RESUMEN
Interleukin (IL)-1 family cytokines potently regulate inflammation, with the majority of the IL-1 family proteins being secreted from immune cells via unconventional pathways. In many cases, secretion of IL-1 cytokines appears to be closely coupled to cell death, yet the secretory mechanisms involved remain poorly understood. Here, we studied the secretion of the three best-characterized members of the IL-1 superfamily, IL-1α, IL-1ß, and IL-18, in a range of conditions and cell types, including murine bone marrow-derived and peritoneal macrophages, human monocyte-derived macrophages, HeLa cells, and mouse embryonic fibroblasts. We discovered that IL-1ß and IL-18 share a common secretory pathway that depends upon membrane permeability and can operate in the absence of complete cell lysis and cell death. We also found that the pathway regulating the trafficking of IL-1α is distinct from the pathway regulating IL-1ß and IL-18. Although the release of IL-1α could also be dissociated from cell death, it was independent of the effects of the membrane-stabilizing agent punicalagin, which inhibited both IL-1ß and IL-18 release. These results reveal that in addition to their role as danger signals released from dead cells, IL-1 family cytokines can be secreted in the absence of cell death. We propose that models used in the study of IL-1 release should be considered context-dependently.
Asunto(s)
Células de la Médula Ósea/metabolismo , Interleucina-18/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Macrófagos Peritoneales/metabolismo , Animales , Células de la Médula Ósea/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Taninos Hidrolizables/farmacología , Macrófagos Peritoneales/citología , Ratones , Transporte de Proteínas/efectos de los fármacosRESUMEN
IL-1ß is a potent pro-inflammatory cytokine produced in response to infection or injury. It is synthesized as an inactive precursor that is activated by the protease caspase-1 within a cytosolic molecular complex called the inflammasome. Assembly of this complex is triggered by a range of structurally diverse damage or pathogen associated stimuli, and the signaling pathways through which these act are poorly understood. Ubiquitination is a post-translational modification essential for maintaining cellular homeostasis. It can be reversed by deubiquitinase enzymes (DUBs) that remove ubiquitin moieties from the protein thus modifying its fate. DUBs present specificity toward different ubiquitin chain topologies and are crucial for recycling ubiquitin molecules before protein degradation as well as regulating key cellular processes such as protein trafficking, gene transcription, and signaling. We report here that small molecule inhibitors of DUB activity inhibit inflammasome activation. Inhibition of DUBs blocked the processing and release of IL-1ß in both mouse and human macrophages. DUB activity was necessary for inflammasome association as DUB inhibition also impaired ASC oligomerization and caspase-1 activation without directly blocking caspase-1 activity. These data reveal the requirement for DUB activity in a key reaction of the innate immune response and highlight the therapeutic potential of DUB inhibitors for chronic auto-inflammatory diseases.
Asunto(s)
Caspasa 1/metabolismo , Endopeptidasas/fisiología , Interleucina-1beta/metabolismo , Animales , Carboxipeptidasas/metabolismo , Endopeptidasas/química , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Hidrazonas/farmacología , Hidroxiurea/análogos & derivados , Hidroxiurea/farmacología , Inmunidad Innata , Inflamación , Interleucina-1alfa/metabolismo , Interleucinas/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Ubiquitina TiolesterasaRESUMEN
AZD0171 (INN: Falbikitug) is being developed as a humanized monoclonal antibody (mAb), immunoglobulin G subclass 1 (IgG1), which binds specifically to the immunosuppressive human cytokine leukemia inhibitory factor (LIF) and inhibits downstream signaling by blocking recruitment of glycoprotein 130 (gp130) to the LIF receptor (LIFR) subunit (gp190) and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and is intended to treat adult participants with advanced solid tumors. LIF is a pleiotropic cytokine (and a member of the IL-6 family of cytokines) involved in many physiological and pathological processes and is highly expressed in a subset of solid tumors, including non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer. The aim of this work was to develop a mechanistic PK/PD model to investigate the effect of AZD0171 on tumor LIF levels, predict the level of downstream signaling complex (LIF:LIFR:gp130) inhibition, and examine the dose-response relationship to support dose selection for a Phase II clinical study. Modeling results show that tumor LIF is inhibited in a dose-dependent manner with >90% inhibition for 95% of patients at the Phase II clinical dose of 1500 mg Q2W.
RESUMEN
Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that regulates inflammatory responses to injury and infection. IL-1ß secretion requires the protease caspase-1, which is activated following recruitment to inflammasomes. Endogenous danger-associated molecular patterns (DAMPs) released from necrotic cells activate caspase-1 through an NLRP3-inflammasome. Here, we show that the endogenous lipid metabolite sphingosine (Sph) acts as a DAMP by inducing the NLRP3-inflammasome-dependent secretion of IL-1ß from macrophages. This process was dependent upon serine/threonine protein phosphatases since the PP1/PP2A inhibitors okadaic acid and calyculin A inhibited Sph-induced IL-1ß release. IL-1ß release induced by other well-characterized NLRP3-inflammasome activators, such as ATP and uric acid crystals, in addition to NLRC4 and AIM2 inflammasome activators was also blocked by these inhibitors. Thus, we propose Sph as a new DAMP, and that a serine/threonine phosphatase (PP1/PP2A)-dependent signal is central to the endogenous host mechanism through which diverse stimuli regulate inflammasome activation.
