RESUMEN
Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.
Asunto(s)
Predisposición Genética a la Enfermedad , Lupus Eritematoso Sistémico , Receptor Toll-Like 7 , Lupus Eritematoso Sistémico/genética , Humanos , Animales , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Ratones , Niño , Femenino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Masculino , Edad de Inicio , Variación Genética , FN-kappa B/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Adolescente , Células THP-1 , Interferón Tipo I/metabolismoRESUMEN
SARS-CoV-2 infection causes more severe disease in pregnant women compared to age-matched non-pregnant women. Whether maternal infection causes changes in the transfer of immunity to infants remains unclear. Maternal infections have previously been associated with compromised placental antibody transfer, but the mechanism underlying this compromised transfer is not established. Here, we used systems serology to characterize the Fc profile of influenza-, pertussis-, and SARS-CoV-2-specific antibodies transferred across the placenta. Influenza- and pertussis-specific antibodies were actively transferred. However, SARS-CoV-2-specific antibody transfer was significantly reduced compared to influenza- and pertussis-specific antibodies, and cord titers and functional activity were lower than in maternal plasma. This effect was only observed in third-trimester infection. SARS-CoV-2-specific transfer was linked to altered SARS-CoV-2-antibody glycosylation profiles and was partially rescued by infection-induced increases in IgG and increased FCGR3A placental expression. These results point to unexpected compensatory mechanisms to boost immunity in neonates, providing insights for maternal vaccine design.
Asunto(s)
Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Inmunoglobulina G/inmunología , Intercambio Materno-Fetal/inmunología , Placenta/inmunología , Complicaciones Infecciosas del Embarazo/inmunología , SARS-CoV-2/inmunología , Adulto , Femenino , Humanos , Recién Nacido , Embarazo , Tercer Trimestre del Embarazo/inmunología , Receptores de IgG/inmunología , Células THP-1RESUMEN
The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , ADN-Topoisomerasas de Tipo I/metabolismo , SARS-CoV-2/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Topotecan/farmacología , Animales , COVID-19/enzimología , COVID-19/patología , Chlorocebus aethiops , Humanos , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/patología , Inflamación/virología , Mesocricetus , Ratones , Ratones Transgénicos , Células THP-1 , Células VeroRESUMEN
COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.
Asunto(s)
COVID-19/inmunología , COVID-19/patología , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/patología , Interferón gamma/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Muerte Celular , Modelos Animales de Enfermedad , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/inmunología , Inflamación/patología , Linfohistiocitosis Hemofagocítica/inducido químicamente , Masculino , Ratones , Ratones Transgénicos , Células THP-1RESUMEN
Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Bacterias/inmunología , Movimiento Celular/inmunología , Células Asesinas Naturales/inmunología , Trofoblastos/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Células Dendríticas/inmunología , Femenino , Células HeLa , Humanos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Placenta/inmunología , Placenta/microbiología , Embarazo , Ratas , Células THP-1 , Trofoblastos/microbiologíaRESUMEN
Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages.
Asunto(s)
Macrófagos/microbiología , Macrófagos/patología , Mitocondrias/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Tuberculosis/inmunología , Tuberculosis/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Calcio/metabolismo , Retículo Endoplásmico/microbiología , Humanos , Lisosomas/microbiología , Potencial de la Membrana Mitocondrial , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium marinum , Mycobacterium tuberculosis , Necrosis , Especies Reactivas de Oxígeno/metabolismo , Células THP-1 , Pez CebraRESUMEN
The presence of DNA in the cytoplasm is normally a sign of microbial infections and is quickly detected by cyclic GMP-AMP synthase (cGAS) to elicit anti-infection immune responses. However, chronic activation of cGAS by self-DNA leads to severe autoimmune diseases for which no effective treatment is available yet. Here we report that acetylation inhibits cGAS activation and that the enforced acetylation of cGAS by aspirin robustly suppresses self-DNA-induced autoimmunity. We find that cGAS acetylation on either Lys384, Lys394, or Lys414 contributes to keeping cGAS inactive. cGAS is deacetylated in response to DNA challenges. Importantly, we show that aspirin can directly acetylate cGAS and efficiently inhibit cGAS-mediated immune responses. Finally, we demonstrate that aspirin can effectively suppress self-DNA-induced autoimmunity in Aicardi-Goutières syndrome (AGS) patient cells and in an AGS mouse model. Thus, our study reveals that acetylation contributes to cGAS activity regulation and provides a potential therapy for treating DNA-mediated autoimmune diseases.
