Proteomic analysis of necroptotic extracellular vesicles.

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.

Necroptotic extracellular vesicles - present and future.

Necroptosis is a receptor-interacting protein kinase 3 (RIPK3)-/mixed lineage kinase domain-like(MLKL)-dependent and caspase-independent form of cell death that promotes inflammation. We, and others, have recently shown that necroptotic cells release extracellular vesicles (EVs). This finding has highlighted that the impact of necroptosis extends well beyond its cell death function. In this review, we summarize the general characteristics, biogenesis, and function of EVs, as well as their role in cancer. In addition, we outline our current knowledge regarding necroptotic EVs, including their recently discovered and analyzed proteome. We examine the accumulating evidence for a role for necroptosis in anti-tumor immunity. Finally, we suggest that necroptotic EVs play an important role in the necroptosis-induced immune response and may, therefore, be exploited in cancer immunotherapy applications.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

Flipping the dogma - phosphatidylserine in non-apoptotic cell death.

The exposure of phosphatidylserine (PS) on the outer plasma membrane has long been considered a unique feature of apoptotic cells. Together with other "eat me" signals, it enables the recognition and phagocytosis of dying cells (efferocytosis), helping to explain the immunologically-silent nature of apoptosis. Recently, however, PS exposure has also been reported in non-apoptotic forms of regulated inflammatory cell death, such as necroptosis, challenging previous dogma. In this review, we outline the evidence for PS exposure in non-apoptotic cells and extracellular vesicles (EVs), and discuss possible mechanisms based on our knowledge of apoptotic-PS exposure. In addition, we examine the outcomes of non-apoptotic PS exposure, including the reversibility of cell death, efferocytosis, and consequent inflammation. By examining PS biology, we challenge the established approach of distinguishing apoptosis from other cell death pathways by AnnexinV staining of PS externalization. Finally, we re-evaluate how PS exposure is thought to define apoptosis as an immunologically silent process distinct from other non-apoptotic and inflammatory cell death pathways. Ultimately, we suggest that a complete understanding of how regulated cell death processes affect the immune system is far from being fully elucidated.

A genetic system for biasing the sex ratio in mice.

Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.

Macrophages, rather than DCs, are responsible for inflammasome activity in the GM-CSF BMDC model.

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.

Necroptosis directly induces the release of full-length biologically active IL-33 in vitro and in an inflammatory disease model.

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.

Distinguishing Necroptosis from Apoptosis.

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.

Phosphatidylserine externalization, "necroptotic bodies" release, and phagocytosis during necroptosis.

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.

Mechanisms of RIPK3-induced inflammation.

Receptor-interacting protein kinase 3 (RIP3/RIPK3) is a multifunctional regulator of cell death and inflammation. It controls signalling downstream of the tumor necrosis factor (TNF) receptor family, DNA-dependent activator of IFN-regulatory factors (DAI) and toll-like receptors (TLRs). Today, it is also widely recognized as a component of caspase-independent cell death known as necroptosis, and cytokine production via activation of the inflammasome. Its role in inflammasome activation, in particular, make the interpretation of its role in vivo more complex. In this review, we focus on divergent roles for RIPK3 in cell death and inflammation.