Wulf Tonnus

The in vivo evidence for regulated necrosis

Necrosis is a hallmark of several widespread diseases or their direct complications. In the past decade, we learned that necrosis can be a regulated process that is potentially druggable. RIPK3‐ and MLKL‐mediated necroptosis represents by far the best studied pathway of regulated necrosis. During necroptosis, the release of damage‐associated molecular patterns (DAMPs) drives a phenomenon referred to as necroinflammation, a common consequence of necrosis. However, most studies of regulated necrosis investigated cell lines in vitro in a cell autonomous manner, which represents a non‐physiological situation. Conclusions based on such work might not necessarily be transferrable to disease states in which synchronized, non‐cell autonomous effects occur. Here, we summarize the current knowledge of the pathophysiological relevance of necroptosis in vivo, and in light of this understanding, we reassess the morphological classification of necrosis that is generally used by pathologists. Along these lines, we discuss the paucity of data implicating necroptosis in human disease. Finally, the in vivo relevance of non‐necroptotic forms of necrosis, such as ferroptosis, is addressed.

Origin and Consequences of Necroinflammation

When cells undergo necrotic cell death in either physiological or pathophysiological settings in vivo, they release highly immunogenic intracellular molecules and organelles into the interstitium and thereby represent the strongest known trigger of the immune system. With our increasing understanding of necrosis as a regulated and genetically determined process (RN, regulated necrosis), necrosis and necroinflammation can be pharmacologically prevented. This review discusses our current knowledge about signaling pathways of necrotic cell death as the origin of necroinflammation. Multiple pathways of RN such as necroptosis, ferroptosis, and pyroptosis have been evolutionary conserved most likely because of their differences in immunogenicity. As the consequence of necrosis, however, all necrotic cells release damage associated molecular patterns (DAMPs) that have been extensively investigated over the last two decades. Analysis of necroinflammation allows characterizing specific signatures for each particular pathway of cell death. While all RN-pathways share the release of DAMPs in general, most of them actively regulate the immune system by the additional expression and/or maturation of either pro- or anti-inflammatory cytokines/chemokines. In addition, DAMPs have been demonstrated to modulate the process of regeneration. For the purpose of better understanding of necroinflammation, we introduce a novel classification of DAMPs in this review to help detect the relative contribution of each RN-pathway to certain physiological and pathophysiological conditions.

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.

P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

α-haemolysin (HlyA)-producing Escherichia coli commonly inflict severe urinary tract infections, including pyelonephritis, which comprises substantial risk for sepsis. In vitro, the cytolytic effect of HlyA is mainly mediated by ATP release through the HlyA pore and subsequent P2X1/P2X7 receptor activation. This amplification of the lytic process is not unique to HlyA but is observed by many other pore-forming proteins including complement-induced haemolysis. Since free hemoglobin in the blood is known to be associated with a worse outcome in sepsis one could speculate that inhibition of P2X receptors would ameliorate the course of sepsis. Surprisingly, this study demonstrates that P2X1−/− and P2X4−/− mice are exceedingly sensitive to sepsis with uropathogenic E. coli. These mice have markedly lower survival, higher cytokine levels and activated intravascular coagulation. Quite the reverse is seen in P2X1−/− mice, which had markedly lower cytokine levels and less coagulation activation compared to controls after exposure to uropathogenic E. coli. The high cytokine levels in the P2X7−/− mouse are unexpected, since P2X7 is implicated in caspase-1-dependent IL-1β production. Here, we demonstrate that IL-1β production during sepsis with uropathogenic E. coli is mediated by caspase-8, since caspase-8 and RIPK3 double knock out mice show substantially lower cytokine during sepsis and increased survival after injection of TNFα. These data support that P2X7 and P2X4 receptor activation has a protective effect during severe E. coli infection.

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.

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.

Die later with ESCRT

The consequences of necroptosis depend on immunomodulatory molecules, the expression of which requires time before the burst of a cell. Gong et al. now provide evidence for ESCRT-III-mediated plasma membrane repair to extend the time to death during necroptosis. Regulated cell death is not restricted to apoptosis, but includes several forms of regulated necrosis. The best characterized signaling pathway of regulated necrosis is necroptosis. Diverse signaling pathways, such as TNFR1-signaling, TLR-signaling and others may result in receptor-interacting protein kinase 3 (RIPK3)dependent phosphorylation and oligomerization of the pseudokinase mixed-lineage kinase domain-like (MLKL). It has been proposed that pMLKL may form pores in the plasma membrane, but the actual processes following plasma membrane translocation remain unclear [1]. In a recent report published in Cell, Gong et al. discovered the ESCRT-III-machinery as an active counterpart of pMLKLassociated membrane damage [2]. As necroptosis actively shapes the immune response [3], ESCRT-III indirectly controls the immunogenicity of necroptotically dying cells. Applying a system to artificially dimerize RIPK3 or MLKL, Gong et al. detected cells that rapidly expose phosphatidylserine (PS) at the outer leaflet of the plasma membrane, a cell death feature that had been associated with apoptosis for the last decades. Single cell analysis revealed the shedding of PS-positive “bubbles”. Unlike apoptotic bodies, these “bubbles” did not contain cytosolic remnants but consisted of broken membranes, as they are permeable to 10 kDa dextran-NH2. These bubbles formed at sites of pMLKL-accumulation, quite similar to what had been previously reported about viral budding in dependence of the ESCRT complex machinery [4]. Indeed, the ESCRT-III-protein CHMP4B co-localized with MLKL at the basis of “bubbles”. A previous study demonstrated ESCRT-mediated repair of laser-damaged membranes [4], but a link to necroptosis was not provided. In line with this, silencing of ESCRT-III-proteins CHMP2A or CHMP4B resulted in spontaneous necroptosis which was prevented by silencing of RIPK3 or MLKL, or by addition of RIPK1-inhibitor Nec-1s. Conversely, silencing of ESCRT-III-proteins CHMP4B, VPS4B, CHMP2A or ESCRT-I-proteins TSG101 or VPS37B sensitized cells to TNF-induced necroptosis even with active caspase-8 which appears to prevent necroptosis independently of this mechanism. Therefore, this study demonstrates the complex interplay between the ESCRT-III machinery and necroptosis execution, prompting the hypothesis of ESCRT-III to delay the time to plasma membrane rupture and release of damage associated molecular patterns (DAMPs). Indeed, even hardly detectable levels of pMLKL were sufficient to induce necroptosis if ESCRT-III was compromised. Along similar lines, active ESCRT-III delayed membrane breakdown resulting in less chemokine production and less efficient cross-priming of T cells as a consequence of a delay in DAMP release. MLKL was recently demonstrated to trigger processing and release of both anti-inflammatory cytokines, such as IL-33 and CXCL1, as well as the proinflammatory cytokine IL-1β [5]. It will be of interest to precisely unravel the proand anti-inflammatory components released by necroptotically dying cells, especially in comparison with cellular cytokines released during distinct pathways of regulated necrosis, such Editorial: Autophagy and Cell Death

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.

Regulated Necrosis and Its Immunogenicity

Abstract Regulated cell death (RCD) plays an integral role in the growth, development, and homeostasis of multicellular organisms. For decades, the term apoptosis was used synonymously with RCD. This term was used to emphasize the difference between RCD and accidental (and therefore unregulated) necrosis. In 2008, however, it became clear that necrosis is not always accidental or inevitable. It can be part of a genetically determined process now termed regulated necrosis (RN) and can be antagonized under specific conditions. Current research efforts are focused on understanding of the physiological and pathophysiological modalities that lead to cell death and to determine why our genes encode several different pathways that lead to RN. These studies have led to a better appreciation of the consequences of tissue injury and inflammation to immunogenicity. Here we review processes that lead to necroinflammation and their contribution to pathologies, such as cancer, transplant rejection, and autoimmune diseases.

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.