RESUMO
Excessive release of heme from RBCs is a key pathophysiological feature of several disease states, including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and the endothelium. In this study, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1ß. Heme-induced IL-1ß release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4 and caspase-5 are essential for heme-induced IL-1ß release, whereas caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme is a damage-associated molecular pattern that can engage canonical and noncanonical inflammasome activation as a key mediator of inflammation in macrophages.
Assuntos
Anemia Falciforme/metabolismo , Caspases Iniciadoras/metabolismo , Caspases/metabolismo , Eritrócitos/fisiologia , Inflamassomos/metabolismo , Inflamação/metabolismo , Macrófagos/imunologia , Alarminas/metabolismo , Morte Celular , Células Cultivadas , Heme/metabolismo , Hemólise , Humanos , Interleucina-1beta/metabolismo , Regulação para CimaRESUMO
The PIDDosome (PIDD-RAIDD-caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2-dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function.
Assuntos
Apoptose , Caspase 2/metabolismo , Nucléolo Celular/enzimologia , Cisteína Endopeptidases/metabolismo , Dano ao DNA , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/genética , Cisteína Endopeptidases/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Ativação Enzimática , Genótipo , Células HEK293 , Células HeLa , Humanos , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Microscopia de Vídeo , Complexos Multiproteicos , Proteínas Nucleares/genética , Nucleofosmina , Fenótipo , Ligação Proteica , Interferência de RNA , Transdução de Sinais , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 min. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings.