Sublethal cytochrome c release generates drug-tolerant persister cells.

Drug-tolerant persister cells (persisters) evade apoptosis upon targeted and conventional cancer therapies and represent a major non-genetic barrier to effective cancer treatment. Here, we show that cells that survive treatment with pro-apoptotic BH3 mimetics display a persister phenotype that includes colonization and metastasis in vivo and increased sensitivity toward ferroptosis by GPX4 inhibition. We found that sublethal mitochondrial outer membrane permeabilization (MOMP) and holocytochrome c release are key requirements for the generation of the persister phenotype. The generation of persisters is independent of apoptosome formation and caspase activation, but instead, cytosolic cytochrome c induces the activation of heme-regulated inhibitor (HRI) kinase and engagement of the integrated stress response (ISR) with the consequent synthesis of ATF4, all of which are required for the persister phenotype. Our results reveal that sublethal cytochrome c release couples sublethal MOMP to caspase-independent initiation of an ATF4-dependent, drug-tolerant persister phenotype.

Oleic acid availability impacts thymocyte preprogramming and subsequent peripheral Treg cell differentiation.

The nature of activation signals is essential in determining T cell subset differentiation; however, the features that determine T cell subset preference acquired during intrathymic development remain elusive. Here we show that naive CD4+ T cells generated in the mouse thymic microenvironment lacking Scd1, encoding the enzyme catalyzing oleic acid (OA) production, exhibit enhanced regulatory T (Treg) cell differentiation and attenuated development of experimental autoimmune encephalomyelitis. Scd1 deletion in K14+ thymic epithelia recapitulated the enhanced Treg cell differentiation phenotype of Scd1-deficient mice. The dearth of OA permitted DOT1L to increase H3K79me2 levels at the Atp2a2 locus of thymocytes at the DN2-DN3 transition stage. Such epigenetic modification persisted in naive CD4+ T cells and facilitated Atp2a2 expression. Upon T cell receptor activation, ATP2A2 enhanced the activity of the calcium-NFAT1-Foxp3 axis to promote naive CD4+ T cells to differentiate into Treg cells. Therefore, OA availability is critical for preprogramming thymocytes with Treg cell differentiation propensities in the periphery.

Polyamine metabolism is a central determinant of helper T cell lineage fidelity.

Polyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4+ helper T cells (TH) to polarize into different functional fates. Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity.

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.

The Coming Decade of Cell Death Research: Five Riddles.

Active cell death, in its many forms, is a fundamental biological process. Studies over the past several decades have explored the functions and consequences of cellular demise and elucidated several of the key cell death pathways. Here, I pose five questions, or riddles, that might provide a guide to the next decade of cell death research. Focusing mainly on four types of active cell death (apoptosis, necroptosis, pyroptosis, and ferroptosis) mainly in mammals, this Perspective explores the possible research directions that might answer these riddles, or at least prompt new ones.

Autophagy-Independent Functions of the Autophagy Machinery.

Macroautophagy (herein referred to as autophagy) is an evolutionary ancient mechanism that culminates with the lysosomal degradation of superfluous or potentially dangerous cytosolic entities. Over the past 2 decades, the molecular mechanisms underlying several variants of autophagy have been characterized in detail. Accumulating evidence suggests that most, if not all, components of the molecular machinery for autophagy also mediate autophagy-independent functions. Here, we discuss emerging data on the non-autophagic functions of autophagy-relevant proteins.

LC3-Associated Endocytosis Facilitates ß-Amyloid Clearance and Mitigates Neurodegeneration in Murine Alzheimer's Disease.

The expression of some proteins in the autophagy pathway declines with age, which may impact neurodegeneration in diseases, including Alzheimer's Disease. We have identified a novel non-canonical function of several autophagy proteins in the conjugation of LC3 to Rab5+, clathrin+ endosomes containing ß-amyloid in a process of LC3-associated endocytosis (LANDO). We found that LANDO in microglia is a critical regulator of immune-mediated aggregate removal and microglial activation in a murine model of AD. Mice lacking LANDO but not canonical autophagy in the myeloid compartment or specifically in microglia have a robust increase in pro-inflammatory cytokine production in the hippocampus and increased levels of neurotoxic ß-amyloid. This inflammation and ß-amyloid deposition were associated with reactive microgliosis and tau hyperphosphorylation. LANDO-deficient AD mice displayed accelerated neurodegeneration, impaired neuronal signaling, and memory deficits. Our data support a protective role for LANDO in microglia in neurodegenerative pathologies resulting from ß-amyloid deposition.