Asunto(s)
Proteínas Portadoras/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Peritonitis/inmunología , Esfingosina/inmunología , Animales , Western Blotting , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Macrófagos Peritoneales/inmunología , Toxinas Marinas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Peritonitis/enzimología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/inmunología , Distribución AleatoriaRESUMEN
Pro-inflammatory members of the interleukin-1 (IL-1) family of cytokines (IL-1alpha and beta) are important mediators of host defense responses to infection but can also exacerbate the damaging inflammation that contributes to major human diseases. IL-1alpha and beta are produced by cells of the innate immune system, such as macrophages, and act largely after their secretion by binding to the type I IL-1 receptor on responsive cells. There is evidence that IL-1alpha is also a nuclear protein that can act intracellularly. In this study, we report that both IL-1alpha and IL-1beta produced by microglia (central nervous system macrophages) in response to an inflammatory challenge are distributed between the cytosol and the nucleus. Using IL-1-beta-galactosidase and IL-1-green fluorescent protein chimeras (analyzed by fluorescence recovery after photobleaching), we demonstrate that nuclear import of IL-1alpha is exclusively active, requiring a nuclear localization sequence and Ran, while IL-1beta nuclear import is entirely passive. These data provide valuable insights into the dynamic regulation of intracellular cytokine trafficking.
Asunto(s)
Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Interleucina-1alfa/genética , Interleucina-1beta/genética , Ratones , Microglía/metabolismo , Datos de Secuencia Molecular , Señales de Localización Nuclear , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
BACKGROUND: Cerebral ischemia is a devastating condition in which the outcome is heavily influenced by inflammatory processes, which can augment primary injury caused by reduced blood supply. The cytokines interleukin-1α (IL-1α) and IL-1ß are key contributors to ischemic brain injury. However, there is very little evidence that IL-1 expression occurs at the protein level early enough (within hours) to influence brain damage after stroke. In order to determine this we investigated the temporal and spatial profiles of IL-1α and IL-1ß expression after cerebral ischemia. FINDINGS: We report here that in mice, as early as 4 h after reperfusion following ischemia induced by occlusion of the middle cerebral artery, IL-1α, but not IL-1ß, is expressed by microglia-like cells in the ischemic hemisphere, which parallels an upregulation of IL-1α mRNA. 24 h after ischemia IL-1α expression is closely associated with areas of focal blood brain barrier breakdown and neuronal death, mostly near the penumbra surrounding the infarct. The sub-cellular distribution of IL-1α in injured areas is not uniform suggesting that it is regulated. CONCLUSIONS: The early expression of IL-1α in areas of focal neuronal injury suggests that it is the major form of IL-1 contributing to inflammation early after cerebral ischemia. This adds to the growing body of evidence that IL-1α is a key mediator of the sterile inflammatory response.
Asunto(s)
Lesiones Encefálicas/inmunología , Lesiones Encefálicas/patología , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Interleucina-1alfa/inmunología , Interleucina-1beta/inmunología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/patología , Receptor 1 de Quimiocinas CX3C , Infarto de la Arteria Cerebral Media , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/citología , Microglía/inmunología , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunologíaRESUMEN
Sterile inflammation is a host response to tissue injury that is mediated by damage-associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro-inflammatory cytokine IL-1 alpha is reported to be a damage-associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL-1 alpha is produced by microglia, the resident brain macrophages. We found that IL-1 alpha is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL-1 alpha to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygen-glucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL-1 alpha released significantly less IL-1 alpha than microglia with predominantly cytosolic IL-1 alpha. The remaining IL-1 alpha was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL-1 alpha may serve to limit inflammation following cell death.
Asunto(s)
Núcleo Celular/metabolismo , Inflamación/metabolismo , Interleucina-1alfa/metabolismo , Necrosis/metabolismo , Animales , Humanos , Microglía/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
Asunto(s)
Afinidad de Anticuerpos , Arginasa/química , Anticuerpos de Cadena Única/química , Regulación Alostérica , Cristalografía por Rayos X , HumanosRESUMEN
NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.
Asunto(s)
Boro/química , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Animales , Células de la Médula Ósea/citología , Boro/farmacología , Compuestos de Boro/química , Compuestos de Boro/metabolismo , Compuestos de Boro/farmacología , Calcio/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Conformación Molecular , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Relación Estructura-ActividadRESUMEN
Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies (anti-CTLA-4, anti-PD-L1, anti-PD-1), which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Antígenos CD40/agonistas , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Microambiente Tumoral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígeno B7-H1/inmunología , Antígenos CD40/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/patología , Distribución AleatoriaRESUMEN
Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.