Asunto(s)
ADN/inmunología , Nucleotidiltransferasas/metabolismo , Autotolerancia/inmunología , Acetilación , Secuencia de Aminoácidos , Animales , Aspirina/farmacología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Autoinmunidad , Línea Celular , ADN/genética , ADN/metabolismo , Modelos Animales de Enfermedad , Exodesoxirribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutación , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Células THP-1RESUMEN
Despite the worldwide success of vaccination, newborns remain vulnerable to infections. While neonatal vaccination has been hampered by maternal antibody-mediated dampening of immune responses, enhanced regulatory and tolerogenic mechanisms, and immune system immaturity, maternal pre-natal immunization aims to boost neonatal immunity via antibody transfer to the fetus. However, emerging data suggest that antibodies are not transferred equally across the placenta. To understand this, we used systems serology to define Fc features associated with antibody transfer. The Fc-profile of neonatal and maternal antibodies differed, skewed toward natural killer (NK) cell-activating antibodies. This selective transfer was linked to digalactosylated Fc-glycans that selectively bind FcRn and FCGR3A, resulting in transfer of antibodies able to efficiently leverage innate immune cells present at birth. Given emerging data that vaccination may direct antibody glycosylation, our study provides insights for the development of next-generation maternal vaccines designed to elicit antibodies that will most effectively aid neonates.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/metabolismo , Placenta/metabolismo , Polisacáridos/metabolismo , Receptores Fc/inmunología , Receptores Fc/metabolismo , Adolescente , Adulto , Bélgica , Degranulación de la Célula , Estudios de Cohortes , Femenino , Glicosilación , Humanos , Recién Nacido , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Masculino , Embarazo , Receptores de IgG/metabolismo , Células THP-1 , Estados Unidos , Vacunación , Adulto JovenRESUMEN
B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.
Asunto(s)
Mapeo Epitopo/métodos , Epítopos/química , Receptores de Antígenos de Linfocitos B/química , Análisis de Secuencia de ADN/métodos , Análisis de la Célula Individual/métodos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Antígenos/química , Antígenos/inmunología , Células Cultivadas , Epítopos/inmunología , Células HEK293 , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Receptores de Antígenos de Linfocitos B/inmunología , Células THP-1RESUMEN
Metabolic inflammation is closely linked to obesity, and is implicated in the pathogenesis of metabolic diseases. FTO harbors the strongest genetic association with polygenic obesity, and IRX3 mediates the effects of FTO on body weight. However, in what cells and how IRX3 carries out this control are poorly understood. Here we report that macrophage IRX3 promotes metabolic inflammation to accelerate the development of obesity and type 2 diabetes. Mice with myeloid-specific deletion of Irx3 were protected against diet-induced obesity and metabolic diseases via increasing adaptive thermogenesis. Mechanistically, macrophage IRX3 promoted proinflammatory cytokine transcription and thus repressed adipocyte adrenergic signaling, thereby inhibiting lipolysis and thermogenesis. JNK1/2 phosphorylated IRX3, leading to its dimerization and nuclear translocation for transcription. Further, lipopolysaccharide stimulation stabilized IRX3 by inhibiting its ubiquitination, which amplified the transcriptional capacity of IRX3. Together, our findings identify a new player, macrophage IRX3, in the control of body weight and metabolic inflammation, implicating IRX3 as a therapeutic target.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Obesidad/metabolismo , Factores de Transcripción/metabolismo , Adipocitos/metabolismo , Adulto , Animales , Peso Corporal/fisiología , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Dieta/métodos , Células HEK293 , Humanos , Masculino , Enfermedades Metabólicas/metabolismo , Ratones , Células RAW 264.7 , Células THP-1 , Termogénesis/fisiología , Transcripción Genética/fisiología , Adulto JovenRESUMEN
Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.
