ABSTRACT
Caspase-11 detection of intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria mediates noncanonical activation of the NLRP3 inflammasome. While avirulent bacteria do not invade the cytosol, their presence in tissues necessitates clearance and immune system mobilization. Despite sharing LPS, only live avirulent Gram-negative bacteria activate the NLRP3 inflammasome. Here, we found that bacterial mRNA, which signals bacterial viability, was required alongside LPS for noncanonical activation of the NLRP3 inflammasome in macrophages. Concurrent detection of bacterial RNA by NLRP3 and binding of LPS by pro-caspase-11 mediated a pro-caspase-11-NLRP3 interaction before caspase-11 activation and inflammasome assembly. LPS binding to pro-caspase-11 augmented bacterial mRNA-dependent assembly of the NLRP3 inflammasome, while bacterial viability and an assembled NLRP3 inflammasome were necessary for activation of LPS-bound pro-caspase-11. Thus, the pro-caspase-11-NLRP3 interaction nucleated a scaffold for their interdependent activation explaining their functional reciprocal exclusivity. Our findings inform new vaccine adjuvant combinations and sepsis therapy.
Subject(s)
Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Caspase 1/metabolism , Caspases , Gram-Negative Bacteria , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Lipopolysaccharides , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , RNA, MessengerABSTRACT
Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 2/metabolism , ATP-Binding Cassette Transporters/metabolism , CD8-Positive T-Lymphocytes/immunology , Cross-Priming , Dendritic Cells/immunology , Histocompatibility Antigens Class I/immunology , Influenza A virus/immunology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/virology , ATP Binding Cassette Transporter, Subfamily B, Member 2/genetics , ATP-Binding Cassette Transporters/genetics , Animals , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , Cell Proliferation , Cells, Cultured , Coculture Techniques , Dendritic Cells/metabolism , Dendritic Cells/virology , Disease Models, Animal , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Female , Golgi Apparatus/immunology , Golgi Apparatus/metabolism , Golgi Apparatus/virology , Histocompatibility Antigens Class I/metabolism , Host-Pathogen Interactions , Humans , Influenza A virus/pathogenicity , Lymphocyte Activation , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Orthomyxoviridae Infections/geneticsABSTRACT
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.
Subject(s)
Gram-Positive Bacteria/physiology , Gram-Positive Bacterial Infections/immunology , Membrane Proteins/metabolism , Phagocytes/immunology , Animals , Autophagy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Female , Male , Mice , Pathogen-Associated Molecular Pattern Molecules/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
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.
Subject(s)
Caspase 8/genetics , Disease Susceptibility , Fas-Associated Death Domain Protein/metabolism , Inflammation/etiology , Inflammation/metabolism , Necroptosis/genetics , Animals , Apoptosis/genetics , Biomarkers , Caspase 8/chemistry , Caspase 8/metabolism , Disease Models, Animal , Disease Progression , Fluorescent Antibody Technique , Gene Expression Regulation , Inflammasomes/metabolism , Inflammation/mortality , Inflammation/pathology , Lipopolysaccharides/adverse effects , Lipopolysaccharides/immunology , Mice , Mice, Knockout , Mortality , Phenotype , Protein MultimerizationABSTRACT
Notch signaling is critical for development and adult tissue physiology, controlling cell fate in a context-dependent manner. Upon ligand binding, the transmembrane Notch receptor undergoes two ordered proteolytic cleavages releasing Notch intracellular domain, which regulates the transcription of Notch target genes. The strength of Notch signaling is of crucial importance and depends notably on the quantity of Notch receptor at the cell surface. Using an shRNA library screen monitoring Notch trafficking and degradation in the absence of ligand, we identified mammalian USP12 and its Drosophila melanogaster homolog as novel negative regulators of Notch signaling. USP12 silencing specifically interrupts Notch trafficking to the lysosomes and, as a consequence, leads to an increased amount of receptor at the cell surface and to a higher Notch activity. At the biochemical level, USP12 with its activator UAF1 deubiquitinate the nonactivated form of Notch in cell culture and in vitro. These results characterize a new level of conserved regulation of Notch signaling by the ubiquitin system.
Subject(s)
Drosophila Proteins/metabolism , Nuclear Proteins/metabolism , Proteolysis , Receptors, Notch/metabolism , Signal Transduction/physiology , Ubiquitin Thiolesterase/metabolism , Animals , Cell Line, Tumor , Drosophila Proteins/genetics , Drosophila melanogaster , Humans , Nuclear Proteins/genetics , Protein Transport/physiology , Receptors, Notch/genetics , Ubiquitin Thiolesterase/geneticsABSTRACT
Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.
Subject(s)
Enzymes/metabolism , Eukaryotic Initiation Factor-3/physiology , Receptors, Notch/metabolism , Ubiquitin/metabolism , Cell Line , Eukaryotic Initiation Factor-3/genetics , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Signal TransductionABSTRACT
The very conserved Notch pathway is used iteratively during development and adulthood to regulate cell fates. Notch activation relies on interactions between neighboring cells, through the binding of Notch receptors to their ligands, both transmembrane molecules. This inter-cellular contact initiates a cascade of events eventually transforming the cell surface receptor into a nuclear factor acting on the transcription of specific target genes. This review highlights how the various processes undergone by Notch receptors and ligands that regulate the pathway are linked to ubiquitination events.
ABSTRACT
The pathogenesis of hepatic fibrosis is driven by dysregulated metabolism precipitated by chronic inflammation. Rho-associated coiled-coil-containing protein kinases (ROCKs) have been implicated in these processes, however the ability of selective ROCK2 inhibition to target simultaneously profibrotic, pro-inflammatory and metabolic pathways remains undocumented. Here we show that therapeutic administration of GV101, a selective ROCK2 inhibitor with more than 1000-fold selectivity over ROCK1, attenuates established liver fibrosis induced by thioacetamide (TAA) in combination with high-fat diet in mice. GV101 treatment significantly reduces collagen levels in liver, associated with downregulation of pCofilin, pSTAT3, pAkt, while pSTAT5 and pAMPK levels are increased in tissues of treated mice. In vitro, GV101 inhibits profibrogenic markers expression in fibroblasts, adipogenesis in primary adipocytes and TLR-induced cytokine secretion in innate immune cells via targeting of Akt-mTOR-S6K signaling axis, further uncovering the ROCK2-specific complex mechanism of action and therapeutic potential of highly selective ROCK2 inhibitors in liver fibrosis.
Subject(s)
Inflammation , Liver Cirrhosis , Mice , Animals , Fibrosis , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Inflammation/drug therapy , Inflammation/pathology , Signal TransductionABSTRACT
The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.
Subject(s)
Endocytosis/physiology , Protein Serine-Threonine Kinases/metabolism , Receptors, Notch/metabolism , Adaptor Proteins, Signal Transducing , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Membrane/genetics , Cell Membrane/metabolism , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Metalloproteases/genetics , Metalloproteases/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Stability , Receptors, Notch/genetics , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolismABSTRACT
Inflammasomes are multiprotein complexes that assemble upon detection of danger signals to activate the inflammatory enzyme caspase-1, trigger secretion of the highly proinflammatory cytokine IL-1ß, and induce an inflammatory cell death called pyroptosis. Distinctiveness of the nucleotide-binding oligomerization (NOD), Leucine-rich repeat (LRR)-containing protein (NLRP3) inflammasome resides in the diversity of molecules that induce its activation, indicating a certain intricacy. Furthermore, besides the canonical activation of NLRP3 in response to various stimuli, caspase-11-dependent detection of intracellular LPS activates NLRP3 through a noncanonical pathway. Several aspects of the NLRP3 inflammasome are not characterized or remain unclear. In this review, we summarize the different modes of NLRP3 activation. We describe recent insights into post-translational and cellular regulation that confer further complexity to NLRP3 inflammasomes.