An Element of Life.

While it has been known for decades that the essential function of selenium was in the form of its incorporation as selenocysteine into selenoproteins-including the enzyme glutathione peroxidase-4-now, Ingold et al. (2018) reveal the precise role of selenolate-based catalysis by this enzyme.

LC3-Associated Phagocytosis in Myeloid Cells Promotes Tumor Immune Tolerance.

Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here, we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single-cell RNA sequencing (RNA-seq) analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.

Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection.

Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.

The clearance of dead cells by efferocytosis.

Multiple modes of cell death have been identified, each with a unique function and each induced in a setting-dependent manner. As billions of cells die during mammalian embryogenesis and daily in adult organisms, clearing dead cells and associated cellular debris is important in physiology. In this Review, we present an overview of the phagocytosis of dead and dying cells, a process known as efferocytosis. Efferocytosis is performed by macrophages and to a lesser extent by other 'professional' phagocytes (such as monocytes and dendritic cells) and 'non-professional' phagocytes, such as epithelial cells. Recent discoveries have shed light on this process and how it functions to maintain tissue homeostasis, tissue repair and organismal health. Here, we outline the mechanisms of efferocytosis, from the recognition of dying cells through to phagocytic engulfment and homeostatic resolution, and highlight the pathophysiological consequences that can arise when this process is abrogated.

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.

STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum.

Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of the mechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms to promote immunity and survival after infection.

A BH3 Mimetic for Killing Cancer Cells.

Venetoclax is a BH3 mimetic approved for treating chronic lymphocytic leukemia. Cancer cells are resistant to apoptosis but "primed for death" by elevated BCL-2, which binds to pro-apoptotic proteins and holds them in check. Venetoclax releases this antagonism and is the first approved drug to target a protein-protein interaction.

BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Localization of a TORC1-eIF4F translation complex during CD8+ T cell activation drives divergent cell fate.

Activated CD8+ T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct T cell fate trajectories can be established during the first division.

The evolution of regulated cell death pathways in animals and their evasion by pathogens.

The coevolution of host-pathogen interactions underlies many human physiological traits associated with protection from or susceptibility to infections. Among the mechanisms that animals utilize to control infections are the regulated cell death pathways of pyroptosis, apoptosis, and necroptosis. Over the course of evolution these pathways have become intricate and complex, coevolving with microbes that infect animal hosts. Microbes, in turn, have evolved strategies to interfere with the pathways of regulated cell death to avoid eradication by the host. Here, we present an overview of the mechanisms of regulated cell death in Animalia and the strategies devised by pathogens to interfere with these processes. We review the molecular pathways of regulated cell death, their roles in infection, and how they are perturbed by viruses and bacteria, providing insights into the coevolution of host-pathogen interactions and cell death pathways.

Caspase-8-Dependent Inflammatory Responses Are Controlled by Its Adaptor, FADD, and Necroptosis.

Cell death pathways regulate various homeostatic processes. Autoimmune lymphoproliferative syndrome (ALPS) in humans and lymphoproliferative (LPR) disease in mice result from abrogated CD95-induced apoptosis. Because caspase-8 mediates CD95 signaling, we applied genetic approaches to dissect the roles of caspase-8 in cell death and inflammation. Here, we describe oligomerization-deficient Caspase-8F122GL123G/F122GL123G and non-cleavable Caspase-8D387A/D387A mutant mice with defective caspase-8-mediated apoptosis. Although neither mouse developed LPR disease, removal of the necroptosis effector Mlkl from Caspase-8D387A/D387A mice revealed an inflammatory role of caspase-8. Ablation of one allele of Fasl, Fadd, or Ripk1 prevented the pathology of Casp8D387A/D387AMlkl-/- animals. Removing both Fadd alleles from these mice resulted in early lethality prior to post-natal day 15 (P15), which was prevented by co-ablation of either Ripk1 or Caspase-1. Our results suggest an in vivo role of the inflammatory RIPK1-caspase-8-FADD (FADDosome) complex and reveal a FADD-independent inflammatory role of caspase-8 that involves activation of an inflammasome.