Asunto(s)
Artritis Juvenil/inmunología , Diferenciación Celular/inmunología , Epigénesis Genética/inmunología , Histonas/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/inmunología , Adolescente , Artritis Juvenil/sangre , Artritis Juvenil/genética , Sistemas CRISPR-Cas/genética , Catepsina G/genética , Catepsina G/metabolismo , Diferenciación Celular/genética , Núcleo Celular/metabolismo , Niño , Preescolar , Cromatina/metabolismo , Pruebas de Enzimas , Epigenómica , Femenino , Técnicas de Inactivación de Genes , Humanos , Células Jurkat , Elastasa de Leucocito/genética , Elastasa de Leucocito/metabolismo , Leucocitos Mononucleares/inmunología , Macrófagos/metabolismo , Masculino , Mieloblastina/genética , Mieloblastina/metabolismo , Cultivo Primario de Células , Proteolisis , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células THP-1 , Adulto JovenRESUMEN
CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.
Asunto(s)
Sistemas CRISPR-Cas , Citometría de Flujo/métodos , Genómica/métodos , Espectrometría de Masas/métodos , Análisis de la Célula Individual/métodos , Animales , Epítopos/química , Epítopos/clasificación , Epítopos/genética , Células HEK293 , Humanos , Inmunofenotipificación/métodos , Células Jurkat , Ratones Endogámicos BALB C , Proteoma/química , Proteoma/clasificación , Proteoma/genética , Células THP-1RESUMEN
Aberrant activation of innate immune receptors can cause a spectrum of immune disorders, such as Aicardi-Goutières syndrome (AGS). One such receptor is MDA5, a viral dsRNA sensor that induces antiviral immune response. Using a newly developed RNase-protection/RNA-seq approach, we demonstrate here that constitutive activation of MDA5 in AGS results from the loss of tolerance to cellular dsRNAs formed by Alu retroelements. While wild-type MDA5 cannot efficiently recognize Alu-dsRNAs because of its limited filament formation on imperfect duplexes, AGS variants of MDA5 display reduced sensitivity to duplex structural irregularities, assembling signaling-competent filaments on Alu-dsRNAs. Moreover, we identified an unexpected role of an RNA-rich cellular environment in suppressing aberrant MDA5 oligomerization, highlighting context dependence of self versus non-self discrimination. Overall, our work demonstrates that the increased efficiency of MDA5 in recognizing dsRNA comes at a cost of self-recognition and implicates a unique role of Alu-dsRNAs as virus-like elements that shape the primate immune system.
Asunto(s)
Elementos Alu/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Helicasa Inducida por Interferón IFIH1/inmunología , Malformaciones del Sistema Nervioso/inmunología , Multimerización de Proteína/inmunología , ARN Bicatenario/inmunología , Autotolerancia , Células A549 , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/patología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Helicasa Inducida por Interferón IFIH1/genética , Muramidasa , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/patología , Fragmentos de Péptidos , Multimerización de Proteína/genética , ARN Bicatenario/genética , Células THP-1RESUMEN
Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.
Asunto(s)
Herpes Simple/inmunología , Proteínas de la Membrana/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Animales , Carbolinas/farmacología , Células Cultivadas , ADN Viral/inmunología , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Femenino , Fibroblastos , Aparato de Golgi/metabolismo , Células HEK293 , Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Homeostasis/inmunología , Humanos , Inmunidad Innata , Peroxidación de Lípido/genética , Peroxidación de Lípido/inmunología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , Nucleotidiltransferasas/metabolismo , Oxidación-Reducción , Oximas/farmacología , Fosfolípido Hidroperóxido Glutatión Peroxidasa/antagonistas & inhibidores , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Cultivo Primario de Células , Carbonilación Proteica/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Sulfonamidas/farmacología , Células THP-1 , Replicación Viral/inmunologíaRESUMEN
Toxoplasma gondii is a common protozoan parasite that infects up to one third of the world's population. Notably, very little is known about innate immune sensing mechanisms for this obligate intracellular parasite by human cells. Here, by applying an unbiased biochemical screening approach, we show that human monocytes recognized the presence of T. gondii infection by detecting the alarmin S100A11 protein, which is released from parasite-infected cells via caspase-1-dependent mechanisms. S100A11 induced a potent chemokine response to T. gondii by engaging its receptor RAGE, and regulated monocyte recruitment in vivo by inducing expression of the chemokine CCL2. Our experiments reveal a sensing system for T. gondii by human cells that is based on the detection of infection-mediated release of S100A11 and RAGE-dependent induction of CCL2, a crucial chemokine required for host resistance to the parasite.
Asunto(s)
Quimiocina CCL2/metabolismo , Inmunidad Innata , Proteínas S100/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/inmunología , Animales , Antígenos de Neoplasias/metabolismo , Caspasa 1/metabolismo , Quimiotaxis , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN Interferente Pequeño/genética , Proteínas S100/genética , Células THP-1RESUMEN
Succinate is a signaling metabolite sensed extracellularly by succinate receptor 1 (SUNCR1). The accumulation of succinate in macrophages is known to activate a pro-inflammatory program; however, the contribution of SUCNR1 to macrophage phenotype and function has remained unclear. Here we found that activation of SUCNR1 had a critical role in the anti-inflammatory responses in macrophages. Myeloid-specific deficiency in SUCNR1 promoted a local pro-inflammatory phenotype, disrupted glucose homeostasis in mice fed a normal chow diet, exacerbated the metabolic consequences of diet-induced obesity and impaired adipose-tissue browning in response to cold exposure. Activation of SUCNR1 promoted an anti-inflammatory phenotype in macrophages and boosted the response of these cells to type 2 cytokines, including interleukin-4. Succinate decreased the expression of inflammatory markers in adipose tissue from lean human subjects but not that from obese subjects, who had lower expression of SUCNR1 in adipose-tissue-resident macrophages. Our findings highlight the importance of succinate-SUCNR1 signaling in determining macrophage polarization and assign a role to succinate in limiting inflammation.
Asunto(s)
Inflamación/inmunología , Macrófagos/inmunología , Obesidad/inmunología , Receptores Acoplados a Proteínas G/inmunología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/inmunología , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Inflamación/genética , Inflamación/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Obesidad/genética , Obesidad/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Ácido Succínico/inmunología , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Células THP-1RESUMEN
Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1ß and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.
Asunto(s)
Caspasas/inmunología , Mediadores de Inflamación/inmunología , Inflamación/inmunología , Serpinas/inmunología , Animales , Caspasas/genética , Caspasas/metabolismo , Línea Celular , Células Cultivadas , Activación Enzimática/inmunología , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Piroptosis/efectos de los fármacos , Piroptosis/inmunología , Células RAW 264.7 , Interferencia de ARN , Serina Proteasas/inmunología , Serina Proteasas/metabolismo , Serpinas/genética , Serpinas/metabolismo , Células THP-1 , Células U937RESUMEN
The unique cell biology of Toll-like receptor 4 (TLR4) allows it to initiate two signal-transduction cascades: a signal dependent on the adaptors TIRAP (Mal) and MyD88 that begins at the cell surface and regulates proinflammatory cytokines, and a signal dependent on the adaptors TRAM and TRIF that begins in the endosomes and drives the production of type I interferons. Negative feedback circuits to limit TLR4 signals from both locations are necessary to balance the inflammatory response. We describe a negative feedback loop driven by autocrine-paracrine prostaglandin E2 (PGE2) and the PGE2 receptor EP4 that restricted TRIF-dependent signals and the induction of interferon-ß through the regulation of TLR4 trafficking. Inhibition of PGE2 production or antagonism of EP4 increased the rate at which TLR4 translocated to endosomes and amplified TRIF-dependent activation of the transcription factor IRF3 and caspase-8. This PGE2-driven mechanism restricted TLR4-TRIF signaling in vitro after infection of macrophages by the Gram-negative pathogens Escherichia coli or Citrobacter rodentium and protected mice against mortality induced by Salmonella enteritidis serovar Typhimurium. Thus, PGE2 restricted TLR4-TRIF signaling specifically in response to lipopolysaccharide.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/inmunología , Dinoprostona/inmunología , Inmunidad Innata/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Animales , Infecciones Bacterianas/inmunología , Retroalimentación Fisiológica/fisiología , Humanos , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Células THP-1RESUMEN
Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.
Asunto(s)
Gasderminas , Lipoilación , Proteínas de Unión a Fosfato , Especies Reactivas de Oxígeno , Animales , Femenino , Humanos , Masculino , Ratones , Aciltransferasas/metabolismo , Microscopía por Crioelectrón , Cisteína/metabolismo , Gasderminas/química , Gasderminas/metabolismo , Inflamasomas/metabolismo , Liposomas/metabolismo , Liposomas/química , Mitocondrias/metabolismo , Proteínas de Unión a Fosfato/química , Proteínas de Unión a Fosfato/metabolismo , Piroptosis , Especies Reactivas de Oxígeno/metabolismo , Células THP-1RESUMEN
Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology.