RESUMEN
The Nod-like receptor protein 3 (NLRP3) inflammasome is activated by stimuli that induce perturbations in cell homeostasis, which commonly converge on cellular potassium efflux. NLRP3 has thus emerged as a sensor for ionic flux. Here, we identify forchlorfenuron (FCF) as an inflammasome activator that triggers NLRP3 signaling independently of potassium efflux. FCF triggers the rearrangement of septins, key cytoskeletal proteins that regulate mitochondrial function. We report that FCF triggered the rearrangement of SEPT2 into tubular aggregates and stimulated SEPT2-independent NLRP3 inflammasome signaling. Similar to imiquimod, FCF induced the collapse of the mitochondrial membrane potential and mitochondrial respiration. FCF thereby joins the imidazoquinolines as a structurally distinct class of molecules that triggers NLRP3 inflammasome signaling independent of potassium efflux, likely by inducing mitochondrial damage.
Asunto(s)
Mitocondrias , Proteína con Dominio Pirina 3 de la Familia NLR , Compuestos de Fenilurea , Potasio , Septinas , Animales , Humanos , Ratones , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/química , Potasio/metabolismo , Piridinas/farmacología , Piridinas/química , Septinas/efectos de los fármacos , Septinas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Inflammasomes are supramolecular complexes that form in the cytosol in response to pathogen-associated and damage-associated stimuli, as well as other danger signals that perturb cellular homoeostasis, resulting in host defence responses in the form of cytokine release and programmed cell death (pyroptosis). Inflammasome activity is closely associated with numerous human disorders, including rare genetic syndromes of autoinflammation, cardiovascular diseases, neurodegeneration and cancer. In recent years, a range of inflammasome components and their functions have been discovered, contributing to our knowledge of the overall machinery. Here, we review the latest advances in inflammasome biology from the perspective of structural and mechanistic studies. We focus on the most well-studied components of the canonical inflammasome - NAIP-NLRC4, NLRP3, NLRP1, CARD8 and caspase-1 - as well as caspase-4, caspase-5 and caspase-11 of the noncanonical inflammasome, and the inflammasome effectors GSDMD and NINJ1. These structural studies reveal important insights into how inflammasomes are assembled and regulated, and how they elicit the release of IL-1 family cytokines and induce membrane rupture in pyroptosis.
Asunto(s)
Inflamasomas , Piroptosis , Inflamasomas/inmunología , Inflamasomas/metabolismo , Humanos , Piroptosis/inmunología , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas Adaptadoras de Señalización CARD/inmunología , Proteínas Adaptadoras de Señalización CARD/genética , Proteína Inhibidora de la Apoptosis Neuronal/metabolismo , Proteína Inhibidora de la Apoptosis Neuronal/inmunología , Proteína Inhibidora de la Apoptosis Neuronal/genética , Proteínas de Unión a Fosfato/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Caspasas/metabolismo , Caspasas/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas NLR/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/inmunología , GasderminasRESUMEN
Caspase-1 location in cells has been studied with fluorochrome-labeled inhibitors of caspase-1 (FLICA reagents). We report that FLICA reagents have limited cell-membrane permeability. This impacts experimental design as cells with intact membranes, including caspase-1 knockout cells, are not appropriate controls for cells with inflammasome-induced gasdermin D membrane pores.
Asunto(s)
Caspasa 1 , Inhibidores de Caspasas , Permeabilidad de la Membrana Celular , Colorantes Fluorescentes , Inflamasomas , Macrófagos , Caspasa 1/metabolismo , Animales , Macrófagos/inmunología , Macrófagos/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ratones , Inflamasomas/metabolismo , Inhibidores de Caspasas/farmacología , Ratones Noqueados , Proteínas de Unión a Fosfato/metabolismo , HumanosRESUMEN
Inflammasomes are multiprotein complexes that drive inflammation and contribute to protective immunity against pathogens and immune pathology in autoinflammatory diseases. Inflammasomes assemble when an inflammasome scaffold protein senses an activating signal and forms a signaling platform with the inflammasome adaptor protein ASC. The NLRP subfamily of NOD-like receptors (NLRs) includes inflammasome nucleators (such as NLRP3) and also NLRP12, which is genetically linked to familial autoinflammatory disorders that resemble diseases caused by gain-of-function NLRP3 mutants that generate a hyperactive NLRP3 inflammasome. We performed a screen to identify ASC inflammasome-nucleating proteins among NLRs that have the canonical pyrin-NACHT-LRR domain structure. Only NLRP3 and NLRP6 could initiate ASC polymerization to form "specks," and NLRP12 failed to nucleate ASC polymerization. However, wild-type NLRP12 inhibited ASC inflammasome assembly induced by wild-type and gain-of-function mutant NLRP3, an effect not seen with disease-associated NLRP12 mutants. The capacity of NLRP12 to suppress NLRP3 inflammasome assembly was limited to human NLRP3 and was not observed for wild-type murine NLRP3. Furthermore, peripheral blood mononuclear cells from patients with an NLRP12 mutant-associated inflammatory disorder produced increased amounts of the inflammatory cytokine IL-1ß in response to NLRP3 stimulation. Thus, our findings provide insights into NLRP12 biology and suggest that NLRP3 inhibitors in clinical trials for NLRP3-driven diseases may also be effective in treating NLRP12-associated autoinflammatory diseases.
Asunto(s)
Enfermedades Autoinflamatorias Hereditarias , Inflamasomas , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales , Péptidos y Proteínas de Señalización Intracelular , Leucocitos Mononucleares , Proteína con Dominio Pirina 3 de la Familia NLR , SíndromeRESUMEN
The biology of the NACHT domain and leucine-rich repeat (NLR) and pyrin domain-containing 1 (NLRP1) inflammasome has perplexed researchers since this inflammasome was first described about two decades ago. The identification of oxidized thioredoxin 1 (TRX1) as a suppressor of NLRP1 recently linked cellular redox homeostasis to NLRP1 inflammasome signaling. Now, Zhang et al. present a molecular structure of TRX1-bound NLRP1 with unprecedented detail. This structure gives key insight into regulatory mechanisms governing NLRP1 activation and offers enormous potential for structure-based anti-inflammatory drug design.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Inflamasomas , Inflamasomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas NLR , Transducción de SeñalRESUMEN
Apoptosis is traditionally considered to be an immunologically silent form of cell death. Multiple mechanisms exist to ensure that apoptosis does not stimulate the immune system to cause inflammation or autoimmunity. Against this expectation, we now report that epithelia are programmed to provoke, rather than suppress, inflammation in response to apoptosis. We found that an acute inflammatory response led by neutrophils occurs in zebrafish and cell culture when apoptotic epithelial cells cannot be expelled from the monolayer by apical extrusion. This reflects an intrinsic circuit where ATP released from apoptotic cells stimulates epithelial cells in the immediate vicinity to produce interleukin-8 (IL-8). Apical extrusion therefore prevents inappropriate epithelial inflammation by physically eliminating apoptotic cells before they can activate this pro-inflammatory circuit. This carries the implication that epithelia may be predisposed to inflammation, elicited by sporadic or induced apoptosis, if apical extrusion is compromised.
Asunto(s)
Apoptosis , Pez Cebra , Animales , Apoptosis/fisiología , Epitelio , Muerte Celular , InflamaciónRESUMEN
The noncanonical inflammasome is a signalling complex critical for cell defence against cytosolic Gram-negative bacteria. A key step in the human noncanonical inflammasome pathway involves unleashing the proteolytic activity of caspase-4 within this complex. Caspase-4 induces inflammatory responses by cleaving gasdermin-D (GSDMD) to initiate pyroptosis; however, the molecular mechanisms that activate caspase-4 and govern its capacity to cleave substrates remain poorly defined. Caspase-11, the murine counterpart of caspase-4, acquires protease activity within the noncanonical inflammasome by forming a dimer that self-cleaves at D285 to cleave GSDMD. These cleavage events trigger signalling via the NLRP3-ASC-caspase-1 axis, leading to downstream cleavage of the pro-IL-1ß cytokine precursor. Here, we show that caspase-4 first dimerises then self-cleaves at two sites-D270 and D289-in the interdomain linker to acquire full proteolytic activity, cleave GSDMD, and induce cell death. Surprisingly, caspase-4 dimerisation and self-cleavage at D289 generate a caspase-4 p34/p9 protease species that directly cleaves pro-IL-1ß, resulting in its maturation and secretion independently of the NLRP3 inflammasome in primary human myeloid and epithelial cells. Our study thus elucidates the key molecular events that underpin signalling by the caspase-4 inflammasome and identifies IL-1ß as a natural substrate of caspase-4.