Subject(s)
Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Animals , Humans , Models, Immunological , Protein Processing, Post-Translational , Subcellular Fractions/metabolismABSTRACT
Phagocytes cope with the threat of living bacteria via detection of vita-PAMPs, a specific class of pathogen-associated molecular patterns (PAMPs) that denotes microbial viability and trigger a commensurate innate response. Prokaryotic mRNA and cyclic-di-adenosine monophosphate (c-di-AMP) serve as vita-PAMPs for Gram-negative and Gram-positive bacteria, respectively, and elicit heightened proinflammatory responses not warranted for dead bacteria. The innate sensor TMEM173/STING detects c-di-AMP produced by internalized live Gram-positive bacteria, and quickly mobilizes interdependent pre-formed cell-autonomous responses including endoplasmic reticulum (ER) stress, MTOR inactivation, and reticulophagy. In turn, reticulophagy serves a dual role in restoring phagocyte homeostasis and orchestrating a type I IFN response. ER-stress induced macroautophagy/autophagy sequesters stressed ER, resolves ER stress and prevents apoptosis in response to live bacteria. Reticulophagy relocalizes ER-resident TMEM173/STING to phagophores, which then act as TMEM173/STING-signaling compartments. Here, we discuss our findings in the context of innate immunity and cell homeostasis.
Subject(s)
Autophagy , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Immunity, Innate , Microbial ViabilityABSTRACT
The innate immune system directly senses microbial viability via the detection of a special class of viability-associated pathogen-associated molecular patterns (vita-PAMPs), such as prokaryotic messenger RNA. In the case of Gram-negative bacteria, detection of bacterial viability by phagocytes leads to a unique activation of inflammasome and type I interferon pathways, resulting in a robust pro-inflammatory innate response and a vigorous adaptive immune response. This protocol describes the methods required to study activation of both inflammasome and type I interferon pathways after stimulation of mouse bone marrow-derived macrophages with live or killed Gram-negative and Gram-positive bacteria. It covers the generation and handling of bone marrow-derived macrophages, the culture and killing of bacteria, the preparation of bacterial messenger RNA, and the stimulation of macrophages with live or killed bacteria. Lastly, this protocol describes the techniques employed to measure the hallmarks of inflammasome (secretion of interleukin-1ß) and type I interferon (activation of TBK1, IRF3 and secretion of type I interferon) pathways.
Subject(s)
Bacteria/immunology , Inflammasomes/analysis , Interferon Type I/analysis , Macrophages/immunology , Microbial Viability/immunology , Animals , Bacteria/growth & development , Immunity, Innate , Inflammasomes/immunology , Interferon Type I/immunology , Mice , Signal TransductionABSTRACT
The innate immune response of phagocytes to microbes has long been known to depend on the core signaling cascades downstream of pattern recognition receptors (PRRs), which lead to expression and production of inflammatory cytokines that counteract infection and induce adaptive immunity. Cell-autonomous responses have recently emerged as important mechanisms of innate immunity. Either IFN-inducible or constitutive, these processes aim to guarantee cell homeostasis but have also been shown to modulate innate immune response to microbes and production of inflammatory cytokines. Among these constitutive cell-autonomous responses, autophagy is prominent and its role in innate immunity has been well characterized. Other stress responses, such as metabolic stress, the ER stress/unfolded protein response, mitochondrial stress, or the DNA damage response, seem to also be involved in innate immunity, although the precise mechanisms by which they regulate the innate immune response are not yet defined. Of importance, these distinct constitutive cell-autonomous responses appear to be interconnected and can also be modulated by microbes and PRRs, which add further complexity to the interplay between innate immune signaling and cell-autonomous responses in the mediation of an efficient innate immune response.
