RESUMO
Pyroptosis is a proinflammatory mode of lytic cell death mediated by accumulation of plasma membrane (PM) macropores composed of gasdermin-family (GSDM) proteins. It facilitates two major functions in innate immunity: (i) elimination of intracellular replicative niches for pathogenic bacteria; and (ii) non-classical secretion of IL-1 family cytokines that amplify host-beneficial inflammatory responses to microbial infection or tissue damage. Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL-1ß release during inflammasome signaling have been extensively characterized in macrophages. This involves cleavage of GSDMD by caspase-1 to generate GSDMD macropores that mediate IL-1ß efflux and progression to pyroptotic lysis. Neutrophils, which rapidly accumulate in large numbers at sites of tissue infection or damage, become the predominant local source of IL-1ß in coordination with their potent microbiocidal capacity. Similar to macrophages, neutrophils express GSDMD and utilize the same spectrum of diverse inflammasome platforms for caspase-1-mediated cleavage of GSDMD. Distinct from macrophages, neutrophils possess a remarkable capacity to resist progression to GSDMD-dependent pyroptotic lysis to preserve their viability for efficient microbial killing while maintaining GSDMD-dependent mechanisms for export of bioactive IL-1ß. Rather, neutrophils employ cell-specific mechanisms to conditionally engage GSDMD-mediated pyroptosis in response to bacterial pathogens that use neutrophils as replicative niches. GSDMD and pyroptosis have also been mechanistically linked to induction of NETosis, a signature neutrophil pathway that expels decondensed nuclear DNA into extracellular compartments for immobilization and killing of microbial pathogens. This review summarizes a rapidly growing number of recent studies that have produced new insights, unexpected mechanistic nuances, and some controversies regarding the regulation of, and roles for, neutrophil inflammasomes, pyroptosis, and GSDMs in diverse innate immune responses.
Assuntos
Inflamassomos , Piroptose , Humanos , Piroptose/fisiologia , Inflamassomos/metabolismo , Neutrófilos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Gasderminas , Caspase 1/metabolismo , Transdução de SinaisRESUMO
When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.
Assuntos
Gasderminas , Piroptose , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Membrana Celular/metabolismo , Inflamassomos/metabolismo , Engenharia de ProteínasRESUMO
Pyroptosis is a mechanism of programmed, necrotic cell death mediated by gasdermins, a family of pore-forming proteins. Caspase-1 activates gasdermin D (GSDMD) under inflammatory conditions, whereas caspase-3 activates GSDME under apoptotic conditions, such as those induced by chemotherapy. These pathways are thought to be separate. However, we found that they are part of an integrated network of gatekeepers that enables pyroptotic cell death. We observed that GSDMD was the primary pyroptotic mediator in cultured blood cells in response to doxorubicin and etoposide, two common chemotherapies for hematopoietic malignancies. Upon treatment, the channel protein pannexin-1 (PANX1), which is stimulated by the initiation of apoptosis, increased membrane permeability to induce K+ efflux-driven activation of the NLRP3 inflammasome and GSDMD. However, either PANX1 or GSDME could also be the primary mediator of chemotherapy-induced pyroptosis when present at higher amounts. The most abundant pore-forming protein in acute myeloid leukemias from patients predicted the cell death pathway in response to chemotherapy. This interconnected network, a multistep switch that converts apoptosis to pyroptosis, could be clinically titratated to modulate cell death with regard to antitumor immunity or tumor lysis syndrome in patients.
Assuntos
Antineoplásicos , Neoplasias Hematológicas , Humanos , Gasderminas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Apoptose , Necrose , Inflamassomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Conexinas/genética , Conexinas/metabolismoRESUMO
Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1ß. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1ß-containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro-IL-1ß, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1ß colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63+ sEVs containing IL-1ß, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1ß-containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1ß sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.
Assuntos
Colite/metabolismo , Células Epiteliais/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Animais , Linhagem Celular , Colite/genética , Colite/patologia , Células Epiteliais/patologia , Exossomos/genética , Exossomos/metabolismo , Exossomos/patologia , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Interleucina-1beta/genética , Mucosa Intestinal/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas de Ligação a Fosfato/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismoRESUMO
Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1ß release. In contrast, we report that although N-GSDMD is required for IL-1ß secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3+ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1ß via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.
Assuntos
Membrana Celular/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neutrófilos/metabolismo , Organelas/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/genética , Caspase 1/metabolismo , Permeabilidade da Membrana Celular/genética , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Transporte Proteico , Piroptose/genéticaRESUMO
The efficacy of cancer chemotherapy is enhanced by induction of sustainable anti-tumor immune responses. Such responses involve accumulation of immunogenic mediators, such as extracellular ATP and ATP metabolites, within the tumor microenvironment. Recent studies have identified nucleotide-permeable plasma membrane channels or pores that are activated as early downstream consequences of different regulated cell death pathways: pannexin-1 channels in apoptosis, MLKL pores in necroptosis, and gasdermin-family pores in pyroptosis. This chapter describes the use of highly quantitative and semi-high-throughput methods based on the ATP sensor luciferase to measure dynamic changes in extracellular ATP, ADP, and AMP in tissue/cell culture models of cancer cells during various modes of regulated cell death in response to chemotherapeutic drugs, death receptors, or metabolic perturbation.
Assuntos
Trifosfato de Adenosina/análise , Luciferases/química , Neoplasias/tratamento farmacológico , Difosfato de Adenosina/análise , Difosfato de Adenosina/imunologia , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/análise , Monofosfato de Adenosina/imunologia , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/imunologia , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Humanos , Morte Celular Imunogênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Cultura Primária de Células , Piroptose/efeitos dos fármacos , Piroptose/imunologia , RatosRESUMO
Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40+ endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.
Assuntos
Encéfalo/parasitologia , Antígenos CD40/fisiologia , Células Endoteliais/imunologia , Retina/parasitologia , Toxoplasma/patogenicidade , Animais , Autofagia , Movimento Celular , Proteínas de Choque Térmico HSP70/fisiologia , Leucócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Orthopaedic wear particles activate the NLRP3 inflammasome to produce active interleukin 1ß (IL1ß). However, the NLRP3 inflammasome must be primed before it can be activated, and it is unknown whether wear particles induce priming. Toll-like receptors (TLRs) are thought to mediate particle bioactivity. It remains controversial whether pathogen-associated molecular patterns (PAMPs) and/or alarmins are responsible for TLR activation by wear particles. QUESTIONS/PURPOSES: (1) Does priming of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? (2) Does priming of the NLRP3 inflammasome by wear particles depend on TLRs and TIRAP/Mal? (3) Does priming of the NLRP3 inflammasome by wear particles depend on cognate TLRs? (4) Does activation of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? METHODS: Immortalized murine macrophages were stimulated by as-received titanium particles with adherent bacterial debris, endotoxin-free titanium particles, or titanium particles with adherent ultrapure lipopolysaccharide. To study priming, NLRP3 and IL1ß mRNA and IL1ß protein levels were assessed in wild-type, TLR4, TLR2, and TIRAP/Mal macrophages. To study activation, IL1ß protein secretion was assessed in wild-type macrophages preprimed with ultrapure lipopolysaccharide. RESULTS: Compared with titanium particles with adherent bacterial debris, endotoxin-free titanium particles induced 86% less NLRP3 mRNA (0.05 ± 0.03 versus 0.35 ± 0.01 NLRP3/GAPDH, p < 0.001) and 91% less IL1ß mRNA (0.02 ± 0.01 versus 0.22 ± 0.03 IL1ß/GAPDH, p < 0.001). ProIL1ß protein level was robustly increased in wild-type macrophages stimulated by particles with adherent PAMPs but was not detectably produced in macrophages stimulated by endotoxin-free particles. Adherence of ultrapure lipopolysaccharide to endotoxin-free particles reconstituted stimulation of NLRP3 and IL1ß mRNA. Particles with adherent bacterial debris induced 79% less NLRP3 mRNA (0.09 ± 0.004 versus 0.43 ± 0.13 NLRP3/GAPDH, p < 0.001) and 40% less IL1ß mRNA (0.09 ± 0.04 versus 0.15 ± 0.03 IL1ß/GAPDH, p = 0.005) in TLR4 macrophages than in wild-type. Similarly, those particles induced 49% less NLRP3 mRNA (0.22 ± 0.10 versus 0.43 ± 0.13 NLRP3/GAPDH, p = 0.004) and 47% less IL1ß mRNA (0.08 ± 0.02 versus 0.15 ± 0.03 IL1ß/GAPDH, p = 0.012) in TIRAP/Mal macrophages than in wild-type. Particles with adherent ultrapure lipopolysaccharide induced 96% less NLRP3 mRNA (0.012 ± 0.001 versus 0.27 ± 0.05 NLRP3/GAPDH, p = 0.003) and 91% less IL1ß mRNA (0.03 ± 0.01 versus 0.34 ± 0.07 IL1ß/GAPDH, p < 0.001) expression in TLR4 macrophages than in wild-type. In contrast, those particles did not induce less NLRP3 and IL1ß mRNA in TLR2 macrophages. IL1ß protein secretion was equivalently induced by particles with adherent bacterial debris or by endotoxin-free particles in a time-dependent manner in wild-type macrophages. For example, particles with adherent bacterial debris induced 99% ± 2% of maximal IL1ß secretion after 12 hours, whereas endotoxin-free particles induced 92% ± 11% (p > 0.5). CONCLUSIONS: This cell culture study showed that adherent PAMPs are required for priming of the NLRP3 inflammasome by wear particles and this process is dependent on their cognate TLRs and TIRAP/Mal. In contrast, activation of the NLRP3 inflammasome by titanium particles is not dependent on adherent PAMPs. Animal and implant retrieval studies are needed to determine whether wear particles have similar effects on the NLRP3 inflammasome in vivo. CLINICAL RELEVANCE: Our findings, together with recent findings that aseptic loosening associates with polymorphisms in the TIRAP/Mal locus, support that adherent PAMPs may contribute to aseptic loosening in patients undergoing arthroplasty.
Assuntos
Apresentação Cruzada/efeitos dos fármacos , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Moléculas com Motivos Associados a Patógenos/metabolismo , Titânio/farmacologia , Receptores Toll-Like/metabolismo , Animais , Interleucina-1beta/metabolismo , CamundongosRESUMO
Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1ß, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.
Assuntos
Acrilamidas/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Sulfonamidas/farmacologia , Acrilamidas/uso terapêutico , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Citocinas/genética , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas de Ligação a Fosfato , Pirina/fisiologia , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/imunologia , Salmonella typhimurium , Sepse/tratamento farmacológico , Sepse/imunologia , Sulfonamidas/uso terapêutico , Células THP-1RESUMO
The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1ß release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme-inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.
Assuntos
Proteínas Reguladoras de Apoptose/química , Inibidores de Caspase/química , Proteínas de Neoplasias/química , Peptídeos/química , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/química , Caspase 3/metabolismo , Inibidores de Caspase/metabolismo , Domínio Catalítico , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Camundongos , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Proteínas de Ligação a Fosfato , Estrutura Secundária de Proteína , Células RAW 264.7 , Células THP-1RESUMO
Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The molecular mechanisms of autoinhibition for GSDMD are poorly characterized. Here we report the crystal structures of the human and murine GSDMD C-terminal domains, which differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis. Our results suggest that GSDMDs may employ a distinct mode of intramolecular domain interaction and autoinhibition, which may be relevant to its unique role in pyroptosis downstream of inflammasome activation.
Assuntos
Proteínas Reguladoras de Apoptose/química , Mutação , Proteínas de Neoplasias/química , Animais , Proteínas Reguladoras de Apoptose/genética , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Moleculares , Proteínas de Neoplasias/genética , Proteínas de Ligação a Fosfato , Domínios Proteicos , Estrutura Secundária de Proteína , PiroptoseRESUMO
Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12-/-) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor-mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMß2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12-/- mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12-/- hosts was sufficient to correct the neutrophil migration defect in F12-/- mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.
Assuntos
Fator XII/metabolismo , Neutrófilos/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Cicatrização , Animais , Cálcio/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Armadilhas Extracelulares , Feminino , Humanos , Inflamação , Leucócitos/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peritonite/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.
Assuntos
Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Caspases/metabolismo , Inflamassomos/metabolismo , Macrófagos/citologia , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Piroptose , Substituição de Aminoácidos , Animais , Linhagem Celular Transformada , Células HEK293 , Humanos , Inflamassomos/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microscopia de Fluorescência , Microscopia de Vídeo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a Fosfato , Mutação Puntual , Multimerização Proteica , Transporte Proteico , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor-α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and αß-methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels drives accumulation of immunostimulatory ATP versus immunosuppressive adenosine within the tumor microenvironment.
Assuntos
Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Antineoplásicos/toxicidade , Conexinas/metabolismo , Proteína de Domínio de Morte Associada a Fas/deficiência , Proteínas do Tecido Nervoso/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Humanos , Células Jurkat , Proteínas de Ligação a RNA , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohn's disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.
Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Transtornos Linfoproliferativos/metabolismo , Mutação , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Linhagem Celular , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/patologia , Proteína Adaptadora de Sinalização NOD2/genética , Domínios Proteicos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
Müller cells and macrophages/microglia are likely important for the development of diabetic retinopathy; however, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger proinflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated retinal tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), intracellular adhesion molecule 1 (ICAM-1), and nitric oxide synthase (NOS2), developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1ß secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40+ Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C-dependent ATP release that caused P2X7-dependent production of TNF-α and IL-1ß by macrophages. P2X7-/- mice and mice treated with a P2X7 inhibitor were protected from diabetes-induced TNF-α, IL-1ß, ICAM-1, and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy and that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X7 pathway.
Assuntos
Antígenos CD40/imunologia , Citocinas/imunologia , Diabetes Mellitus Experimental/imunologia , Retinopatia Diabética/imunologia , Células Ependimogliais/imunologia , Macrófagos/imunologia , Microglia/imunologia , Receptores Purinérgicos P2X7/imunologia , Animais , Antígenos CD40/genética , Capilares , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/etiologia , Inflamação , Molécula 1 de Adesão Intercelular/imunologia , Interleucina-1beta/imunologia , Leucostasia/imunologia , Masculino , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fosfolipases Tipo C/imunologiaRESUMO
Purpose: Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox. Methods: Human RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry. Results: Ligation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1ß secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7-mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner. Conclusions: Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.
Assuntos
Apoptose , Ligante de CD40/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Diabetes Mellitus Experimental , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Endoteliais/patologia , Ensaio de Imunoadsorção Enzimática , Células Ependimogliais/patologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas do Receptor Purinérgico P2X/farmacologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P2X7/metabolismo , Retina/metabolismo , Retina/patologia , Tetrazóis/farmacologiaRESUMO
Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd)-dependent lytic cell death called pyroptosis that promotes antimicrobial host defense but may contribute to sepsis. The nature of the caspase-1-dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium(2+) (Pro(2+)) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDMs) as an indicator of this PM permeabilization. BMDMs were characterized by rapid Pro(2+) influx after initiation of NLRP3 or Pyrin inflammasomes by nigericin (NG) or Clostridium difficile toxin B (TcdB), respectively. No Pro(2+) uptake in response to NG or TcdB was observed in Casp1(-/-) or Asc(-/-) BMDMs. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro(2+) influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro(2+) influx and pyroptotic lysis. Extracellular La(3+) and Gd(3+) rapidly and reversibly blocked the induced Pro(2+) influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome assembly and caspase-1 activation. Thus, caspase-1-driven pyroptosis requires induction of initial prelytic pores in the PM that are dependent on Gsdmd expression. These PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca(2+) Although lanthanides and Gsdmd deletion both suppressed PM pore activity and pyroptotic lysis, robust IL-1ß release was observed in lanthanide-treated BMDMs but not in Gsdmd-deficient cells. This suggests roles for Gsdmd in both passive IL-1ß release secondary to pyroptotic lysis and in nonlytic/nonclassical IL-1ß export.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspase 1/metabolismo , Piroptose/fisiologia , Animais , Membrana Celular/patologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Elementos da Série dos Lantanídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a FosfatoRESUMO
Interleukin 1ß (IL-1ß) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1ß during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1ß production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1ß, whereas ATP stimulation triggered T cell production of IL-1ß via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1ß. Together these data reveal a critical role for IL-1ß produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Células Th17/imunologia , Trifosfato de Adenosina/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Caspase 8/genética , Caspase 8/imunologia , Caspase 8/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Citometria de Fluxo , Expressão Gênica/imunologia , Immunoblotting , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
Although extracellular ATP is abundant at sites of inflammation, its role in activating inflammasome signalling in neutrophils is not well characterized. In the current study, we demonstrate that human and murine neutrophils express functional cell-surface P2X7R, which leads to ATP-induced loss of intracellular K(+), NLRP3 inflammasome activation and IL-1ß secretion. ATP-induced P2X7R activation caused a sustained increase in intracellular [Ca(2+)], which is indicative of P2X7R channel opening. Although there are multiple polymorphic variants of P2X7R, we found that neutrophils from multiple donors express P2X7R, but with differential efficacies in ATP-induced increase in cytosolic [Ca(2+)]. Neutrophils were also the predominant P2X7R-expressing cells during Streptococcus pneumoniae corneal infection, and P2X7R was required for bacterial clearance. Given the ubiquitous presence of neutrophils and extracellular ATP in multiple inflammatory conditions, ATP-induced P2X7R activation and IL-1ß secretion by neutrophils likely has a significant, wide ranging clinical impact.