Andreas Linkermann

Regulated necrosis: the expanding network of non-apoptotic cell death pathways

Cell death research was revitalized by the understanding that necrosis can occur in a highly regulated and genetically controlled manner. Although RIPK1 (receptor-interacting protein kinase 1)- and RIPK3–MLKL (mixed lineage kinase domain-like)-mediated necroptosis is the most understood form of regulated necrosis, other examples of this process are emerging, including cell death mechanisms known as parthanatos, oxytosis, ferroptosis, NETosis, pyronecrosis and pyroptosis. Elucidating how these pathways of regulated necrosis are interconnected at the molecular level should enable this process to be therapeutically targeted.

Two independent pathways of regulated necrosis mediate ischemia–reperfusion injury

Regulated necrosis (RN) may result from cyclophilin (Cyp)D-mediated mitochondrial permeability transition (MPT) and receptor-interacting protein kinase (RIPK)1-mediated necroptosis, but it is currently unclear whether there is one common pathway in which CypD and RIPK1 act in or whether separate RN pathways exist. Here, we demonstrate that necroptosis in ischemia–reperfusion injury (IRI) in mice occurs as primary organ damage, independent of the immune system, and that mice deficient for RIPK3, the essential downstream partner of RIPK1 in necroptosis, are protected from IRI. Protection of RIPK3-knockout mice was significantly stronger than of CypD-deficient mice. Mechanistically, in vivo analysis of cisplatin-induced acute kidney injury and hyperacute TNF-shock models in mice suggested the distinctness of CypD-mediated MPT from RIPK1/RIPK3-mediated necroptosis. We, therefore, generated CypD-RIPK3 double-deficient mice that are viable and fertile without an overt phenotype and that survived prolonged IRI, which was lethal to each single knockout. Combined application of the RIPK1 inhibitor necrostatin-1 and the MPT inhibitor sanglifehrin A confirmed the results with mutant mice. The data demonstrate the pathophysiological coexistence and corelevance of two separate pathways of RN in IRI and suggest that combination therapy targeting distinct RN pathways can be beneficial in the treatment of ischemic injury.

Rip1 (Receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury

Loss of kidney function in renal ischemia/reperfusion injury is due to programmed cell death, but the contribution of necroptosis, a newly discovered form of programmed necrosis, has not been evaluated. Here, we identified the presence of death receptor-mediated but caspase-independent cell death in murine tubular cells and characterized it as necroptosis by the addition of necrostatin-1, a highly specific receptor-interacting protein kinase 1 inhibitor. The detection of receptor-interacting protein kinase 1 and 3 in whole-kidney lysates and freshly isolated murine proximal tubules led us to investigate the contribution of necroptosis in a mouse model of renal ischemia/reperfusion injury. Treatment with necrostatin-1 reduced organ damage and renal failure, even when administered after reperfusion, resulting in a significant survival benefit in a model of lethal renal ischemia/reperfusion injury. Unexpectedly, specific blockade of apoptosis by zVAD, a pan-caspase inhibitor, did not prevent the organ damage or the increase in urea and creatinine in vivo in renal ischemia/reperfusion injury. Thus, necroptosis is present and has functional relevance in the pathophysiological course of ischemic kidney injury and shows the predominance of necroptosis over apoptosis in this setting. Necrostatin-1 may have therapeutic potential to prevent and treat renal ischemia/reperfusion injury.

Synchronized renal tubular cell death involves ferroptosis

Significance Cell death by regulated necrosis causes tremendous tissue damage in a wide variety of diseases, including myocardial infarction, stroke, sepsis, and ischemia–reperfusion injury upon solid organ transplantation. Here, we demonstrate that an iron-dependent form of regulated necrosis, referred to as ferroptosis, mediates regulated necrosis and synchronized death of functional units in diverse organs upon ischemia and other stimuli, thereby triggering a detrimental immune response. We developed a novel third-generation inhibitor of ferroptosis that is the first compound in this class that is stable in plasma and liver microsomes and that demonstrates high efficacy when supplied alone or in combination therapy. Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia–reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the first-in-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.

Regulated cell death and inflammation: an auto-amplification loop causes organ failure

Regulated cell death (RCD) is either immunologically silent or immunogenic. RCD in parenchymal cells may lead to the release of damage- associated molecular patterns that drive both tissue inflammation and the activation of further pathways of RCD. Following an initial event of regulated necrosis, RCD and inflammation can induce each other and drive a local auto-amplification loop that leads to exaggerated cell death and inflammation. In this Opinion article, we propose that such crosstalk between pro-inflammatory and RCD pathways has pathophysiological relevance in solid organ failure, transplantation and cancer. In our opinion, clinicians should not only prescribe immunosuppressive treatments to disrupt this circuit, but also implement the neglected therapeutic option of adding compounds that interfere with RCD.