Subject(s)
Immunity, Innate , Stress, Physiological/immunology , Animals , Autophagy/immunology , DNA Damage , Endoplasmic Reticulum Stress , Humans , TOR Serine-Threonine Kinases/metabolismABSTRACT
A decade of work shows that the core function of phagocytosis in engulfment and destruction of microorganisms is only a small facet of the full spectrum of roles for phagocytosis in the immune system. The regulation of phagocytosis and its outcomes by inflammatory pattern recognition receptors (PRRs) is now followed by new studies strengthening this concept and adding further complexity to the relationship between phagocytosis and innate immune signaling. Phagocytosis forms the platform for activation of distinct members of the Toll-like receptor family, and even dictates their signaling outcomes. In many cases, phagocytosis is a necessary precedent to the activation of cytosolic PRRs and assembly of canonical and non-canonical inflammasomes, leading to strong pro-inflammatory responses and inflammatory cell death.
Subject(s)
Immunity, Innate , Inflammasomes/metabolism , Phagocytes/immunology , Phagocytosis/immunology , Receptors, Pattern Recognition/metabolism , Animals , Cell Death/immunology , Cytosol/immunology , Cytosol/metabolism , Cytosol/microbiology , Endosomes/immunology , Endosomes/metabolism , Endosomes/microbiology , Humans , Nucleic Acids/metabolism , Phagocytes/metabolism , Phagocytes/microbiology , Phagosomes/immunology , Phagosomes/metabolism , Phagosomes/microbiology , Receptors, Pattern Recognition/physiology , Signal Transduction/immunologyABSTRACT
The activity of Notch ligands is tightly regulated by trafficking events occurring both before and after ligand-receptor interaction. In particular endocytosis and recycling have been shown to be required for full signaling activity of the ligands before they encounter the Notch receptor. However little is known about the precise endocytic processes that contribute to ligand internalization. Here we demonstrate that endocytosis contributes to Dll1 signaling activity by preserving the ligand from shedding and degradation. We further show that the glycosphingolipid-binding motif originally identified in Drosophila Notch ligands is conserved in mammals and is necessary for Dll1 internalization. Mutation of its conserved tryptophan residue results in a Dll1 molecule which is rapidly inactivated by shedding and degradation, does not recycle to the cell surface and does not activate Notch signaling. Finally, silencing in the signal-sending cells of glucosylceramide synthase, the enzyme implicated in the initial phase of glycosphingolipid synthesis, down-regulates Notch activation. Our data indicate that glycosphingolipids, by interacting with Dll1, may act as functional co-factors to promote its biological activity.
Subject(s)
Glycosphingolipids/metabolism , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Protein Interaction Domains and Motifs , Receptors, Notch/metabolism , Amino Acid Sequence , Animals , Cell Membrane/chemistry , Cell Membrane/metabolism , Gene Silencing , Glucosyltransferases/genetics , Glycosphingolipids/chemistry , Humans , Ligands , Membrane Lipids/metabolism , Mice , Molecular Sequence Data , Protein Binding , Protein Stability , Protein Transport , RNA Interference , Signal TransductionABSTRACT
The metalloprotease ADAM10/Kuzbanian catalyzes the ligand-dependent ectodomain shedding of Notch receptors and activates Notch. Here, we show that the human tetraspanins of the evolutionary conserved TspanC8 subfamily (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33) directly interact with ADAM10, regulate its exit from the endoplasmic reticulum, and that four of them regulate ADAM10 surface expression levels. In an independent RNAi screen in Drosophila, two TspanC8 genes were identified as Notch regulators. Functional analysis of the three Drosophila TspanC8 genes (Tsp3A, Tsp86D, and Tsp26D) indicated that these genes act redundantly to promote Notch signaling. During oogenesis, TspanC8 genes were up-regulated in border cells and regulated Kuzbanian distribution, Notch activity, and cell migration. Furthermore, the human TspanC8 tetraspanins Tspan5 and Tspan14 positively regulated ligand-induced ADAM10-dependent Notch1 signaling. We conclude that TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch receptor activation.