RESUMEN
Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.
Asunto(s)
Muerte Celular/inmunología , Vesículas Extracelulares/metabolismo , Inmunidad/inmunología , Necroptosis/inmunología , Proteómica/métodos , HumanosRESUMEN
Inflammasomes are one of the most important mechanisms for innate immune defense against microbial infection but are also known to drive various inflammatory disorders via processing and release of the cytokine IL-1ß. As research into the regulation and effects of inflammasomes in disease has rapidly expanded, a variety of cell types, including dendritic cells (DCs), have been suggested to be inflammasome competent. Here we describe a major fault in the widely used DC-inflammasome model of bone marrow-derived dendritic cells (BMDCs) generated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that among GM-CSF bone marrow-derived cell populations, monocyte-derived macrophages, rather than BMDCs, were responsible for inflammasome activation and IL-1ß secretion. Therefore, GM-CSF bone marrow-derived cells should not be used to draw conclusions about DC-dependent inflammasome biology, although they remain a useful tool for analysis of inflammasome responses in monocytes-macrophages.
Asunto(s)
Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Inflamasomas/metabolismo , Macrófagos/inmunología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células Cultivadas , Interleucina-1beta/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos InmunológicosRESUMEN
Interleukin-33 (IL-33) is a pro-inflammatory cytokine that plays a significant role in inflammatory diseases by activating immune cells to induce type 2 immune responses upon its release. Although IL-33 is known to be released during tissue damage, its exact release mechanism is not yet fully understood. Previously, we have shown that cleaved IL-33 can be detected in the plasma and epithelium of Ripk1-/- neonates, which succumb to systemic inflammation driven by spontaneous receptor-interacting protein kinase-3 (RIPK3)-dependent necroptotic cell death, shortly after birth. Thus, we hypothesized that necroptosis, a RIPK3/mixed lineage kinase-like protein (MLKL)-dependent, caspase-independent cell death pathway controls IL-33 release. Here, we show that necroptosis directly induces the release of nuclear IL-33 in its full-length form. Unlike the necroptosis executioner protein, MLKL, which was released in its active phosphorylated form in extracellular vesicles, IL-33 was released directly into the supernatant. Importantly, full-length IL-33 released in response to necroptosis was found to be bioactive, as it was able to activate basophils and eosinophils. Finally, the human and murine necroptosis inhibitor, GW806742X, blocked necroptosis and IL-33 release in vitro and reduced eosinophilia in Aspergillus fumigatus extract-induced asthma in vivo, an allergic inflammation model that is highly dependent on IL-33. Collectively, these data establish for the first time, necroptosis as a direct mechanism for IL-33 release, a finding that may have major implications in type 2 immune responses.
Asunto(s)
Apoptosis/inmunología , Asma/inmunología , Interleucina-33/inmunología , Necrosis/inmunología , Proteínas Quinasas/inmunología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Aspergillus fumigatus/química , Aspergillus fumigatus/inmunología , Asma/inducido químicamente , Asma/tratamiento farmacológico , Asma/genética , Basófilos/efectos de los fármacos , Basófilos/inmunología , Basófilos/patología , Línea Celular , Mezclas Complejas/administración & dosificación , Mezclas Complejas/química , Mezclas Complejas/inmunología , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Inmunidad Innata/efectos de los fármacos , Interleucina-33/genética , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Queratinocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Necrosis/genética , Necrosis/patología , Necrosis/prevención & control , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de SeñalRESUMEN
Apoptosis was the first programmed cell death to be defined-highly regulated and immunologically silent, as apoptotic bodies are being removed without triggering inflammation. Few decades later, necroptosis was discovered-uniquely regulated but inflammatory. As these two programmed cell death pathways may be initiated via similar pathways (death receptors and intracellular receptors) while being differently regulated and resulting in distinctive physiological consequences, the need for distinguishing apoptosis from necroptosis is required. Here we describe a series of distinguishing assays that use apoptotic- and necroptotic-distinct response to pharmacological interventions with specific death inhibitors, morphology and death-specific proteins involvement. The procedure includes cell death kinetics assessment and morphology monitoring of stimulated and pharmacologically treated-cells using flow cytometry and live imaging, with the detection of death-specific proteins using Immunoblot. The procedure described here is simple and thus can be adjusted to various experimental systems, enabling apoptosis to be distinguished from necroptosis in one's system of interest, without the need for more complex reagents such as genetic knockout models.
Asunto(s)
Apoptosis , Inflamación/patología , Necrosis , Animales , Caspasas/metabolismo , Humanos , Inflamación/etiología , Proteínas Quinasas/metabolismoRESUMEN
Necroptosis is a regulated, nonapoptotic form of cell death initiated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins. It is considered to be a form of regulated necrosis, and, by lacking the "find me" and "eat me" signals that are a feature of apoptosis, necroptosis is considered to be inflammatory. One such "eat me" signal observed during apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane. Here, we demonstrate that necroptotic cells also expose PS after phosphorylated mixed lineage kinase-like (pMLKL) translocation to the membrane. Necroptotic cells that expose PS release extracellular vesicles containing proteins and pMLKL to their surroundings. Furthermore, inhibition of pMLKL after PS exposure can reverse the process of necroptosis and restore cell viability. Finally, externalization of PS by necroptotic cells drives recognition and phagocytosis, and this may limit the inflammatory response to this nonapoptotic form of cell death. The exposure of PS to the outer membrane and to extracellular vesicles is therefore a feature of necroptotic cell death and may serve to provide an immunologically-silent window by generating specific "find me" and "eat me" signals.
Asunto(s)
Membrana Celular/metabolismo , Necrosis/metabolismo , Fagocitosis , Fosfatidilserinas/metabolismo , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/inmunología , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Humanos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Necrosis/inmunología , Necrosis/patología , Necrosis/prevención & control , Fagocitosis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Propiedades de Superficie/efectos de los fármacosRESUMEN
Organic Te(IV) compounds (organotelluranes) differing in their labile ligands exhibited anti-integrin activities in vitro and anti-metastatic properties in vivo. They underwent ligand substitution with l-cysteine, as a thiol model compound. Unlike inorganic Te(IV) compounds, the organotelluranes did not form a stable complex with cysteine, but rather immediately oxidized it. The organotelluranes inhibited integrin functions, such as adhesion, migration, and metalloproteinase secretion mediation in B16F10 murine melanoma cells. In comparison, a reduced derivative with no labile ligand inhibited adhesion of B16F10 cells to a significantly lower extent, thus pointing to the importance of the labile ligands of the Te(IV) atom. One of the organotelluranes inhibited circulating cancer cells in vivo, possibly by integrin inhibition. Our results extend the current knowledge on the reactivity and mechanism of organotelluranes with different labile ligands and highlight their clinical potential.