RESUMEN
Immunosenescence poses a significant challenge to tumor immunotherapy in elderly individuals. In this issue of Cell, Zhivaki et al. elucidate that dendritic cells "hyperactivated" by specific adjuvants elicit TH1-skewed CD4+ T cell responses in a manner contingent on the NLRP3 inflammasome, which can eliminate tumors in aged mice.
Asunto(s)
Células Dendríticas , Animales , Células Dendríticas/inmunología , Ratones , Neoplasias/inmunología , Neoplasias/terapia , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Inflamasomas/inmunología , Inmunoterapia/métodos , Linfocitos T CD4-Positivos/inmunología , Humanos , Envejecimiento/inmunología , Células TH1/inmunología , InmunosenescenciaRESUMEN
Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.
Asunto(s)
Adaptación Biológica , Evolución Biológica , Peces/genética , Secuenciación Completa del Genoma , Aletas de Animales/anatomía & histología , Aletas de Animales/fisiología , Animales , Extremidades/anatomía & histología , Extremidades/fisiología , Peces/anatomía & histología , Peces/clasificación , Peces/fisiología , Filogenia , Fenómenos Fisiológicos Respiratorios , Sistema Respiratorio/anatomía & histología , Vertebrados/genéticaRESUMEN
The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.
Asunto(s)
Inflamasomas/ultraestructura , Complejos Multiproteicos/ultraestructura , Proteínas de Unión a Fosfato/ultraestructura , Piroptosis/genética , Animales , Caspasa 1/química , Caspasa 1/genética , Caspasa 1/ultraestructura , Caspasas Iniciadoras/química , Caspasas Iniciadoras/genética , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inflamasomas/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Proteínas de Unión a Fosfato/química , Proteínas de Unión a Fosfato/genética , Conformación Proteica en Lámina beta/genética , Dominios Proteicos/genética , Procesamiento Proteico-Postraduccional/genética , ProteolisisRESUMEN
Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Bacterianas/genética , Macroautofagia , Salmonella/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Factores de Virulencia/genética , ADP-Ribosilación , Proteínas Relacionadas con la Autofagia/deficiencia , Proteínas Relacionadas con la Autofagia/genética , Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Salmonella/patogenicidad , Sistemas de Secreción Tipo III/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Factores de Virulencia/metabolismoRESUMEN
A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEß7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation.
Asunto(s)
Interleucina-33 , Mastocitos , Proteínas de Unión a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Animales , Ratones , Comunicación Celular/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Interleucina-33/metabolismo , Interleucina-33/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/inmunología , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMEN
This SnapShot depicts how the noncanonical inflammasome pathway is initiated and activated, as well as its effector mechanism in triggering pyroptosis and immune defenses.
Asunto(s)
Infecciones/inmunología , Inflamasomas/inmunología , Animales , Caspasas/metabolismo , Humanos , Inmunidad Innata , Infecciones/metabolismo , Inflamasomas/metabolismo , Transducción de SeñalRESUMEN
Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1ß and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.
Asunto(s)
Caspasas/inmunología , Citosol/inmunología , Inmunidad Innata/inmunología , Lipopolisacáridos/inmunología , Animales , Caspasas/metabolismo , Citosol/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/metabolismo , Modelos Inmunológicos , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Fosfato , Piroptosis/inmunologíaRESUMEN
The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.
Asunto(s)
Barrera Hematoencefálica , Encéfalo , Células Endoteliales , Gasderminas , Inflamación , Animales , Femenino , Humanos , Masculino , Ratones , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/ultraestructura , Barrera Hematoencefálica/virología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Caspasas Iniciadoras/metabolismo , Dependovirus , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Gasderminas/antagonistas & inhibidores , Gasderminas/metabolismo , Inflamación/patología , Inflamación/metabolismo , Klebsiella pneumoniae/fisiología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Piroptosis , Sepsis/metabolismo , Sepsis/patología , Sepsis/microbiología , Análisis de la Célula Individual , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructuraRESUMEN
The canonical (caspase-1) and noncanonical (comprising caspases 4, 5 and 11; hereafter, caspase-4/5/11) inflammasomes both cleave gasdermin D (GSDMD) to induce pyroptosis1,2. Whereas caspase-1 processes IL-1ß and IL-18 for maturation3-6, no cytokine target has been firmly established for lipopolysaccharide-activated caspase-4/5/117-9. Here we show that activated human caspase-4, but not mouse caspase-11, directly and efficiently processes IL-18 in vitro and during bacterial infections. Caspase-4 cleaves the same tetrapeptide site in pro-IL-18 as caspase-1. The crystal structure of the caspase-4-pro-IL-18 complex reveals a two-site (binary) substrate-recognition mechanism; the catalytic pocket engages the tetrapeptide, and a unique exosite that critically recognizes GSDMD10 similarly binds to a specific structure formed jointly by the propeptide and post-cleavage-site sequences in pro-IL-18. This binary recognition is also used by caspase-5 as well as caspase-1 to process pro-IL-18. In caspase-11, a structural deviation around the exosite underlies its inability to target pro-IL-18, which is restored by rationally designed mutations. The structure of pro-IL-18 features autoinhibitory interactions between the propeptide and the post-cleavage-site region, preventing recognition by the IL-18Rα receptor. Cleavage by caspase-1, -4 or -5 induces substantial conformational changes of IL-18 to generate two critical receptor-binding sites. Our study establishes IL-18 as a target of lipopolysaccharide-activated caspase-4/5. The finding is paradigm shifting in the understanding of noncanonical-inflammasome-mediated defences and also the function of IL-18 in immunity and disease.
Asunto(s)
Inflamasomas , Péptidos y Proteínas de Señalización Intracelular , Humanos , Inflamasomas/metabolismo , Interleucina-18 , Lipopolisacáridos/farmacología , Caspasas/metabolismo , Caspasa 1/metabolismo , PiroptosisRESUMEN
Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein1,2, to trigger target cell pyroptosis3. GSDMB and the charter gasdermin family member GSDMD4,5 have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. 6,7). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently6,8. Here we report the crystal structure of the IpaH7.8-GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins9,10. GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies6,8. Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms.
Asunto(s)
Gasderminas , Proteínas Citotóxicas Formadoras de Poros , Animales , Humanos , Ratones , Línea Celular Tumoral , Microscopía por Crioelectrón , Cristalografía por Rayos X , Gasderminas/química , Gasderminas/genética , Gasderminas/metabolismo , Gasderminas/ultraestructura , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/ultraestructura , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Citotóxicas Formadoras de Poros/ultraestructura , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructura , Piroptosis , Shigella flexneri , Especificidad de la Especie , Empalme AlternativoRESUMEN
Using a forward-genetic screening of macrophages from randomly mutagenized mice, Kayagaki et al. (2021) identify NINJ1 that mediates plasma membrane rupture following various types of programmed cell death, an event previously thought to be passive.
Asunto(s)
Moléculas de Adhesión Celular Neuronal , Factores de Crecimiento Nervioso , Animales , Membrana Celular , Células Endoteliales , Macrófagos , RatonesRESUMEN
Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gram-negative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis.
Asunto(s)
Coagulación Sanguínea , Inflamasomas/metabolismo , Piroptosis , Trombosis/etiología , Trombosis/metabolismo , Animales , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/microbiología , Biomarcadores , Caspasas/metabolismo , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/metabolismo , Modelos Animales de Enfermedad , Humanos , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Monocitos/inmunología , Monocitos/metabolismo , Transducción de Señal , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/mortalidadRESUMEN
Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Shigella flexneri/patogenicidad , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Autofagia , Disentería Bacilar/inmunología , Disentería Bacilar/microbiología , Escherichia coli Enteropatógena/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Fibroblastos/metabolismo , Interleucina-8/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Shigella flexneri/metabolismo , Virulencia , Factores de Virulencia/químicaRESUMEN
Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD1-5. This non-canonical inflammasome defends against Gram-negative bacteria6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/metabolismo , Caspasas Iniciadoras/metabolismo , Evasión Inmune , Piroptosis , Shigella flexneri/patogenicidad , Adenosina Difosfato/metabolismo , Animales , Disentería Bacilar/inmunología , Disentería Bacilar/microbiología , Femenino , Inmunidad Humoral , Inflamasomas/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , NAD/metabolismo , Piroptosis/efectos de los fármacos , Vacunas contra la Shigella , Shigella flexneri/inmunología , VirulenciaRESUMEN
Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.
Asunto(s)
Escherichia coli Enteropatógena/genética , Proteínas de Escherichia coli/química , Interacciones Huésped-Patógeno/genética , Conformación Proteica , Factores de Virulencia/química , Animales , Apoptosis/genética , Arginina/química , Arginina/genética , Coenzima A Ligasas/química , Coenzima A Ligasas/genética , Cristalografía por Rayos X , Dominio de Muerte/genética , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/genética , Guanidina/química , Humanos , Manganeso/química , Ratones , Mutagénesis , Proteína de Dominio de Muerte Asociada a Receptor de TNF/química , Proteína de Dominio de Muerte Asociada a Receptor de TNF/genética , Factores de Virulencia/genéticaRESUMEN
Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1-5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and 'cleaves' a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody-drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein-including an active gasdermin-from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.
Asunto(s)
Preparaciones de Acción Retardada/administración & dosificación , Neoplasias Mamarias Experimentales/inmunología , Piroptosis/inmunología , Animales , Cumarinas/administración & dosificación , Cumarinas/química , Cumarinas/metabolismo , Cumarinas/farmacocinética , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/metabolismo , Preparaciones de Acción Retardada/farmacocinética , Femenino , Proteínas Fluorescentes Verdes/administración & dosificación , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/farmacocinética , Células HeLa , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Inmunoconjugados/farmacocinética , Inflamasomas/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/farmacocinética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas/administración & dosificación , Proteínas/química , Proteínas/metabolismo , Proteínas/farmacocinética , Silanos/administración & dosificación , Silanos/química , Silanos/metabolismo , Silanos/farmacocinética , Linfocitos T/inmunología , Trastuzumab/administración & dosificación , Trastuzumab/química , Trastuzumab/metabolismo , Trastuzumab/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1ß (IL1ß) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.
Asunto(s)
Gasderminas , Infecciones por Salmonella , Animales , Ratones , Infecciones por Salmonella/prevención & control , Salmonella typhimurium , Inflamación , Células Epiteliales , InflamasomasRESUMEN
Microbial pathogens have evolved complex mechanisms to interface with host cells in order to evade host defenses and replicate. However, mammalian innate immune receptors detect the presence of molecules unique to the microbial world or sense the activity of virulence factors, activating antimicrobial and inflammatory pathways. We focus on how studies of the major virulence factor of one group of microbial pathogens, the type III secretion system (T3SS) of human pathogenic Yersinia, have shed light on these important innate immune responses. Yersinia are largely extracellular pathogens, yet they insert T3SS cargo into target host cells that modulate the activity of cytosolic innate immune receptors. This review covers both the host pathways that detect the Yersinia T3SS and the effector proteins used by Yersinia to manipulate innate immune signaling.
Asunto(s)
Citosol/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Sistemas de Secreción Tipo III/inmunología , Yersinia/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Citosol/microbiología , Humanos , Inflamasomas , Piroptosis , Transducción de Señal , Factores de Virulencia/metabolismo , Yersinia/metabolismo , Yersinia/patogenicidadRESUMEN
Electrochemical reduction of CO(2) to value-added chemicals and fuels is a promising strategy to sustain pressing renewable energy demands and to address climate change issues. Direct observation of reaction intermediates during the CO(2) reduction reaction will contribute to mechanistic understandings and thus promote the design of catalysts with the desired activity, selectivity, and stability. Herein, we combined in situ electrochemical shell-isolated nanoparticle-enhanced Raman spectroscopy and ab initio molecular dynamics calculations to investigate the CORR process on Cu single-crystal surfaces in various electrolytes. Competing redox pathways and coexistent intermediates of CO adsorption (*COatop and *CObridge), dimerization (protonated dimer *HOCCOH and its dehydrated *CCO), oxidation (*CO2- and *CO32-), and hydrogenation (*CHO), as well as Cu-Oad/Cu-OHad species at Cu-electrolyte interfaces, were simultaneously identified using in situ spectroscopy and further confirmed with isotope-labeling experiments. With AIMD simulations, we report accurate vibrational frequency assignments of these intermediates based on the calculated vibrational density of states and reveal the corresponding species in the electrochemical CO redox landscape on Cu surfaces. Our findings provide direct insights into key intermediates during the CO(2)RR and offer a full-spectroscopic tool (40-4,000 cm-1) for future mechanistic studies.
RESUMEN
This study aims to explore and characterize the role of pediatric sedation via rectal route. A pediatric physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) model of midazolam gel was built and validated to support dose selection for pediatric clinical trials. Before developing the rectal PBPK model, an intravenous PBPK model was developed to determine drug disposition, specifically by describing the ontogeny model of the metabolic enzyme. Pediatric rectal absorption was developed based on the rectal PBPK model of adults. The improved Weibull function with permeability, surface area, and fluid volume parameters was used to extrapolate pediatric rectal absorption. A logistic regression model was used to characterize the relationship between the free concentrations of midazolam and the probability of sedation. All models successfully described the PK profiles with absolute average fold error (AAFE) < 2, especially our intravenous PBPK model that extended the predicted age to preterm. The simulation results of the PD model showed that when the free concentrations of midazolam ranged from 3.9 to 18.4 ng/mL, the probability of "Sedation" was greater than that of "Not-sedation" states. Combined with the rectal PBPK model, the recommended sedation doses were in the ranges of 0.44-2.08 mg/kg for children aged 2-3 years, 0.35-1.65 mg/kg for children aged 4-7 years, 0.24-1.27 mg/kg for children aged 8-12 years, and 0.20-1.10 mg/kg for adolescents aged 13-18 years. Overall, this model mechanistically quantified drug disposition and effect of midazolam gel in the pediatric population, accurately predicted the observed clinical data, and simulated the drug exposure for sedation that will inform dose selection for following pediatric clinical trials.