RESUMO
Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.
Assuntos
Vesículas Extracelulares , Gasderminas , Humanos , Monócitos , Diferenciação Celular , Fibroblastos , CaspasesRESUMO
Caspase-1 activation is a central event in innate immune responses to many pathogenic infections and tissue damage. The NLRP3 inflammasome, a multiprotein scaffolding complex that assembles in response to two distinct steps, priming and activation, is required for caspase-1 activation. However, the detailed mechanisms of these steps remain poorly characterized. To investigate the process of LPS-mediated NLRP3 inflammasome priming, we used constitutively present pro-IL-18 as the caspase-1-specific substrate to allow study of the early events. We analyzed human monocyte caspase-1 activity in response to LPS priming, followed by activation with ATP. Within minutes of endotoxin priming, the NLRP3 inflammasome is licensed for ATP-induced release of processed IL-18, apoptosis-associated speck-forming complex containing CARD, and active caspase-1, independent of new mRNA or protein synthesis. Moreover, extracellular signal-regulated kinase 1 (ERK1) phosphorylation is central to the priming process. ERK inhibition and small interfering RNA-mediated ERK1 knockdown profoundly impair priming. In addition, proteasome inhibition prevents ERK phosphorylation and blocks priming. Scavenging reactive oxygen species with diphenylene iodonium also blocks both priming and ERK phosphorylation. These findings suggest that ERK1-mediated posttranslational modifications license the NLRP3 inflammasome to respond to the second signal ATP by inducing posttranslational events that are independent of new production of pro-IL-1ß and NOD-like receptor components.
Assuntos
Inflamassomos , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade Inata , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Monócitos/imunologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Oxidantes/farmacologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The key to further improving outcomes in sepsis lies in understanding and abrogating the dysfunctional immune response that leads to organ failure. Activation of gasdermin-D, a pore-forming protein within the inflammasome cascade, has recently been recognized as the critical step in pyroptosis and organ dysfunction. In this study, we sought to investigate the presence of gasdermin-D in critically ill subjects. DESIGN SETTING AND PATIENTS: Prospective pilot study comparing microparticulate active gasdermin-D levels in critically ill patients admitted to the medical ICU at The Ohio State University Medical Center to healthy donors and clinical outcomes. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma was collected from subjects upon consent and microparticles were isolated by ultracentrifugation. Proteins of interest were identified by immunoblot analysis of microparticle lysates. Quantification was accomplished by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD). Investigators were then unblinded and compared microparticulate active gasdermin-D levels to physician adjudicated clinical diagnoses and outcomes. No appreciable levels of active gasdermin-D were observed in microparticles from healthy volunteers and nonseptic critically ill patients. However, elevated levels of gasdermin-D were noted in microparticles from the septic cohort of critically ill patients. Furthermore, a significant positive correlation by linear regression was noted when microparticulate active gasdermin-D levels were compared with microparticulate levels of CD63, an exosomal marker, CD14, a monocyte marker, and CD69, a marker of monocyte activation (R 2 = 0.37, p = 0.0011, R 2 = 0.85, p < 0.0001, and R2 = 0.43, p = 0.0003, respectively). CONCLUSIONS: This is the first study to demonstrate circulating active gasdermin-D in septic patients in the intensive care setting. Our findings also suggest that active gasdermin-D in septic patients is encapsulated in exosomes derived from activated monocytes. Further characterization in the clinical setting is warranted.
RESUMO
OBJECTIVE: Immune dysregulation during sepsis is poorly understood, however, lymphocyte apoptosis has been shown to correlate with poor outcomes in septic patients. The inflammasome, a molecular complex which includes caspase-1, is essential to the innate immune response to infection and also important in sepsis induced apoptosis. Our group has recently demonstrated that endotoxin-stimulated monocytes release microvesicles (MVs) containing caspase-1 that are capable of inducing apoptosis. We sought to determine if MVs containing caspase-1 are being released into the blood during human sepsis and induce apoptosis.. DESIGN: Single-center cohort study. MEASUREMENTS: 50 critically ill patients were screened within 24 hours of admission to the intensive care unit and classified as either a septic or a critically ill control. Circulatory MVs were isolated and analyzed for the presence of caspase-1 and the ability to induce lymphocyte apoptosis. Patients remaining in the ICU for 48 hours had repeated measurement of caspase-1 activity on ICU day 3. MAIN RESULTS: Septic patients had higher microvesicular caspase-1 activity 0.05 (0.04, 0.07) AFU versus 0.0 AFU (0, 0.02) (p<0.001) on day 1 and this persisted on day 3, 0.12 (0.1, 0.2) versus 0.02 (0, 0.1) (p<0.001). MVs isolated from septic patients on day 1 were able to induce apoptosis in healthy donor lymphocytes compared with critically ill control patients (17.8±9.2% versus 4.3±2.6% apoptotic cells, p<0.001) and depletion of MVs greatly diminished this apoptotic signal. Inhibition of caspase-1 or the disruption of MV integrity abolished the ability to induce apoptosis. CONCLUSION: These findings suggest that microvesicular caspase-1 is important in the host response to sepsis, at least in part, via its ability to induce lymphocyte apoptosis. The ability of microvesicles to induce apoptosis requires active caspase-1 and intact microvesicles.