RESUMO
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.
Assuntos
Mitocôndrias , Proteína 3 que Contém Domínio de Pirina da Família NLR , Compostos de Fenilureia , Potássio , Septinas , Animais , Humanos , Camundongos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/química , Potássio/metabolismo , Piridinas/farmacologia , Piridinas/química , Septinas/efeitos dos fármacos , Septinas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Caspase 1 , Inibidores de Caspase , Permeabilidade da Membrana Celular , Corantes Fluorescentes , Inflamassomos , Macrófagos , Caspase 1/metabolismo , Animais , Macrófagos/imunologia , Macrófagos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Camundongos , Inflamassomos/metabolismo , Inibidores de Caspase/farmacologia , Camundongos Knockout , Proteínas de Ligação a Fosfato/metabolismo , HumanosRESUMO
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.
Assuntos
Doenças Hereditárias Autoinflamatórias , Inflamassomos , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal , Peptídeos e Proteínas de Sinalização Intracelular , Leucócitos Mononucleares , Proteína 3 que Contém Domínio de Pirina da Família NLR , SíndromeRESUMO
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.
Assuntos
Caspases Iniciadoras , Gasderminas , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Humanos , Camundongos , Caspase 1/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Caspases Iniciadoras/metabolismo , Gasderminas/metabolismoRESUMO
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.
Assuntos
Astrócitos , Microglia , Animais , Encéfalo , Citometria de Fluxo/métodos , Camundongos , NeurogliaRESUMO
Immune cells are armed with Toll-like receptors (TLRs) for sensing and responding to pathogens and other danger cues. The role of extracellular-signal-regulated kinases 1/2 (Erk1/2) in TLR signaling remains enigmatic, with both pro- and anti-inflammatory functions described. We reveal here that the immune-specific transmembrane adaptor SCIMP is a direct scaffold for Erk1/2 in TLR pathways, with high-resolution, live-cell imaging revealing that SCIMP guides the spatial and temporal recruitment of Erk2 to membrane ruffles and macropinosomes for pro-inflammatory TLR4 signaling. SCIMP-deficient mice display defects in Erk1/2 recruitment to TLR4, c-Fos activation, and pro-inflammatory cytokine production, with these effects being phenocopied by Erk1/2 signaling inhibition. Our findings thus delineate a selective role for SCIMP as a key scaffold for the membrane recruitment of Erk1/2 kinase to initiate TLR-mediated pro-inflammatory responses in macrophages.
Assuntos
Macrófagos/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/fisiologia , Receptores Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos Transgênicos , Fosforilação , Receptor 4 Toll-Like/metabolismoRESUMO
To maintain homeostasis, an organism must detect and resolve sterile tissue damage. The NLRP3 inflammasome coordinates such processes to clear tissue damage and induce repair. Dysregulated NLRP3 inflammasome activity, however, drives many conditions including Alzheimer's disease (AD). Recent reports posit that ß-amyloid and tau aggregates trigger destructive NLRP3 inflammasome signalling in the brain, leading to AD pathophysiology and cognitive decline. Other endogenous molecules (e.g. TNF, ATP, serum amyloid A), as well as dysbiosis, can induce peripheral or central inflammation and thereby promote microglial NLRP3 inflammasome signalling and resultant AD. The NLRP3 inflammasome is thus emerging as a critical driver of sterile neuroinflammation and the resultant pathogenesis and progression of AD.
Assuntos
Doença de Alzheimer/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , HumanosRESUMO
Environmental factors, such as cigarette smoking or lung infections, may influence chronic obstructive pulmonary disease (COPD) progression by modifying the respiratory tract microbiome. However, whether the disease itself induces or maintains dysbiosis remains undefined. In this longitudinal study, we investigated the oropharyngeal microbiota composition and disease progression of mice (in cages of 5-10 mice per cage) before, during and up to 3 months after chronic cigarette smoke exposure or exposure to room air for 6 months. Cigarette smoke exposure induced pulmonary emphysema measurable at the end of exposure for 6 months, as well as 3 months following smoke exposure cessation. Using both classical culture methods and 16S rRNA sequencing, we observed that cigarette smoke exposure altered the relative composition of the oropharyngeal microbiota and reduced its diversity (P <0.001). More than 60 taxa were substantially reduced after 6 months of smoke exposure (P <0.001) However, oropharyngeal microbiota disordering was reversed 3 months after smoke exposure cessation and no significant difference was observed compared to age-matched control mice. The effects of lung infection with Streptococcus pneumoniae on established smoke-induced emphysema and on the oropharyngeal microbiota were also evaluated. Inoculation with S. pneumoniae induced lung damage and altered the microbiota composition for a longer time compared to control groups infected but not previously exposed to smoke (P=0.01). Our data demonstrate effects of cigarette smoke and pneumococcus infection leading to altered microbiota and emphysema development. The reversal of the disordering of the microbiota composition, but not lung damage, following smoke exposure cessation and after clearance of infection suggest that changes in lung structure are not sufficient to sustain a disordered microbiota in mice. Whether changes in the airway microbiota contribute to inducing emphysema requires further investigation.
