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BACKGROUND & AIMS: Gut bacterial translocation contributes to immune dysfunction and spontaneous bacterial peritonitis (SBP) in cirrhosis. We hypothesized that exposure of peritoneal macrophages (PMs) to bacterial DNA results in type-I interferon (IFN) production, shaping subsequent immune responses, inflammasome activation, and the release of damage-associated molecular patterns (DAMPs). METHODS: PMs from patients with cirrhosis were stimulated with E. coli single-stranded DNA (ssDNA), lipopolysaccharide and IFN, or infected with E. coli, S. aureus, and Group B streptococcus in vitro. Cytokine release, inflammasome activation, and DAMP release were quantified by quantitative-PCR, ELISA, western blots, and reporter cells employing primary PMs, monocytes, and caspase-deficient THP-1 macrophages. Serum progranulin concentration was correlated with transplant-free survival in 77 patients with SBP. RESULTS: E. coli ssDNA induced strong type-I IFN activity in PMs and monocytes, priming them for enhanced lipopolysaccharide-mediated tumor necrosis factor production without inducing toll-like receptor 4 tolerance. During in vitro macrophage bacterial infection, type-I IFN release aligned with upregulated expression of IFN-regulatory factors (IRF)1/2 and guanylate binding proteins (GBP)2/5. PMs upregulated inflammasome-associated proteins and type-I IFN upon E. coli ssDNA exposure and released interleukin-1ß upon bacterial infection. Proteomic screening in mouse macrophages revealed progranulin release as being caspase-11-dependent during E. coli infection. PMs and THP-1 macrophages released significant amounts of progranulin when infected with S. aureus or E. coli via gasdermin D in a type-I IFN- and caspase-5-dependent manner. During SBP, PMs upregulated IRF1, GBP2/5 and caspase-5 and higher serum progranulin concentrations were indicative of lower 90-day transplant-free survival after SBP. CONCLUSIONS: Type-I IFN shapes peritoneal immune responses and regulates caspase-5-mediated progranulin release during SBP. IMPACT AND IMPLICATIONS: Patients with cirrhosis exhibit impaired immune responses and increased susceptibility to bacterial infections. This study reveals that type-I interferon responses, triggered by pathogen-associated molecular patterns, are crucial in regulating macrophage activation and priming them for inflammatory responses. Additionally, we elucidate the mechanisms by which type-I interferons promote the release of progranulin from macrophages during spontaneous bacterial peritonitis. Our findings enhance understanding of how bacterial translocation affects immune responses, identify novel biomarkers for inflammasome activation during infections, and point to potential therapeutic targets.
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Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance.
Asunto(s)
Vesículas Extracelulares , Inflamasomas , Humanos , Animales , Ratones , Inflamasomas/metabolismo , Lipopolisacáridos , Caspasas/metabolismo , Piroptosis , Citosol , Vesículas Extracelulares/metabolismoRESUMEN
Macrophages facilitate critical functions in regulating pathogen clearance and immune homeostasis in tissues. The remarkable functional diversity exhibited by macrophage subsets is dependent on tissue environment and the nature of the pathological insult. Our current knowledge of the mechanisms that regulate the multifaceted counter-inflammatory responses mediated by macrophages remains incomplete. Here, we report that CD169+ macrophage subsets are necessary for protection under excessive inflammatory conditions. We show that in the absence of these macrophages, even under mild septic conditions, mice fail to survive and exhibit increased production of inflammatory cytokines. Mechanistically, CD169+ macrophages control inflammatory responses via interleukin-10 (IL-10), as CD169+ macrophage-specific deletion of IL-10 was lethal during septic conditions, and recombinant IL-10 treatment reduced lipopolysaccharide (LPS)-induced lethality in mice lacking CD169+ macrophages. Collectively, our findings show a pivotal homeostatic role for CD169+ macrophages and suggest they may serve as an important target for therapy under damaging inflammatory conditions.
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Interleucina-10 , Sepsis , Animales , Ratones , Citocinas , Lipopolisacáridos/farmacología , MacrófagosRESUMEN
Intracellular lipopolysaccharide (LPS) sensing by the noncanonical inflammasome comprising caspase-4 or -11 governs antibacterial host defense. How LPS gains intracellular access in vivo is largely unknown. Here, we show that CD14-an LPS-binding protein with a well-documented role in TLR4 activation-plays a vital role in intracellular LPS sensing in vivo. By generating Cd14-/- and Casp11-/- mice strains on a Tlr4-/- background, we dissociate CD14's known role in TLR4 signaling from its role in caspase-11 activation and show a TLR4-independent role for CD14 in GSDMD activation, pyroptosis, alarmin release, and the lethality driven by cytosolic LPS. Mechanistically, CD14 enables caspase-11 activation by mediating cytosolic localization of LPS in a TLR4-independent manner. Overall, our findings attribute a critical role for CD14 in noncanonical inflammasome sensing of LPS in vivo and establish-together with previous literature-CD14 as an essential proximal component of both TLR4-based extracellular and caspase-11-based intracellular LPS surveillance.
