Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair.

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

Rubicon-deficiency sensitizes mice to mixed lineage kinase domain-like (MLKL)-mediated kidney ischemia-reperfusion injury.

The cytosolic protein rubicon (RUBCN) has been implicated in the removal of necrotic debris and autoimmunity. However, the role of RUBCN in models of acute kidney injury (AKI), a condition that typically involves necrotic kidney tubules, was not investigated. Here, we demonstrate that RUBCN-deficient mice are hypersensitive to renal damage induced by ischemia-reperfusion injury (IRI) and cisplatin-induced AKI. Combined deficiency of RUBCN and mixed lineage kinase domain-like (MLKL) partially reversed the sensitivity in the IRI model suggesting that the absence of RUBCN sensitizes to necroptosis in that model. Necroptosis is known to contribute to TNFα-induced severe inflammatory response syndrome (SIRS), but we detected no statistically significant difference in overall survival following injection of TNFα in RUBCN-deficient mice. We additionally generated RUBCN-deficient mice which lack gasdermin D (GSDMD), the terminal mediator of pyroptosis, but no reversal of the AKI phenotype was observed. Finally, and in contrast to the previous understanding of the role of RUBCN, we did not find a significant autoimmune phenotype in RUBCN-deficient mice, but detected chronic kidney injury (CKD) in aged RUBCN-deficient mice of both sexes. In summary, our data indicate that RUBCN-deficient mice are hypersensitive to kidney injury.

Dexamethasone sensitizes to ferroptosis by glucocorticoid receptor-induced dipeptidase-1 expression and glutathione depletion.

Dexamethasone is widely used as an immunosuppressive therapy and recently as COVID-19 treatment. Here, we demonstrate that dexamethasone sensitizes to ferroptosis, a form of iron-catalyzed necrosis, previously suggested to contribute to diseases such as acute kidney injury, myocardial infarction, and stroke, all of which are triggered by glutathione (GSH) depletion. GSH levels were significantly decreased by dexamethasone. Mechanistically, we identified that dexamethasone up-regulated the GSH metabolism regulating protein dipeptidase-1 (DPEP1) in a glucocorticoid receptor (GR)-dependent manner. DPEP1 knockdown reversed the phenotype of dexamethasone-induced ferroptosis sensitization. Ferroptosis inhibitors, the DPEP1 inhibitor cilastatin, or genetic DPEP1 inactivation reversed the dexamethasone-induced increase in tubular necrosis in freshly isolated renal tubules. Our data indicate that dexamethasone sensitizes to ferroptosis by a GR-mediated increase in DPEP1 expression and GSH depletion. Together, we identified a previously unknown mechanism of glucocorticoid-mediated sensitization to ferroptosis bearing clinical and therapeutic implications.

Dysfunction of the key ferroptosis-surveilling systems hypersensitizes mice to tubular necrosis during acute kidney injury.

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.

Caspase-8-dependent gasdermin D cleavage promotes antimicrobial defense but confers susceptibility to TNF-induced lethality.

Gasdermin D (GSDMD) is a pore-forming protein that promotes pyroptosis and release of proinflammatory cytokines. Recent studies revealed that apoptotic caspase-8 directly cleaves GSDMD to trigger pyroptosis. However, the molecular requirements for caspase-8-dependent GSDMD cleavage and the physiological impact of this signaling axis are unresolved. Here, we report that caspase-8-dependent GSDMD cleavage confers susceptibility to tumor necrosis factor (TNF)-induced lethality independently of caspase-1 and that GSDMD activation provides host defense against Yersinia infection. We further demonstrate that GSDMD inactivation by apoptotic caspases at aspartate 88 (D88) suppresses TNF-induced lethality but promotes anti-Yersinia defense. Last, we show that caspase-8 dimerization and autoprocessing are required for GSDMD cleavage, and provide evidence that the caspase-8 autoprocessing and activity on various complexes correlate with its ability to directly cleave GSDMD. These findings reveal GSDMD as a potential therapeutic target to reduce inflammation associated with mutations in the death receptor signaling machinery.

