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Necrosis of a cell is defined by the loss of its plasma membrane integrity. Morphologically, necrosis occurs in several forms such as coagulative necrosis, colliquative necrosis, caseating necrosis, fibrinoid necrosis, and others. Biochemically, necrosis was demonstrated to represent a number of genetically determined signalling pathways. These include (i) kinase-mediated necroptosis, which depends on receptor interacting protein kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL); (ii) gasdermin-mediated necrosis downstream of inflammasomes, also referred to as pyroptosis; and (iii) an iron-catalysed mechanism of highly specific lipid peroxidation named ferroptosis. Given the molecular understanding of the nature of these pathways, specific antibodies may allow direct detection of regulated necrosis and correlation with morphological features. Necroptosis can be specifically detected by immunohistochemistry and immunofluorescence employing antibodies to phosphorylated MLKL. Likewise, it is possible to generate cleavage-specific antibodies against epitopes in gasdermin protein family members. In ferroptosis, however, specific detection requires quantification of oxidative lipids by mass spectrometry (oxylipidomics). Together with classical cell death markers, such as TUNEL staining and detection of cleaved caspase-3 in apoptotic cells, the extension of the arsenal of necrosis markers will allow pathological detection of specific molecular pathways rather than isolated morphological descriptions. These novel pieces of information will be extraordinarily helpful for clinicians as inhibitors of necroptosis (necrostatins), ferroptosis (ferrostatins), and inflammasomes have emerged in clinical trials. Anatomical pathologists should embrace these novel ancillary tests and the concepts behind them and test their impact on diagnostic precision, prognostication, and the prediction of response to the upcoming anti-necrotic therapies. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.

Acute kidney injury (AKI) is characterized by necrotic tubular cell death and inflammation. The TWEAK/Fn14 axis is a mediator of renal injury. Diverse pathways of regulated necrosis have recently been reported to contribute to AKI, but there are ongoing discussions on the timing or molecular regulators involved. We have now explored the cell death pathways induced by TWEAK/Fn14 activation and their relevance during AKI. In cultured tubular cells, the inflammatory cytokine TWEAK induces apoptosis in a proinflammatory environment. The default inhibitor of necroptosis [necrostatin-1 (Nec-1)] was protective, while caspase inhibition switched cell death to necroptosis. Additionally, folic acid-induced AKI in mice resulted in increased expression of Fn14 and necroptosis mediators, such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage domain-like protein (MLKL). Targeting necroptosis with Nec-1 or by genetic RIPK3 deficiency and genetic Fn14 ablation failed to be protective at early time points (48 h). However, a persistently high cell death rate and kidney dysfunction (72-96 h) were dependent on an intact TWEAK/Fn14 axis driving necroptosis. This was prevented by Nec-1, or MLKL, or RIPK3 deficiency and by Nec-1 stable (Nec-1s) administered before or after induction of AKI. These data suggest that initial kidney damage and cell death are amplified through recruitment of inflammation-dependent necroptosis, opening a therapeutic window to treat AKI once it is established. This may be relevant for clinical AKI, since using current diagnostic criteria, severe injury had already led to loss of renal function at diagnosis.