Knocking 'em Dead: Pore-Forming Proteins in Immune Defense.

Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain-like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.

Programmed necrosis in the cross talk of cell death and inflammation.

Cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. Disease pathologies ensue when these processes are disturbed. A plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity, or immunodeficiency. Programmed necrosis or necroptosis is a form of nonapoptotic cell death driven by the receptor interacting protein kinase 3 (RIPK3) and its substrate, mixed lineage kinase domain-like (MLKL). RIPK3 partners with its upstream adaptors RIPK1, TRIF, or DAI to signal for necroptosis in response to death receptor or Toll-like receptor stimulation, pathogen infection, or sterile cell injury. Necroptosis promotes inflammation through leakage of cellular contents from damaged plasma membranes. Intriguingly, many of the signal adaptors of necroptosis have dual functions in innate immune signaling. This unique signature illustrates the cooperative nature of necroptosis and innate inflammatory signaling pathways in managing cell and organismal stresses from pathogen infection and sterile tissue injury.

Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis.

Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.

Gasdermin and MLKL necrotic cell death effectors: Signaling and diseases.

Diverse inflammatory conditions, from infections to autoimmune disease, are often associated with cellular damage and death. Apoptotic cell death has evolved to minimize its inflammatory potential. By contrast, necrotic cell death via necroptosis and pyroptosis-driven by membrane-damaging MLKL and gasdermins, respectively-can both initiate and propagate inflammatory responses. In this review, we provide insights into the function and regulation of MLKL and gasdermin necrotic effector proteins and drivers of plasma membrane rupture. We evaluate genetic evidence that MLKL- and gasdermin-driven necrosis may either provide protection against, or contribute to, disease states in a context-dependent manner. These cumulative insights using gene-targeted mice underscore the necessity for future research examining pyroptotic and necroptotic cell death in human tissue, as a basis for developing specific necrotic inhibitors with the potential to benefit a spectrum of pathological conditions.

Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

ESCRT-III Acts Downstream of MLKL to Regulate Necroptotic Cell Death and Its Consequences.

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here, we show that, during necroptosis, MLKL-dependent calcium (Ca2+) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM "bubbles" with exposed PS that are released from the surface of the otherwise intact cell. The ESCRT-III machinery is required for formation of these bubbles and acts to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT-III controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT-III, cells undergoing necroptosis can express chemokines and other regulatory molecules and promote antigenic cross-priming of CD8+ T cells.

Roles of Caspases in Necrotic Cell Death.

Caspases were originally identified as important mediators of inflammatory response and apoptosis. Recent discoveries, however, have unveiled their roles in mediating and suppressing two regulated forms of necrotic cell death, termed pyroptosis and necroptosis, respectively. These recent advances have significantly expanded our understanding of the roles of caspases in regulating development, adult homeostasis, and host defense response.

NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis.

Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that Ninj1-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.

O-GlcNAc Transferase Suppresses Inflammation and Necroptosis by Targeting Receptor-Interacting Serine/Threonine-Protein Kinase 3.

Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of ß-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

p53 opens the mitochondrial permeability transition pore to trigger necrosis.

Ischemia-associated oxidative damage leading to necrosis is a major cause of catastrophic tissue loss, and elucidating its signaling mechanism is therefore of paramount importance. p53 is a central stress sensor responding to multiple insults, including oxidative stress to orchestrate apoptotic and autophagic cell death. Whether p53 can also activate oxidative stress-induced necrosis is, however, unknown. Here, we uncover a role for p53 in activating necrosis. In response to oxidative stress, p53 accumulates in the mitochondrial matrix and triggers mitochondrial permeability transition pore (PTP) opening and necrosis by physical interaction with the PTP regulator cyclophilin D (CypD). Intriguingly, a robust p53-CypD complex forms during brain ischemia/reperfusion injury. In contrast, reduction of p53 levels or cyclosporine A pretreatment of mice prevents this complex and is associated with effective stroke protection. Our study identifies the mitochondrial p53-CypD axis as an important contributor to oxidative stress-induced necrosis and implicates this axis in stroke pathology.

Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.

The receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling molecule in the programmed necrosis (necroptosis) pathway. This pathway plays important roles in a variety of physiological and pathological conditions, including development, tissue damage response, and antiviral immunity. Here, we report the identification of a small molecule called (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide--hereafter referred to as necrosulfonamide--that specifically blocks necrosis downstream of RIP3 activation. An affinity probe derived from necrosulfonamide and coimmunoprecipitation using anti-RIP3 antibodies both identified the mixed lineage kinase domain-like protein (MLKL) as the interacting target. MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis. Treating cells with necrosulfonamide or knocking down MLKL expression arrested necrosis at a specific step at which RIP3 formed discrete punctae in cells. These findings implicate MLKL as a key mediator of necrosis signaling downstream of the kinase RIP3.

