The Nlrp3 inflammasome is critical for host immunity, but the mechanisms controlling its activation are enigmatic. Here, we show that loss of FADD or caspase-8 in a RIP3-deficient background - but not RIP3-deficiency alone - hampered transcriptional priming and post-translational activation of the canonical and non-canonical Nlrp3 inflammasome. Deletion of caspase-8 in the presence or absence of RIP3 inhibited caspase-1 and caspase-11 activation by Nlrp3 stimuli, but not the Nlrc4 inflammasome. FADD deletion in addition prevented caspase-8 maturation, positioning FADD upstream of caspase-8. Consequently, FADD- and caspase-8-deficient mice had impaired IL-1β production when challenged with LPS or infected with the enteropathogen C. rodentium. Thus, our results reveal FADD and caspase-8 as apical mediators of canonical and non-canonical Nlrp3 inflammasome priming and activation.
inflammasome; caspase-8; FADD; NLRP3; caspase-1; caspase-11; NLR
Regulated necrosis; RIPK1; RIPK3; MLKL; Necrostatin-1; mitochondrial permeability transition; necrosome
Phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) is fundamental to vision. Autophagy is also responsible for bulk degradation of cellular components but its role in POS degradation is not well understood. We report that the morning burst of RPE phagocytosis coincided with the enzymatic conversion of autophagy protein LC3 to its lipidated form. LC3 then associated with single membrane phagosomes containing engulfed POS in an Atg5 dependent manner that required Beclin1 but not the autophagy pre-initiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted lysosomal processing. These mice also exhibited decreased photoreceptor responses to light stimuli and decreased chromophore levels that were restored with exogenous retinoid supplementation. These results establish that the interplay of phagocytosis and autophagy within the RPE are required for both POS degradation and the maintenance of retinoid levels to support vision.
The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.
autophagy; phagocytosis; vision; visual cycle; 11-cis retinal; vitamin A; all-trans retinol; ATG5; LC3; retinal pigment epithelium; photoreceptors
The type I interferon (IFN) response represents the first line of defence to invading pathogens. Internalized viral ribonucleoproteins (vRNPs) of negative-strand RNA viruses induce an early IFN response by interacting with retinoic acid inducible gene I (RIG-I) and its recruitment to mitochondria. Here we employ three-dimensional stochastic optical reconstruction microscopy (STORM) to visualize incoming influenza A virus (IAV) vRNPs as helical-like structures associated with mitochondria. Unexpectedly, an early IFN induction in response to vRNPs is not detected. A distinct amino-acid motif in the viral polymerases, PB1/PA, suppresses early IFN induction. Mutation of this motif leads to reduced pathogenicity in vivo, whereas restoration increases it. Evolutionary dynamics in these sequences suggest that completion of the motif, combined with viral reassortment can contribute to pandemic risks. In summary, inhibition of the immediate anti-viral response is ‘pre-packaged’ in IAV in the sequences of vRNP-associated polymerase proteins.
It is unclear how incoming influenza viruses counteract the cells’ first line of defence, the interferon (IFN) response. Here Liedmann et al. show that a distinct amino-acid motif in polymerases PB1 and PA, which are packaged in the viral particles, inhibit early IFN induction.
Under conditions of genotoxic stress, human p53 activates the apoptotic effectors BAX or BAK, resulting in mitochondrial outer membrane permeabilization and apoptosis. Anti-apoptotic BCL-2 family member BCL-xL opposes this activity by sequestering cytosolic p53 via association with its DNA-binding domain, an interaction that is enhanced by p53 tetramerization. Here we characterized the BCL-xL – p53 complex using NMR spectroscopy and modulated it through mutagenesis to determine the relative contributions of BCL-xL’s interactions with p53, or with other BCL-2 family proteins, to BCL-xL-dependent inhibition of UV irradiation-induced apoptosis. Under our experimental conditions, one third of the anti-apoptotic activity of BCL-xL was mediated by p53 sequestration and the remaining two thirds through sequestration of pro-apoptotic BCL-2 family members. Our studies define the contributions of cytosolic p53 to UV irradiation-induced apoptosis and provide opportunities to explore its contributions to other, p53-dependent apoptotic signaling pathways.
Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fancony Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.
Sepsis; ATM; Autophagy; Anthracyclines
Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII) mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis, in a novel mechanism of CaMKII activation. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca2+ to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes, but also in Murine oocytes. These findings uncover a novel mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.
Caspase-8 or cFLIP deficiency leads to embryonic lethality in mice due to defects in endothelial tissues. Caspase-8−/−, RIPK3−/−, but not cFLIP−/−, RIPK3−/−, double-knockout animals develop normally, indicating that caspase-8 antagonizes the lethal effects of RIPK3 during development. Here we show that the acute deletion of caspase-8 in the gut of adult mice induces enterocyte death, disruption of tissue homeostasis and inflammation, resulting in sepsis and mortality. Likewise, acute deletion of caspase-8 in a focal region of the skin induces local keratinocyte death, tissue disruption and inflammation. Strikingly, RIPK3 ablation rescues both phenotypes. Acute loss of cFLIP in the skin produces a similar phenotype, which, however, is not rescued by RIPK3 ablation. TNF neutralization protects from either acute loss of caspase-8 or cFLIP. These results demonstrate that caspase-8-mediated suppression of RIPK3-induced death is required not only during development, but also for adult homeostasis. Furthermore, RIPK3-dependent inflammation is dispensable for the skin phenotype.
Necroptosisis mediated by engagement of RIP-kinases and a downstream pseudokinase, MLKL. In this issue of Immunity, Murphy et al. (2013) show that it operates at or close to the final execution mechanism of the death process.
“Fa, fa, fa, fa, fa, fa, fa, fa, fa, fa”David Byrne
Upon activation, quiescent naive T cells undergo a growth phase followed by massive clonal expansion and differentiation that are essential for appropriate immune defense and regulation. Accumulation of cell biomass during the initial growth and rapid proliferation during the expansion phase is associated with dramatically increased bioenergetic and biosynthetic demands. This not only requires a metabolic rewiring during the transition between resting and activation, but also ‘addicts’ active T cells to certain metabolic pathways in ways that naive and memory T cells are not. We consider such addiction in terms of the biological effects of deprivation of metabolic substrates or inhibition of specific pathways in T cells. In this review, we illustrate the relevant metabolic pathways revealed by recent metabolic flux analysis and discuss the consequences of metabolic intervention on specific metabolic pathways in T lymphocytes.
metabolism; T lymphocytes; T-cell activation
The nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signaling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here, we show that more than two thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells—the cell of origin of ependymoma—to form these tumours in mice. Our data identify the first highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.
Sirtuins can promote deacetylation of a wide range of substrates in diverse cellular compartments and regulate many cellular processes1,2. Recently Narayan et al., reported that SIRT2 was required for necroptosis based on their findings that SIRT2 inhibition, knock-down or knock-out prevented necroptosis. We sought to confirm and explore the role of SIRT2 in necroptosis and tested four different sources of the SIRT2 inhibitor AGK2, three independent siRNAs against SIRT2, and cells from two independently generated Sirt2−/− mouse strains, however we were unable to show that inhibiting or depleting SIRT2 protected cells from necroptosis. Furthermore, Sirt2−/− mice succumbed to TNF induced Systemic Inflammatory Response Syndrome (SIRS) more rapidly than wild type mice while Ripk3−/− mice were resistant. Our results therefore question the importance of SIRT2 in the necroptosis cell death pathway.
Signal transduction and metabolism cooperate to control cell fate, but mechanisms that link metabolic substrates to functional decisions are elusive. Now, Chang et al. in Cell provide a mechanism whereby available sugars dictate metabolic pathways in activated T cells and direct a non-metabolic regulatory function of glyceraldehyde-3-phosphate dehydrogenase.
