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1.
Sci Rep ; 12(1): 2005, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132157

RESUMO

The inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 have been emphasised to be essential in the host cell response during urinary tract infection (UTI) by regulating IL-1ß release. Our aim was to investigate how the inflammasome-associated proteins regulate the cell response of bladder epithelial cells during infection with uropathogenic Escherichia coli (UPEC). Human bladder epithelial cells (5637) and CRISPR/Cas9 generated caspase-1, caspase-4 and NLRP3 knockdown cells were stimulated with the UPEC strain CFT073. Using Olink proteomics and real time RT-PCR, we showed that caspase-1, caspase-4 and NLRP3 are vital for the expression of many inflammatory genes and proteins from bladder epithelial cells. When investigating the effect of inflammasome-associated proteins on neutrophils, we found that conditioned medium from UPEC-infected caspase-4 knockdown cells significantly increased phagocytosis of CFT073 and significantly decreased ROS production from neutrophils. In contrast, conditioned medium from UPEC-infected NLRP3 knockdown cells significantly decreased the phagocytosis of CFT073 and significantly increased the ROS production from neutrophils. In conclusion, we showed that the inflammasome-associated proteins contribute to the host cell response during UPEC infection.


Assuntos
Caspase 1/fisiologia , Caspases Iniciadoras/fisiologia , Células Epiteliais/imunologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Infecções Urinárias/genética , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologia , Caspases Iniciadoras/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Bexiga Urinária/citologia
2.
PLoS Pathog ; 16(8): e1008695, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750090

RESUMO

The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida's ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections.


Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Glucose/deficiência , Interações Hospedeiro-Patógeno/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Animais , Células 3T3 BALB , Candida albicans/metabolismo , Candidíase/metabolismo , Candidíase/microbiologia , Caspase 1/fisiologia , Caspases Iniciadoras/fisiologia , Feminino , Hifas , Inflamação/metabolismo , Inflamação/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/fisiologia , Piroptose
3.
Mol Metab ; 37: 100988, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32272237

RESUMO

OBJECTIVE: Maternal high-fat diet (HFD) has been shown to promote the development of insulin resistance (IR) in adult offspring; however, the underlying mechanisms remain unclear. METHODS: Eight-week-old female wild-type mice (C57BL/6) were fed either an HFD or a normal diet (ND), one week prior to mating, and the diet was continued throughout gestation and lactation. Eight-week-old male offspring of both groups were fed an HFD for 8 weeks. RESULTS: Offspring of HFD-fed dams (O-HFD) exhibited significantly impaired insulin sensitivity compared with the offspring of ND-fed dams (O-ND). The adipocyte size of the eWAT increased significantly in O-HFD and was accompanied by abundant crown-like structures (CLSs), as well as a higher concentration of interleukin 1ß (IL-1ß) in the eWAT. Treatment with an inflammasome inhibitor, MCC950, completely abrogated the enhanced IR in O-HFD. However, ex vivo caspase-1 activity in eWAT revealed no difference between the two groups. In contrast, noncanonical inflammasome activation of caspase-11 was significantly augmented in O-HFD compared with O-ND, suggesting that membrane pore formation, but not cleavage of pro-IL-1ß by caspase-1, is augmented in O-HFD. To examine the membrane pore formation, we performed metabolic activation of bone marrow-derived macrophages (BMDMs). The percentage of pore formation assessed by ethidium bromide staining was significantly higher in BMDMs of O-HFD, accompanied by an enhanced active caspase-11 expression. Consistently, the concentration of IL-1ß in culture supernatants was significantly higher in the BMDMs from O-HFD than those from O-ND. CONCLUSIONS: These findings demonstrate that maternal HFD exaggerates diet-induced IR in adult offspring by enhancing noncanonical caspase-11-mediated inflammasome activation.


Assuntos
Caspases Iniciadoras/metabolismo , Inflamassomos/metabolismo , Resistência à Insulina/fisiologia , Animais , Caspases/metabolismo , Caspases Iniciadoras/fisiologia , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Feminino , Inflamassomos/fisiologia , Insulina/metabolismo , Macrófagos/metabolismo , Masculino , Exposição Materna , Camundongos , Camundongos Endogâmicos C57BL , Obesidade , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo
4.
Kidney Blood Press Res ; 44(4): 465-478, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230050

RESUMO

BACKGROUND/AIMS: Acute kidney injury (AKI) is a serious complication of sepsis and has a high morbidity and mortality rate. Caspase-11 induces pyroptosis, a form of programmed cell death that plays a critical role in endotoxic shock, but its role in tubular epithelial cell death and whether it contributes to sepsis-associated AKI remains unknown. METHODS: The caspase-11-/- mouse received an intraperitoneal injection of lipopolysaccharide (LPS, 40 mg/kg body weight). Caspase-11-/- renal tubular epithelial cells (RTECs) form C57BL caspase-11-/- mice were treated with LPS in vitro. The IL-1ß ELISA kit and Scr assay kit were used to measure the level of interleukin-1ß and serum creatinine. Annexin V-FITC assay and TUNEL staining assay were used to detect the cell death in different groups in vitro and in vivo. Western blot was performed to analyze the protein expression of caspase-11 and Gsdmdc1. RESULTS: LPS-induced sepsis results in lytic death of RTECs, accompanied by increased expression of the pyroptosis-related proteins caspase-11 and Gsdmd. However, the increase in pyroptosis-related protein expression induced by LPS was attenuated with caspase-11 knockout, both in vitro and in vivo. Furthermore, when challenged with lethal doses of systemic LPS, pathologic abnormalities in renal structure, increased serum and kidney interleukin-1ß, increased serum creatinine, and animal death were observed in wild-type mice but prevented in caspase-11-/- mice. CONCLUSIONS: Caspase-11-induced pyroptosis of RTECs is a key event during septic AKI, and targeting of caspase-11 in RTECs may serve as a novel therapeutic target in septic AKI.


Assuntos
Injúria Renal Aguda/etiologia , Caspases Iniciadoras/fisiologia , Túbulos Renais/patologia , Piroptose , Sepse/complicações , Injúria Renal Aguda/patologia , Animais , Caspases Iniciadoras/genética , Creatinina/sangue , Células Epiteliais/patologia , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
5.
Cell Rep ; 27(4): 1008-1017.e6, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018119

RESUMO

Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.


Assuntos
Caspases Iniciadoras/fisiologia , Escherichia coli Enteropatogênica/patogenicidade , Macrófagos/microbiologia , Actinas/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Caspase 1/fisiologia , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Inflamassomos/fisiologia , Modelos Biológicos , Polimerização
6.
Artigo em Inglês | MEDLINE | ID: mdl-20875757

RESUMO

OBJECTIVE: Apoptosis is frequently found in oral lichen planus (OLP) lesions, but the pathways leading to apoptosis are unknown. STUDY DESIGN: This study focused on analysis of caspase expression which is essential for apoptosis. Expression of caspases 2, 3, 8, 9, and 12 was studied in 70 biopsy samples from atrophic OLP to identify which cascade pathway, extrinsic or intrinsic, is of importance in apoptosis in OLP. RESULTS: Caspase-2 expression was present in every sample, and >70% of the epithelial cells were positive in 33% of the lesions. More than 70% of the epithelial cells expressed caspase-12 in 84% of the specimens. Caspase-8 expression was shown totally in 87% of the specimens. No caspase-3 expression was found in 57% of the samples, and caspase-9 expression was absent in the entire OLP specimen. CONCLUSIONS: The high frequency of intrinsic apoptotic pathway markers caspases 2 and 12 indicates intracellular stress in atrophic OLP epithelial cells.


Assuntos
Apoptose/fisiologia , Caspases Efetoras/fisiologia , Caspases Iniciadoras/fisiologia , Líquen Plano Bucal/enzimologia , Transdução de Sinais/fisiologia , Adulto , Idoso , Biópsia , Caspase 12/análise , Caspase 2/análise , Caspase 3/análise , Caspase 8/análise , Caspase 9/análise , Caspases Efetoras/análise , Caspases Iniciadoras/análise , Cisteína Endopeptidases/análise , Células Epiteliais/enzimologia , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Líquen Plano Bucal/patologia , Masculino , Pessoa de Meia-Idade , Receptores de Morte Celular/análise
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