Ferrostatins Inhibit Oxidative Lipid Damage and Cell Death in Diverse Disease Models

Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington’s disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.

Widespread Mitochondrial Depletion via Mitophagy Does Not Compromise Necroptosis

Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death.

Molecular mechanisms of regulated necrosis.

It is now clear that apoptosis does not constitute the sole genetically encoded form of cell death. Rather, cells can spontaneously undertake or exogenously be driven into a cell death subroutine that manifests with necrotic features, yet can be inhibited by pharmacological and genetic interventions. As regulated necrosis (RN) plays a major role in both physiological scenarios (e.g., embryonic development) and pathological settings (e.g., ischemic disorders), consistent efforts have been made throughout the last decade toward the characterization of the molecular mechanisms that underlie this cell death modality. Contrarily to initial beliefs, RN does not invariably result from the activation of a receptor interacting protein kinase 3 (RIPK3)-dependent signaling pathway, but may be ignited by distinct molecular networks. Nowadays, various types of RN have been characterized, including (but not limited to) necroptosis, mitochondrial permeability transition (MPT)-dependent RN and parthanatos. Of note, the inhibition of only one of these modules generally exerts limited cytoprotective effects in vivo, underscoring the degree of interconnectivity that characterizes RN. Here, we review the signaling pathways, pathophysiological relevance and therapeutic implications of the major molecular cascades that underlie RN.

Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells

Defects in clearance of dying cells have been proposed to underlie the pathogenesis of systemic lupus erythematosus (SLE). Mice lacking molecules associated with dying cell clearance develop SLE-like disease, and phagocytes from patients with SLE often display defective clearance and increased inflammatory cytokine production when exposed to dying cells in vitro. Previously, we and others described a form of noncanonical autophagy known as LC3-associated phagocytosis (LAP), in which phagosomes containing engulfed particles, including dying cells, recruit elements of the autophagy pathway to facilitate maturation of phagosomes and digestion of their contents. Genome-wide association studies have identified polymorphisms in the Atg5 (ref. 8) and possibly Atg7 (ref. 9) genes, involved in both canonical autophagy and LAP, as markers of a predisposition for SLE. Here we describe the consequences of defective LAP in vivo. Mice lacking any of several components of the LAP pathway show increased serum levels of inflammatory cytokines and autoantibodies, glomerular immune complex deposition, and evidence of kidney damage. When dying cells are injected into LAP-deficient mice, they are engulfed but not efficiently degraded and trigger acute elevation of pro-inflammatory cytokines but not anti-inflammatory interleukin (IL)-10. Repeated injection of dying cells into LAP-deficient, but not LAP-sufficient, mice accelerated the development of SLE-like disease, including increased serum levels of autoantibodies. By contrast, mice deficient in genes required for canonical autophagy but not LAP do not display defective dying cell clearance, inflammatory cytokine production, or SLE-like disease, and, like wild-type mice, produce IL-10 in response to dying cells. Therefore, defects in LAP, rather than canonical autophagy, can cause SLE-like phenomena, and may contribute to the pathogenesis of SLE.

Regulated Cell Death in AKI

AKI is pathologically characterized by sublethal and lethal damage of renal tubules. Under these conditions, renal tubular cell death may occur by regulated necrosis (RN) or apoptosis. In the last two decades, tubular apoptosis has been shown in preclinical models and some clinical samples from patients with AKI. Mechanistically, apoptotic cell death in AKI may result from well described extrinsic and intrinsic pathways as well as ER stress. Central converging nodes of these pathways are mitochondria, which become fragmented and sensitized to membrane permeabilization in response to cellular stress, resulting in the release of cell death-inducing factors. Whereas apoptosis is known to be regulated, tubular necrosis was thought to occur by accident until recent work unveiled several RN subroutines, most prominently receptor-interacting protein kinase-dependent necroptosis and RN induced by mitochondrial permeability transition. Additionally, other cell death pathways, like pyroptosis and ferroptosis, may also be of pathophysiologic relevance in AKI. Combination therapy targeting multiple cell-death pathways may, therefore, provide maximal therapeutic benefits.