The innate immune response is shaped by multiple factors, including both traditional autophagy and LC3-associated phagocytosis (LAP). As the autophagic machinery is engaged during times of nutrient stress, arising from scarcity or pathogens, we examine how autophagy, specifically LAP, and cellular metabolism together influence macrophage function and the innate immune response.
Overcoming platinum drug resistance represents a major clinical challenge in cancer treatment. We discovered a novel drug combination using cisplatin and a class of thioquinazolinone derivatives including mdivi-1 (mitochondrial division inhibitor-1), that induces synergistic apoptosis in platinum resistant tumor cells, including those from cisplatin-refractory endstage ovarian cancer patients. However, through study of the combination effect on Drp1 (the reported target of mdivi-1) knockout MEF cells and the functional analysis of mdivi-1 analogs, we revealed that the synergism between mdivi-1 and cisplatin is Drp1-independent. Mdivi-1 impairs DNA replication and its combination with cisplatin induces a synergistic increase of replication stress and DNA damage, causing a preferential upregulation of a BH3-only protein Noxa. Mdivi-1 also represses mitochondrial respiration independent of Drp1, and the combination of mdivi-1 and cisplatin triggers substantial mitochondrial uncoupling and swelling. Upregulation of Noxa and simultaneous mitochondrial swelling causes synergistic induction of mitochondrial outer membrane permeabilization (MOMP), proceeding robust mitochondrial apoptotic signaling independent of Bax/Bak. Thus, the novel mode of MOMP induction by the combination through the “dual-targeting” potential of mdivi-1 on DNA replication and mitochondrial respiration suggests a novel class of compounds for platinum-based combination option in the treatment of platinum as well as multidrug resistant tumors.
Platinum resistance; mdivi-1; replication stress; Noxa; mitochondrial swelling
To fulfill the bioenergetic and biosynthetic demand of proliferation, T cells reprogram their metabolic pathways from fatty acid β-oxidation and pyruvate oxidation via the TCA cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways. Two of the top-ranked candidate transcription factors potentially responsible for the activation-induced T cell metabolic transcriptome, HIF1α and Myc, were induced upon T cell activation, but only the acute deletion of Myc markedly inhibited activation-induced glycolysis and glutaminolysis in T cells. Glutamine deprivation compromised activation-induced T cell growth and proliferation, and this was partially replaced by nucleotides and polyamines, implicating glutamine as an important source for biosynthetic precursors in active T cells. Metabolic tracer analysis revealed a Myc-dependent metabolic pathway linking glutaminolysis to the biosynthesis of polyamines. Therefore, a Myc-dependent global metabolic transcriptome drives metabolic reprogramming in activated, primary T lymphocytes. This may represent a general mechanism for metabolic reprogramming under patho-physiological conditions.
Caspase-8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase-8 activation involves dimerization and subsequent interdomain autoprocessing of caspase-8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase-8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase-8 deficient mice are shared by mice lacking the dimerization adapter FADD or the caspase paralog FLIPL has led to the hypothesis that FADD-dependent formation of heterodimers between caspase-8 and FLIPL could mediate the developmental role of caspase-8. Using an inducible dimerization system we demonstrate that cleavage of the catalytic domain of caspase-8 is crucial for its activity in the context of activation by homodimerization. However, we find that use of FLIPL as a partner for caspase-8 in dimerization-induced activation rescues the requirement for intersubunit linker proteolysis in both protomers. Moreover, before processing, caspase-8 in complex with FLIPL does not generate a fully active enzyme, but an attenuated species able to process only select natural substrates. Based on these results we propose a mechanism of caspase-8 activation by dimerization in the presence of FLIPL, as well as a mechanism of caspase-8 functional divergence in apoptotic and non-apoptotic pathways.
apoptosis; activation mechanism; protein dimerization
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 TNF-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisol (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Further, while 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.
HIF1α induction by mTOR represents a metabolic checkpoint for the differentiation of TH17 and Treg cells.
Upon antigen stimulation, the bioenergetic demands of T cells increase dramatically over the resting state. Although a role for the metabolic switch to glycolysis has been suggested to support increased anabolic activities and facilitate T cell growth and proliferation, whether cellular metabolism controls T cell lineage choices remains poorly understood. We report that the glycolytic pathway is actively regulated during the differentiation of inflammatory TH17 and Foxp3-expressing regulatory T cells (Treg cells) and controls cell fate determination. TH17 but not Treg cell–inducing conditions resulted in strong up-regulation of the glycolytic activity and induction of glycolytic enzymes. Blocking glycolysis inhibited TH17 development while promoting Treg cell generation. Moreover, the transcription factor hypoxia-inducible factor 1α (HIF1α) was selectively expressed in TH17 cells and its induction required signaling through mTOR, a central regulator of cellular metabolism. HIF1α–dependent transcriptional program was important for mediating glycolytic activity, thereby contributing to the lineage choices between TH17 and Treg cells. Lack of HIF1α resulted in diminished TH17 development but enhanced Treg cell differentiation and protected mice from autoimmune neuroinflammation. Our studies demonstrate that HIF1α–dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells.
The BCL-2 family of proteins regulates apoptosis by controlling mitochondrial outer membrane permeabilization (MOMP). Within the family there are numerous protein-protein interactions that influence MOMP; however, defining the ultimate signal that commits a cell to apoptosis remains controversial. We chose to examine the function of the BH3-only protein, p53 upregulated modulator of apoptosis (PUMA), to define its contribution to MOMP and cooperation with the direct activator proteins. PUMA is a potent regulator of MOMP and our data suggest that this function is attributed to two distinct mechanisms which both rely on PUMA binding to the anti-apoptotic BCL-2 proteins: de-repression and sensitization. Here we will define these interactions and discuss our experiments that suggest PUMA cooperates with direct activator proteins to efficiently induce MOMP and apoptosis.
apoptosis; BCL-2 family; cytochrome c release; MOMP; PUMA
Fas, a tumor necrosis factor family receptor, is activated by the membrane protein Fas ligand (FasL) expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas induced cytokines, the IL-1β family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1β cytokines via caspase-1. The mechanisms, by which Fas regulates IL-1β activation, however, remain unresolved. Here, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells leading to the maturation of IL-1β and IL-18 independently of inflammasomes or Rip3. Hence, Fas controls a novel non-canonical IL-1β activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance and control of infectious diseases.
Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.
NOD2 receptor and the cytosolic protein kinase RIPK2 regulate NF-κB and MAP kinase signaling during bacterial infections, but the role of this immune axis during viral infections has not been addressed. We demonstrate that Nod2−/− and Ripk2−/− mice are hypersusceptible to influenza A virus infection. Ripk2−/− cells displayed defective mitophagy leading to enhanced mitochondrial superoxide production and accumulation of damaged mitochondria resulting in increased NLRP3 inflammasome activation and IL-18 production. RIPK2 regulated mitophagy in a kinase-dependent manner by phosphorylating the mitophagy inducer ULK1. Accordingly, Ulk1−/− cells displayed enhanced mitochondrial superoxide production and caspase-1 activation. These results demonstrate a role for NOD2-RIPK2 signaling in protection against virally triggered immunopathology by negatively regulating NLRP3 inflammasome activation and IL-18 production via ULK1-dependent mitophagy.
RIPK2; NLR; Caspase-1; inflammasome; Influenza; IL-18; autophagy; mitophagy
Cells die by a variety of mechanisms, only some of which have been elucidated in detail. A number of `active' forms of cell death exist in which the cell participates in its own death, including apoptosis, programmed necrosis, mitotic catastrophe, and the recently described ferroptosis, among other processes. Here, we attempt to explain why there are so many different forms of cell death, and propose a distinction between active death that is `suicide' versus `sabotage.'