The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways.

The programmed necrosis induced by TNF-α requires the activities of the receptor-interacting serine-threonine kinases RIP1 and RIP3 and their interaction with the mixed lineage kinase domain-like protein MLKL. We report the identification of RIP1- and RIP3-containing protein complexes that form specifically in response to necrosis induction. One component of these complexes is the mitochondrial protein phosphatase PGAM5, which presents as two splice variants, PGAM5L (long form) and PGAM5S (short form). Knockdown of either form attenuated necrosis induced by TNF-α as well as reactive oxygen species (ROS) and calcium ionophore, whereas knockdown of RIP3 and MLKL blocked only TNF-α-mediated necrosis. Upon necrosis induction, PGAM5S recruited the mitochondrial fission factor Drp1 and activated its GTPase activity by dephosphorylating the serine 637 site of Drp1. Drp1 activation caused mitochondrial fragmentation, an early and obligatory step for necrosis execution. These data defined PGAM5 as the convergent point for multiple necrosis pathways.

ZBP1 activation triggers hematopoietic stem and progenitor cell death resulting in bone marrow failure in mice.

Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBP1's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death.

The role of efferocytosis-fueled macrophage metabolism in the resolution of inflammation.

The phagocytosis of dying cells by macrophages, termed efferocytosis, is a tightly regulated process that involves the sensing, binding, engulfment, and digestion of apoptotic cells. Efferocytosis not only prevents tissue necrosis and inflammation caused by secondary necrosis of dying cells, but it also promotes pro-resolving signaling in macrophages, which is essential for tissue resolution and repair following injury or inflammation. An important factor that contributes to this pro-resolving reprogramming is the cargo that is released from apoptotic cells after their engulfment and phagolysosomal digestion by macrophages. The apoptotic cell cargo contains amino acids, nucleotides, fatty acids, and cholesterol that function as metabolites and signaling molecules to bring about this re-programming. Here, we review efferocytosis-induced changes in macrophage metabolism that mediate the pro-resolving functions of macrophages. We also discuss various strategies, challenges, and future perspectives related to drugging efferocytosis-fueled macrophage metabolism as strategy to dampen inflammation and promote resolution in chronic inflammatory diseases.

Plasma membrane repair empowers the necrotic survivors as innate immune modulators.

The plasma membrane is crucial to the survival of animal cells, and damage to it can be lethal, often resulting in necrosis. However, cells possess multiple mechanisms for repairing the membrane, which allows them to maintain their integrity to some extent, and sometimes even survive. Interestingly, cells that survive a near-necrosis experience can recognize sub-lethal membrane damage and use it as a signal to secrete chemokines and cytokines, which activate the immune response. This review will present evidence of necrotic cell survival in both in vitro and in vivo systems, including in C. elegans, mouse models, and humans. We will also summarize the various membrane repair mechanisms cells use to maintain membrane integrity. Finally, we will propose a mathematical model to illustrate how near-death experiences can transform dying cells into innate immune modulators for their microenvironment. By utilizing their membrane repair activity, the biological effects of cell death can extend beyond the mere elimination of the cells.

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.

Catching a killer: Mechanisms of programmed cell death and immune activation in Amyotrophic Lateral Sclerosis.

In the central nervous system (CNS), execution of programmed cell death (PCD) is crucial for proper neurodevelopment. However, aberrant activation of these pathways in adult CNS leads to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). How a cell dies is critical, as it can drive local immune activation and tissue damage. Classical apoptosis engages several mechanisms to evoke "immunologically silent" responses, whereas other forms of programmed death such as pyroptosis, necroptosis, and ferroptosis release molecules that can potentiate immune responses and inflammation. In ALS, a fatal neuromuscular disorder marked by progressive death of lower and upper motor neurons, several cell types in the CNS express machinery for multiple PCD pathways. The specific cell types engaging PCD, and ultimate mechanisms by which neuronal death occurs in ALS are not well defined. Here, we provide an overview of different PCD pathways implicated in ALS. We also examine immune activation in ALS and differentiate apoptosis from necrotic mechanisms based on downstream immunological consequences. Lastly, we highlight therapeutic strategies that target cell death pathways in the treatment of neurodegeneration and inflammation in ALS.

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.

The Pseudokinase MLKL and the Kinase RIPK3 Have Distinct Roles in Autoimmune Disease Caused by Loss of Death-Receptor-Induced Apoptosis.

The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease, and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice compared to Casp8(-/-)Ripk3(-/-) or Fadd(-/-)Ripk3(-/-) mice, respectively. These results demonstrate that MLKL is an essential effector of aberrant necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. It appears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity, and excess cytokine production that occur when FADD or Caspase-8-mediated apoptosis is abrogated.