RESUMO
All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.
Assuntos
Bactérias , Infecções Bacterianas , Membrana Celular , Proteínas de Ligação ao GTP , Reconhecimento da Imunidade Inata , Humanos , Citocinas/química , Tomografia com Microscopia Eletrônica , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/química , Hidrólise , Imunidade Celular , Microscopia Crioeletrônica , Gasderminas/química , Proteínas de Ligação a Fosfato/química , Conformação Proteica , Membrana Celular/química , Membrana Celular/imunologia , Caspases Iniciadoras/química , Infecções Bacterianas/imunologia , Bactérias/imunologiaRESUMO
Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.
Assuntos
Receptor de Morte Celular Programada 1 , Transdução de Sinais , Receptor de Morte Celular Programada 1/metabolismo , Ligantes , Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Antígeno B7-H1/metabolismoRESUMO
Kinase signaling in the tiered activation of inflammasomes and associated pyroptosis is a prime therapeutic target for inflammatory diseases. While MAPKs subsume pivotal roles during inflammasome priming, specifically the MAP3K7/JNK1/NLRP3 licensing axis, their involvement in successive steps of inflammasome activation is poorly defined. Using live-cell MAPK biosensors to focus on the inflammasome triggering event allowed us to identify a subsequent process of biphasic JNK activation. We find that this biphasic post-trigger JNK signaling initially facilitates the mitochondrial reactive oxygen species generation needed to support core inflammasome formation, then supports the gasdermin-mediated cell permeation required for release of active IL-1ß from human macrophages. We further identify and characterize a xanthine oxidase-ROS activated MAP3K5/JNK2 substrate licensing complex as a novel regulator of the GSDMD mobilization which precedes pyroptosis. We show that inhibitors targeting this MAP3K5 cascade alleviate morbidity in mouse models of colitis and dampen both augmented IL-1ß release and cell permeation in monocytes derived from patients with gain-of-function inflammasomopathies.
Assuntos
Inflamassomos , Piroptose , Animais , Humanos , Camundongos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologia , Transdução de SinaisRESUMO
Biologically active small molecules can impart modulatory effects, in some cases providing extended long-term memory. In a screen of biologically active small molecules for regulators of tumor necrosis factor (TNF) induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing lipopolysaccharide (LPS)-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits ß-glucan-induced H3K4Me3 marks at the promoters of several pro-inflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for ß-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and OC43 coronavirus infection, highlighting a potential application of this molecule and other SYKis as prophylactic treatments for viral susceptibility.
Assuntos
Tratamento Farmacológico da COVID-19 , beta-Glucanas , Citocinas , Humanos , Alcaloides Indólicos , Lipopolissacarídeos , Macrófagos , Quinazolinonas , SARS-CoV-2 , Quinase Syk , Fator de Necrose Tumoral alfaRESUMO
Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.
Assuntos
Inflamassomos , Nucleosídeo Difosfato Quinase D , Animais , Inflamassomos/genética , Inflamassomos/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos , Mitocôndrias/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleosídeo Difosfato Quinase D/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1009395.].
RESUMO
Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.
Assuntos
Apolipoproteínas L/metabolismo , Membrana Celular/metabolismo , Citosol/microbiologia , Bactérias Gram-Negativas/fisiologia , Interferon gama/imunologia , Apolipoproteínas L/química , Apolipoproteínas L/genética , Membrana Externa Bacteriana/metabolismo , Bacteriólise , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Células Cultivadas , Detergentes/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Edição de Genes , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Negativas/ultraestrutura , Humanos , Imunidade Inata , Lipoproteínas/química , Viabilidade Microbiana , Antígenos O/metabolismo , Domínios Proteicos , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Salmonella typhimurium/ultraestrutura , SolubilidadeRESUMO
The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses.
Assuntos
Infecções por Burkholderia/imunologia , Burkholderia cenocepacia/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Macrófagos/imunologia , Animais , Citosol/imunologia , Citosol/microbiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Activation of the TLR4 signaling pathway by lipopolysaccharide (LPS) leads to induction of both inflammatory and interferon-stimulated genes, but the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remain poorly understood. In a genome-wide small interfering RNA (siRNA) screen of the LPS-induced tumor necrosis factor α (TNF-α) response in macrophages, we identify the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identifies a positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced gene classes. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3A-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate a function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Macrófagos/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transdução de Sinais , Complexo Correpressor Histona Desacetilase e Sin3 , Fator de Necrose Tumoral alfa/imunologia , Células U937RESUMO
Traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. Emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interferon-induced GTPases called 'guanylate-binding proteins' (GBPs). Here we investigate the critical roles of interferon-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity to a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential effect of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases.
Assuntos
Proteínas de Ligação ao GTP/imunologia , Inflamassomos/imunologia , Interferons/imunologia , Transdução de Sinais/imunologia , Animais , Resistência à Doença/genética , Resistência à Doença/imunologia , Proteínas de Ligação ao GTP/classificação , Proteínas de Ligação ao GTP/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Infecções/imunologia , Infecções/microbiologia , Infecções/parasitologia , Inflamassomos/genética , Inflamassomos/metabolismo , Interferons/metabolismo , Listeria monocytogenes/imunologia , Listeria monocytogenes/fisiologia , Camundongos , Modelos Imunológicos , Filogenia , Transdução de Sinais/genética , Toxoplasma/imunologia , Toxoplasma/fisiologiaRESUMO
Specialized adaptations for killing microbes are synonymous with phagocytic cells including macrophages, monocytes, inflammatory neutrophils, and eosinophils. Recent genome sequencing of extant species, however, reveals that analogous antimicrobial machineries exist in certain non-immune cells and also within species that ostensibly lack a well-defined immune system. Here we probe the evolutionary record for clues about the ancient and diverse phylogenetic origins of macrophage killing mechanisms and how some of their properties are shared with cells outside the traditional bounds of immunity in higher vertebrates such as mammals.
Assuntos
Evolução Biológica , Macrófagos/imunologia , Viabilidade Microbiana , Fagocitose , Animais , HumanosRESUMO
From plants to humans, the ability to control infection at the level of an individual cell-a process termed cell-autonomous immunity-equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell's interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic, and inflammasome-related antimicrobial activities within the cytosol, as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of guanylate binding proteins (GBPs) with tuberculosis susceptibility and Crohn's colitis.
Assuntos
GTP Fosfo-Hidrolases/imunologia , Interferons/imunologia , Animais , Doença de Crohn/imunologia , Vesículas Citoplasmáticas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Interferons/metabolismo , Tuberculose/imunologiaRESUMO
Rubicon is a protein known to engage the Beclin-1/Vps34-PI3K/UVRAG complex and inhibit endosome and autophagosomal fusion with lysosomes. Yang et al. (2012) uncover new roles for this adaptor protein within noncanonical p22(phox) or CARD9 complexes that regulate oxidative and cytokine responses in activated macrophages, respectively. Both complexes impact pathogen-specific host defense.