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Inflamasomas , Lipopolisacáridos , Animales , Caspasas/metabolismo , Inflamasomas/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Piroptosis , Receptor Toll-Like 4RESUMEN
Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1ß, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c+ cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM+ myeloid cells and MRP8+ neutrophils, but not CD11c+ cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance.
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Caspasas Iniciadoras/genética , Lipopolisacáridos/toxicidad , Macrófagos Peritoneales/inmunología , Neutrófilos/inmunología , Choque Séptico/inmunología , Bazo/inmunología , Animales , Burkholderia/crecimiento & desarrollo , Burkholderia/patogenicidad , Antígenos CD11/genética , Antígenos CD11/inmunología , Calgranulina A/genética , Calgranulina A/inmunología , Caspasas Iniciadoras/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Femenino , Regulación de la Expresión Génica , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Hígado/inmunología , Hígado/microbiología , Macrófagos Peritoneales/microbiología , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Monocitos/inmunología , Monocitos/microbiología , Neutrófilos/microbiología , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/inmunología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunología , Choque Séptico/genética , Choque Séptico/microbiología , Choque Séptico/mortalidad , Transducción de Señal , Bazo/microbiología , Análisis de SupervivenciaRESUMEN
Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.
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Alarminas/metabolismo , Endotoxemia/inmunología , Galectina 1/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Alarminas/deficiencia , Alarminas/genética , Animales , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , Endotoxemia/patología , Femenino , Galectina 1/sangre , Galectina 1/deficiencia , Galectina 1/genética , Células HeLa , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Antígenos Comunes de Leucocito/metabolismo , Lipopolisacáridos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Necroptosis , Proteínas de Unión a Fosfato/deficiencia , Proteínas de Unión a Fosfato/genética , Células RAW 264.7 , Sepsis/sangre , Sepsis/diagnóstico , Transducción de Señal , Regulación hacia ArribaRESUMEN
Inflammatory caspase-dependent cytosolic lipopolysaccharide (LPS) sensing is a critical arm of host defense against bacteria. How pathogens overcome this pathway to establish infections is largely unknown. Enterohemorrhagic Escherichia coli (EHEC) is a clinically important human pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. We found that a bacteriophage-encoded virulence factor of EHEC, Shiga toxin (Stx), suppresses caspase-11-mediated activation of the cytosolic LPS sensing pathway. Stx was essential and sufficient to inhibit pyroptosis and interleukin-1 (IL-1) responses elicited specifically by cytosolic LPS. The catalytic activity of Stx was necessary for suppression of inflammasome responses. Stx impairment of inflammasome responses to cytosolic LPS occurs at the level of gasdermin D activation. Stx also suppresses inflammasome responses in vivo after LPS challenge and bacterial infection. Overall, this study assigns a previously undescribed inflammasome-subversive function to a well-known bacterial toxin, Stx, and reveals a new phage protein-based pathogen blockade of cytosolic immune surveillance.
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Escherichia coli Enterohemorrágica/patogenicidad , Infecciones por Escherichia coli/inmunología , Inflamasomas/inmunología , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Animales , Bacteriófagos/inmunología , Bacteriófagos/metabolismo , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Chlorocebus aethiops , Citrobacter rodentium/inmunología , Citrobacter rodentium/patogenicidad , Modelos Animales de Enfermedad , Escherichia coli Enterohemorrágica/inmunología , Escherichia coli Enterohemorrágica/virología , Infecciones por Escherichia coli/microbiología , Femenino , Humanos , Vigilancia Inmunológica , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión a Fosfato/metabolismo , Toxina Shiga I/inmunología , Toxina Shiga II/inmunología , Células Vero , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
Nucleic acid sensing is a critical mechanism by which the immune system monitors for pathogen invasion. A set of germline-encoded innate immune receptors detect microbial DNA in various compartments of the cell, such as endosomes, the cytosol, and the nucleus. Sensing of microbial DNA through these receptors stimulates, in most cases, interferon regulatory factor-dependent type I IFN synthesis followed by JAK/STAT-dependent interferon-stimulated gene expression. In contrast, the detection of DNA in the cytosol by AIM2 assembles a macromolecular complex called the inflammasome, which unleashes the proteolytic activity of a cysteine protease caspase-1. Caspase-1 cleaves and activates the pro-inflammatory cytokines such as IL-1ß and IL-18 and a pore-forming protein, gasdermin D, which triggers pyroptosis, an inflammatory form of cell death. Research over the past decade has revealed that AIM2 plays essential roles not only in host defense against pathogens but also in inflammatory diseases, autoimmunity, and cancer in inflammasome-dependent and inflammasome-independent manners. This review discusses the latest advancements in our understanding of AIM2 biology and its functions in health and disease.