Asunto(s)
Caspasas Iniciadoras , Gasderminas , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Humanos , Ratones , Caspasa 1/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Caspasas Iniciadoras/metabolismo , Gasderminas/metabolismoRESUMEN
The Nod-like Receptor (NLR) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that sense cytosolic bacterial proteins. NAIP ligation induces its association with NLRC4, leading to the assembly of the NAIP/NLRC4 inflammasome, which induces the activation of the caspase-1 protease. Caspase-1 then cleaves pro-interleukin (IL)-1ß, pro-IL-18, and gasdermin D and induces a form of pro-inflammatory cell death, pyroptosis. These processes culminate in host defense against bacterial infection. Here we describe methods for activating NAIP/NLRC4 inflammasome signalling in human and murine macrophages and quantifying inflammasome-induced cell death.
Asunto(s)
Proteínas de Unión al Calcio , Inflamasomas , Animales , Ratones , Humanos , Inflamasomas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Muerte Celular , Caspasas/metabolismo , Caspasa 1/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismoRESUMEN
Inflammasomes are the ultimate weapon of the macrophage immune arsenal. Inflammasome signalling in macrophages triggers pyroptosis, a lytic cell death pathway that facilitates inflammation-driven pathogen clearance. Imaging-based approaches to investigating cell death have proven useful, revealing cellular remodelling events such as the generation of extracellular vesicles, and continuing to uncover important structural changes in cells involved in inflammatory signalling. Pyroptosis has proved extremely challenging to image, because its lytic nature is incompatible with many well-established imaging approaches employed for other, non-lytic pathways. The complexities of ectopically expressing fluorescent constructs in primary macrophages and the sensitivity of such proteins to drug-based probes compound this difficulty. We and others have demonstrated key differences in pyroptosis induced by canonical versus noncanonical inflammasomes that delineate functional differences between these signalling pathways. Here, we describe a live imaging approach to study and compare canonical versus noncanonical inflammasome signalling and pyroptotic architecture in primary murine macrophages.
Asunto(s)
Inflamasomas , Piroptosis , Animales , Ratones , Inflamasomas/metabolismo , Macrófagos/metabolismo , Muerte Celular , Inflamación/metabolismoRESUMEN
Hexokinase dissociation from mitochondria triggers calcium-induced oligomerization of VDAC within the outer mitochondrial membrane, leading to NLRP3 recruitment and inflammasome signaling (see related Research Article by Baik et al.).
Asunto(s)
Mitocondrias , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Membranas Mitocondriales/metabolismo , Inflamasomas/metabolismo , Transducción de SeñalRESUMEN
We have replicated our original finding of elevated cleaved caspase-1 in mouse brains and neuroprotection by an NLRP3 inflammasome inhibitor in two mouse models of Parkinson's disease.
Asunto(s)
Inflamasomas , Enfermedad de Parkinson , Ratones , Animales , alfa-Sinucleína , Proteína con Dominio Pirina 3 de la Familia NLR , Enfermedad de Parkinson/patología , DopaminaRESUMEN
Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/genética , Citocinas , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Macrófagos/metabolismo , Virión/metabolismoRESUMEN
Mitochondria have long been appreciated as the metabolic hub of cells. Emerging evidence also posits these organelles as hubs for innate immune signalling and activation, particularly in macrophages. Macrophages are front-line cellular defenders against endogenous and exogenous threats in mammals. These cells use an array of receptors and downstream signalling molecules to respond to a diverse range of stimuli, with mitochondrial biology implicated in many of these responses. Mitochondria have the capacity to both divide through mitochondrial fission and coalesce through mitochondrial fusion. Mitochondrial dynamics, the balance between fission and fusion, regulate many cellular functions, including innate immune pathways in macrophages. In these cells, mitochondrial fission has primarily been associated with pro-inflammatory responses and metabolic adaptation, so can be considered as a combative strategy utilised by immune cells. In contrast, mitochondrial fusion has a more protective role in limiting cell death under conditions of nutrient starvation. Hence, fusion can be viewed as a cellular survival strategy. Here we broadly review the role of mitochondria in macrophage functions, with a focus on how regulated mitochondrial dynamics control different functional responses in these cells.
Asunto(s)
Mitocondrias , Dinámicas Mitocondriales , Animales , Dinámicas Mitocondriales/fisiología , Mitocondrias/metabolismo , Muerte Celular , Transducción de Señal , Macrófagos/metabolismo , Proteínas Mitocondriales/metabolismo , Mamíferos/metabolismoRESUMEN
The cellular and molecular sources of elevated IL-1ß and IL-6 in COVID-19 remain unclear. In this issue of Cell Host and Microbe, Barnett et al. determine how immune cells sense SARS-CoV-2 infection in neighboring epithelial cells to trigger inflammasome signaling and IL-1ß release, which in turn promotes IL-6 release.
Asunto(s)
COVID-19 , Inflamasomas , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR , Interleucina-6 , SARS-CoV-2 , Interleucina-1betaRESUMEN
The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.
Asunto(s)
Antiinfecciosos , Triaje , Especies Reactivas de Oxígeno/metabolismo , NADP/metabolismo , Macrófagos/metabolismo , Antiinfecciosos/metabolismo , Vía de Pentosa Fosfato/fisiologíaRESUMEN
Activated microglia foster a neurotoxic, inflammatory environment in the mammalian central nervous system (CNS) that drives the pathology of neurodegenerative diseases including Parkinson's disease (PD). Moreover, mitochondrial fission promotes microglial inflammatory responses in vitro. Given that the NLRP3 inflammasome and mitochondria are central regulators of both inflammation and PD, we explore potential functions for the NLRP3 inflammasome and mitochondrial dynamics in PD. Specifically, we propose that inducible microglial mitochondrial fission can promote NLRP3-dependent neuroinflammation in hereditary and idiopathic PD. Further in-depth exploration of this topic can prompt valuable discoveries of the underlying molecular mechanisms of PD neuroinflammation, identify novel candidate anti-inflammatory therapeutics for PD, and ideally provide better outcomes for PD patients.
Asunto(s)
Inflamasomas , Enfermedad de Parkinson , Animales , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad de Parkinson/patología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Microglía , Mitocondrias , MamíferosRESUMEN
Oxidized mitochondrial DNA (ox-mtDNA) activates NLRP3 inflammasome signaling through an ill-defined mechanism. In this issue of Immunity, Xian et al. reveal FEN1 endonuclease cleaves ox-mtDNA into fragments that escape mitochondria, igniting NLRP3 and cGAS-STING signaling and inflammation.
Asunto(s)
ADN Mitocondrial , Proteína con Dominio Pirina 3 de la Familia NLR , ADN Mitocondrial/genética , Inflamasomas , Mitocondrias/genética , Transducción de SeñalRESUMEN
The nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome has emerged as a key mediator of pathological inflammation in many diseases and is an exciting drug target. Here, we review the molecular basis of NLRP3 inhibition by drug-like small molecules under development as novel therapeutics. We also summarize recent strategies to block pyroptosis as a novel approach to suppress chronic inflammation. Major recent developments in this area include the elucidation of mechanisms of action (MoAs) by which small molecules block NLRP3 inflammasome assembly and gasdermin D (GSDMD)-induced pyroptosis. We also discuss the status of clinical trials using agents that block specific components of the NLRP3 pathway, including their potential clinical applications for the treatment of many diseases.
Asunto(s)
Inflamasomas , Piroptosis , Humanos , Inflamasomas/metabolismo , Inflamación/tratamiento farmacológico , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis/fisiologíaRESUMEN
PURPOSE OF REVIEW: Inflammasomes are multimeric protein structures with crucial roles in host responses against infections and injuries. The importance of inflammasome activation goes beyond host defense as a dysregulated inflammasome and subsequent secretion of IL-1 family members is believed to be involved in the pathogenesis of various diseases, some of which also produce skeletal manifestations. The purpose of this review is to summarize recent developments in the understanding of inflammasome regulation and IL-1 family members in bone physiology and pathology and current therapeutics will be discussed. RECENT FINDINGS: Small animal models have been vital to help understand how the inflammasome regulates bone dynamics. Animal models with gain or loss of function in various inflammasome components or IL-1 family signaling have illustrated how these systems can impact numerous bone pathologies and have been utilized to test new inflammasome therapeutics. It is increasingly clear that a tightly regulated inflammasome is required not only for host defense but for skeletal homeostasis, as a dysregulated inflammasome is linked to diseases of pathological bone accrual and loss. Given the complexities of inflammasome activation and redundancies in IL-1 activation and secretion, targeting these pathways is at times challenging. Ongoing research into inflammasome-mediated mechanisms will allow the development of new therapeutics for inflammasome/IL-1 diseases.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Homeostasis , Humanos , Inflamasomas/metabolismo , Interleucina-1beta , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de SeñalRESUMEN
Microglia and astrocytes are implicated in aging and age-related diseases. Here, we present a protocol to isolate and culture these glia cells from the murine brain. The protocol consists of two parts: magnetic sorting of adult microglia and mechanical/magnetic sorting of adult microglia and astrocytes. We then describe the characterization of these glial cells by flow cytometry and immunohistochemistry. Microglia isolated from aged mice maintain age-related phenotype during culture. These purified glia cells can be applied in ex vivo studies.