Assuntos
Bactérias/classificação , Disbiose/etiologia , Orofaringe/microbiologia , Infecções Pneumocócicas/microbiologia , Enfisema Pulmonar/genética , RNA Ribossômico 16S/genética , Fumaça/efeitos adversos , Animais , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , DNA Ribossômico/genética , Modelos Animais de Doenças , Progressão da Doença , Disbiose/induzido quimicamente , Disbiose/complicações , Disbiose/microbiologia , Feminino , Estudos Longitudinais , Camundongos , Filogenia , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/microbiologia , RNA Bacteriano/genética , Análise de Sequência de DNA/métodos , Produtos do Tabaco/efeitos adversosRESUMO
Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS.
Assuntos
Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/etiologia , Patrimônio Genético , Fumar/efeitos adversos , Idade de Início , Animais , Biópsia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/metabolismo , Imuno-Histoquímica , Camundongos , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Fenótipo , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Proteinase 3 (PR3) is a myeloid serine protease expressed in neutrophils, monocytes, and macrophages. PR3 has a number of well-characterized proinflammatory functions, including cleaving and activating chemokines and controlling cell survival and proliferation. When presented on the surface of apoptotic neutrophils, PR3 can disrupt the normal anti-inflammatory reprogramming of macrophages following the phagocytosis of apoptotic cells. To better understand the function of PR3 in vivo, we generated a human PR3 transgenic mouse (hPR3Tg). During zymosan-induced peritonitis, hPR3Tg displayed an increased accumulation of neutrophils within the peritoneal cavity compared with wild-type control mice, with no difference in the recruitment of macrophages or B or T lymphocytes. Mice were also subjected to cecum ligation and puncture, a model used to induce peritoneal inflammation through infection. hPR3Tg displayed decreased survival rates in acute sepsis, associated with increased neutrophil extravasation. The decreased survival and increased neutrophil accumulation were associated with the cleavage of annexin A1, a powerful anti-inflammatory protein known to facilitate the resolution of inflammation. Additionally, neutrophils from hPR3Tg displayed enhanced survival during apoptosis compared with controls, and this may also contribute to the increased accumulation observed during the later stages of inflammation. Taken together, our data suggest that human PR3 plays a proinflammatory role during acute inflammatory responses by affecting neutrophil accumulation, survival, and the resolution of inflammation.
Assuntos
Mieloblastina/metabolismo , Neutrófilos/imunologia , Cavidade Peritoneal/patologia , Peritonite/imunologia , Sepse/imunologia , Animais , Anexina A1/metabolismo , Apoptose , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mieloblastina/genética , Peritonite/induzido quimicamente , Fagocitose , Sepse/induzido quimicamente , ZimosanRESUMO
Serpinb1 is an inhibitor of neutrophil granule serine proteases cathepsin G, proteinase-3 and elastase. One of its core physiological functions is to protect neutrophils from granule protease-mediated cell death. Mice lacking Serpinb1a (Sb1a-/-), its mouse ortholog, have reduced bone marrow neutrophil numbers due to cell death mediated by cathepsin G and the mice show increased susceptibility to lung infections. Here, we show that conditional deletion of Serpinb1a using the Lyz2-cre and Cebpa-cre knock-in mice effectively leads to recombination-mediated deletion in neutrophils but protein-null neutrophils were only obtained using the latter recombinase-expressing strain. Absence of Serpinb1a protein in neutrophils caused neutropenia and increased granule permeabilization-induced cell death. We then generated transgenic mice expressing human Serpinb1 in neutrophils under the human MRP8 (S100A8) promoter. Serpinb1a expression levels in founder lines correlated positively with increased neutrophil survival when crossed with Sb1a-/- mice, which had their defective neutrophil phenotype rescued in the higher expressing transgenic line. Using new conditional and transgenic mouse models, our study demonstrates the presence of a relatively low Serpinb1a protein threshold in neutrophils that is required for sustained survival. These models will also be helpful in delineating recently described functions of Serpinb1 in metabolism and cancer.