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Proteínas de Unión al ADN , Inflamasomas , Caspasa 1 , Interleucina-18 , PiroptosisRESUMEN
Inflammasome-activated caspase-1 cleaves gasdermin D to unmask its pore-forming activity, the predominant consequence of which is pyroptosis. Here, we report an additional biological role for gasdermin D in limiting cytosolic DNA surveillance. Cytosolic DNA is sensed by Aim2 and cyclic GMP-AMP synthase (cGAS) leading to inflammasome and type I interferon responses, respectively. We found that gasdermin D activated by the Aim2 inflammasome suppressed cGAS-driven type I interferon response to cytosolic DNA and Francisella novicida in macrophages. Similarly, interferon-ß (IFN-ß) response to F. novicida infection was elevated in gasdermin D-deficient mice. Gasdermin D-mediated negative regulation of IFN-ß occurred in a pyroptosis-, interleukin-1 (IL-1)-, and IL-18-independent manner. Mechanistically, gasdermin D depleted intracellular potassium (K+) via membrane pores, and this K+ efflux was necessary and sufficient to inhibit cGAS-dependent IFN-ß response. Thus, our findings have uncovered an additional interferon regulatory module involving gasdermin D and K+ efflux.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Francisella/fisiología , Infecciones por Bacterias Gramnegativas/inmunología , Inflamasomas/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Interferón Tipo I/metabolismo , Interleucina-1/metabolismo , Interleucina-18/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Proteínas de Unión a Fosfato , Potasio/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Glutathione peroxidase 4 (GPX4) is an antioxidant enzyme that protects cells from lipid peroxidation. In this issue of Cell Host & Microbe, Kang et al. (2018) report that GPX4 is a negative regulator of the pyroptotic cell death pathway and plays an important role in inhibiting lethal inflammation associated with sepsis.
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Peroxidación de Lípido , Sepsis , Antioxidantes , Humanos , PiroptosisRESUMEN
Inflammasomes are cytosolic multi-molecular complexes that sense intracellular microbial danger signals and metabolic perturbations. Inflammasome activation leads to the activation of caspase-1 and the release of pro-inflammatory cytokines IL-1ß and IL-18 accompanied by cell death. An inflammasome-based surveillance machinery for Gram-negative bacterial infections has been recently discovered. This noncanonical inflammasome relies on sensing the cytosolic presence of lipopolysaccharide of Gram-negative bacteria via inflammatory caspases such as caspase-4, -5, and -11. This review discusses the recent findings related to the mechanism of activation of the noncanonical inflammasome and its biological functions.
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Caspasas/inmunología , Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Inflamasomas/inmunología , Lipopolisacáridos/inmunología , Animales , Humanos , Interleucina-18/inmunología , Interleucina-1beta/inmunología , Sepsis/inmunologíaRESUMEN
Actin nucleation factors function to organize, shape, and move membrane-bound organelles, yet they remain poorly defined in relation to disease. Galloway-Mowat syndrome (GMS) is an inherited disorder characterized by microcephaly and nephrosis resulting from mutations in the WDR73 gene. This core clinical phenotype appears frequently in the Amish, where virtually all affected individuals harbor homozygous founder mutations in WDR73 as well as the closely linked WHAMM gene, which encodes a nucleation factor. Here we show that patient cells with both mutations exhibit cytoskeletal irregularities and severe defects in autophagy. Reintroduction of wild-type WHAMM restored autophagosomal biogenesis to patient cells, while inactivation of WHAMM in healthy cell lines inhibited lipidation of the autophagosomal protein LC3 and clearance of ubiquitinated protein aggregates. Normal WHAMM function involved binding to the phospholipid PI(3)P and promoting actin nucleation at nascent autophagosomes. These results reveal a cytoskeletal pathway controlling autophagosomal remodeling and illustrate several molecular processes that are perturbed in Amish GMS patients.
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Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Amish/genética , Mutación del Sistema de Lectura , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Fosfatos de Fosfatidilinositol/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/metabolismo , Autofagosomas/metabolismo , Autofagosomas/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Efecto Fundador , Hernia Hiatal/genética , Homocigoto , Humanos , Proteínas de la Membrana/metabolismo , Microcefalia/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Nefrosis/genética , Fosfatos de Fosfatidilinositol/genética , Proteínas/genética , Proteínas/metabolismoRESUMEN
Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11, however, are not cytosolic, and the mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here, we identify outer membrane vesicles (OMVs) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMVs are internalized via endocytosis, and LPS is released into the cytosol from early endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMVs, reveals the importance of OMVs in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMVs in enabling the cytosolic entry of LPS and, consequently, caspase-11 activation during Gram-negative bacterial infections.
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Bacterias Gramnegativas/citología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/microbiología , Lipopolisacáridos/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Citosol/metabolismo , Activación Enzimática , Bacterias Gramnegativas/química , Inmunidad Innata , Inflamación/inmunología , Inflamación/microbiología , Interleucina-1/inmunología , RatonesRESUMEN
Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex-mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein-nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity.