatory potential of 12 types of alginate-based microspheres was assessed in a human whole blood model. The inflammatory potential could be categorized from low to high based on the four main alginate microsphere types; alginate microbeads, liquefied core poly-l-ornithine (PLO)-containing microcapsules, liquefied core poly-l-lysine (PLL)-containing microcapsules, and solid core PLL-containing microcapsules. No complement or inflammatory cytokine activation was detected for the Ca/Ba alginate microbeads. Liquefied core PLO- and PLL-containing microcapsules induced significant fluid phase complement activation (TCC), but with low complement surface deposition (anti-C3c), and a low proinflammatory cytokine secretion, with exception of an elevated MCP-1(CCL2) secretion. The solid core PLL-containing microcapsules generated lower TCC but a marked complement surface deposition and significant induction of the proinflammatory cytokines interleukin (IL-1)ß, TNF, IL-6, the chemokines IL-8 (CXCL8), and MIP-1α (CCL3) and MCP-1(CCL2). Inhibition with compstatin (C3 inhibitor) completely abolished complement surface deposition, leukocyte adhesion and the proinflammatory cytokines. The C5 inhibitions partly lead to a reduction of the proinflammatory cytokines. The leukocyte adhesion was abolished by inhibitory antibodies against CD18 and partly reduced by CD11b, but not by CD11c. Anti-CD18 significantly reduced the (IL-1)ß, TNF, IL-6 and MIP-1α and anti-CD11b significantly reduced the IL-6 and VEGF secretion. MCP-1 was strongly activated by anti-CD18 and anti-CD11b. In conclusion the initial proinflammatory cytokine responses are driven by the microspheres potential to trigger complement C3 (C3b/iC3b) deposition, leukocyte activation and binding through complement receptor CR3 (CD11b/CD18). MCP-1 is one exception dependent on the fluid phase complement activation mediated through CR3.