Gasdermin D and pyroptosis in acute kidney injury.

Cell death is a pathophysiological component of acute tubular necrosis and acute kidney injury. Regulated necrosis, however, comes in several different forms. Although necroptosis and ferroptosis have been recently characterized in acute kidney injury, pyroptosis has not been assessed in detail. In this issue of Kidney International, Miao and Yin et al. investigate the role of gasdermin D, a protein that can form plasma membrane pores.

Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

Don't trick me twice!

Chemotherapy-induced nephrotoxicity limits the success of cancer therapy. Landau et al. now describe a mechanism by which a first dose of cisplatin renders the kidney sensitive to necroptosis mediated by a second dose. Unresolved injury and sustained necrosis, therefore, may represent a pathophysiological means of transition from acute kidney injury to chronic kidney disease.

Assessment of In Vivo Kidney Cell Death: Acute Kidney Injury.

The kidney has been studied as an organ to investigate cell death in vivo for a number of reasons. The unique vasculature that does not contain collateral vessels favors the kidney over other organs for the investigation of ischemia-reperfusion injury. Unilateral uretic obstruction has become the most prominently studied model for fibrosis with impact far beyond postrenal kidney injury. In addition, the tubular elimination mechanisms render the kidney susceptible to toxicity models, such as cisplatin-induced acute kidney injury. During trauma of skeletal muscles, myoglobulin deposition causes tubular cell death in the model of rhabdomyolysis-induced acute kidney injury. Here, we introduce these clinically relevant in vivo models of acute kidney injury (AKI) and critically review the protocols we use to effectively induce them.

Phenytoin inhibits necroptosis.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Sorafenib tosylate inhibits directly necrosome complex formation and protects in mouse models of inflammation and tissue injury.

Necroptosis contributes to the pathophysiology of several inflammatory, infectious and degenerative disorders. TNF-induced necroptosis involves activation of the receptor-interacting protein kinases 1 and 3 (RIPK1/3) in a necrosome complex, eventually leading to the phosphorylation and relocation of mixed lineage kinase domain like protein (MLKL). Using a high-content screening of small compounds and FDA-approved drug libraries, we identified the anti-cancer drug Sorafenib tosylate as a potent inhibitor of TNF-dependent necroptosis. Interestingly, Sorafenib has a dual activity spectrum depending on its concentration. In murine and human cell lines it induces cell death, while at lower concentrations it inhibits necroptosis, without affecting NF-κB activation. Pull down experiments with biotinylated Sorafenib show that it binds independently RIPK1, RIPK3 and MLKL. Moreover, it inhibits RIPK1 and RIPK3 kinase activity. In vivo Sorafenib protects against TNF-induced systemic inflammatory response syndrome (SIRS) and renal ischemia-reperfusion injury (IRI). Altogether, we show that Sorafenib can, next to the reported Braf/Mek/Erk and VEGFR pathways, also target the necroptotic pathway and that it can protect in an acute inflammatory RIPK1/3-mediated pathology.

Gimme a complex! Resident mononuclear phagocytes in the kidney as monitors of circulating antigens and immune complexes.

Over the past decade, researchers have made substantial progress in characterizing a network of mononuclear phagocytes in the kidney, which variously have been referred to as resident macrophages or dendritic cells. Two recent studies, published in Cell and Kidney International, have identified these resident macrophages/dendritic cells as local immune monitors of peritubular capillaries for circulating antigens and immune complexes. These cells appear to represent an early line of defense against circulating infectious particles and immune complexes, but the resulting inflammatory response may also contribute to interstitial inflammation and kidney disease progression.

"Death is my Heir"--Ferroptosis Connects Cancer Pharmacogenomics and Ischemia-Reperfusion Injury.

Although they are key to precision medicine, pharmacokinetics and pharmacogenomics are currently plagued with inconsistent results. In this issue of Cell Chemical Biology, Shimada et al. (2016) use cell line selectivity and appropriate filters to improve the consistency and to identify biomarkers for the selectivity of lethal compounds. These insights may be useful for our understanding of how necrosis and ischemic injury are regulated.