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.

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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.

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.

Despite ongoing efforts to characterize the host response toward SARS-CoV-2, a major gap in our knowledge still exists regarding the magnitude and duration of the humoral response. Analysis of the antibody response in mild versus moderate/severe patients, using our new developed quantitative electrochemiluminescent assay for detecting IgM/IgA/IgG antibodies toward SARS-CoV-2 antigens, revealed a rapid onset of IgG/IgA antibodies, specifically in moderate/severe patients. IgM antibodies against the viral receptor binding domain, but not against nucleocapsid protein, were detected at early stages of the disease. Furthermore, we observed a marked reduction in IgM/IgA antibodies over-time. Adapting our assay for ACE2 binding-competition, demonstrated that the presence of potentially neutralizing antibodies is corelated with IgG/IgA. Finally, analysis of the cytokine profile in COVID-19 patients revealed unique correlation of an IL-12p70/IL33 and IgG seroconversion, which correlated with disease severity. In summary, our comprehensive analysis has major implications on the understanding and monitoring of SARS-CoV-2 infections.