RESUMEN
The stimulator of IFN genes (STING) protein senses cyclic dinucleotides released in response to double-stranded DNA and functions as an adaptor molecule for type I IFN (IFNI) signaling by activating IFNI-stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING-/- mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared with control EC, whereas T cell adhesion was not impaired. STING-/- T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell-expressed molecules ICAM1 and VCAM1, compared with WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α-stimulated STING-/- EC, and genetic loss or pharmacologic antagonisms of IFNI receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC-STING during T cell TEM that is dependent on the ISG CXCL10 and on IFNI/IFNAR signaling.
Asunto(s)
Interferón Tipo I , Proteínas de la Membrana/inmunología , Receptor de Interferón alfa y beta , Linfocitos T , Migración Transendotelial y Transepitelial/inmunología , Animales , Inmunidad Innata , Molécula 1 de Adhesión Intercelular/inmunología , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Ratones , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Inflammation and cell death are closely linked arms of the host immune response to infection, which when carefully balanced ensure host survival. One example of this balance is the tightly regulated transition from TNFR1-associated pro-inflammatory complex I to pro-death complex II. By contrast, here we show that a TRIF-dependent complex containing FADD, RIPK1 and caspase-8 (that we have termed the TRIFosome) mediates cell death in response to Yersinia pseudotuberculosis and LPS. Furthermore, we show that constitutive binding between ZBP1 and RIPK1 is essential for the initiation of TRIFosome interactions, caspase-8-mediated cell death and inflammasome activation, thus positioning ZBP1 as an effector of cell death in the context of bacterial blockade of pro-inflammatory signaling. Additionally, our findings offer an alternative to the TNFR1-dependent model of complex II assembly, by demonstrating pro-death complex formation reliant on TRIF signaling.
Asunto(s)
Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Proteínas de Unión al ARN/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Caspasa 8/metabolismo , Muerte Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , YersiniaRESUMEN
Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are well known for their capacity to drive necroptosis via mixed-lineage kinase-like domain (MLKL). Recently, RIPK1/3 kinase activity has been shown to drive inflammation via activation of MAPK signaling. However, the regulatory mechanisms underlying this kinase-dependent cytokine production remain poorly understood. In the present study, we establish that the kinase activity of RIPK1/3 regulates cytokine translation in mouse and human macrophages. Furthermore, we show that this inflammatory response is downregulated by type I interferon (IFN) signaling, independent of type I IFN-promoted cell death. Specifically, low-level constitutive IFN signaling attenuates RIPK-driven activation of cap-dependent translation initiation pathway components AKT, mTORC1, 4E-BP and eIF4E, while promoting RIPK-dependent cell death. Altogether, these data characterize constitutive IFN signaling as a regulator of RIPK-dependent inflammation and establish cap-dependent translation as a crucial checkpoint in the regulation of cytokine production.
Asunto(s)
Citocinas/metabolismo , Interferones/metabolismo , Biosíntesis de Proteínas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Citocinas/genética , Regulación hacia Abajo , Factor 4E Eucariótico de Iniciación/metabolismo , Femenino , Humanos , Inflamación/patología , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Interferons (IFNs) are critical determinants in immune-competence and autoimmunity, and are endogenously regulated by a low-level constitutive feedback loop. However, little is known about the functions and origins of constitutive IFN. Recently, lipopolysaccharide (LPS)-induced IFN was implicated as a driver of necroptosis, a necrotic form of cell death downstream of receptor-interacting protein (RIP) kinase activation and executed by mixed lineage kinase like-domain (MLKL) protein. We found that the pre-established IFN status of the cell, instead of LPS-induced IFN, is critical for the early initiation of necroptosis in macrophages. This pre-established IFN signature stems from cytosolic DNA sensing via cGAS/STING, and maintains the expression of MLKL and one or more unknown effectors above a critical threshold to allow for MLKL oligomerization and cell death. Finally, we found that elevated IFN-signaling in systemic lupus erythematosus (SLE) augments necroptosis, providing a link between pathological IFN and tissue damage during autoimmunity.
Asunto(s)
Interferón beta/metabolismo , Macrófagos/metabolismo , Necroptosis , Proteínas Quinasas/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citosol/metabolismo , ADN/metabolismo , Técnicas de Inactivación de Genes , Humanos , Interferón beta/genética , Interferón beta/farmacología , Lipopolisacáridos/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Legionella pneumophila elicits caspase-11-driven macrophage pyroptosis through guanylate-binding proteins (GBPs) encoded on chromosome 3. It has been proposed that microbe-driven IFN upregulates GBPs to facilitate pathogen vacuole rupture and bacteriolysis preceding caspase-11 activation. We show here that macrophage death occurred independently of microbial-induced IFN signaling and that GBPs are dispensable for pathogen vacuole rupture. Instead, the host-intrinsic IFN status sustained sufficient GBP expression levels to drive caspase-1 and caspase-11 activation in response to cytosol-exposed bacteria. In addition, endogenous GBP levels were sufficient for the release of DNA from cytosol-exposed bacteria, preceding the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway for Ifnb induction. Mice deficient for chromosome 3 GBPs were unable to mount a rapid IL-1/chemokine (C-X-C motif) ligand 1 (CXCL1) response during Legionella-induced pneumonia, with defective bacterial clearance. Our results show that rapid GBP activity is controlled by host-intrinsic cytokine signaling and that GBP activities precede immune amplification responses, including IFN induction, inflammasome activation, and cell death.
Asunto(s)
ADN Bacteriano/metabolismo , Proteínas de Unión al GTP/metabolismo , Interferones/metabolismo , Legionella/metabolismo , Piroptosis , Animales , Antiinfecciosos/farmacología , Cromosomas de los Mamíferos/metabolismo , Citosol/metabolismo , Femenino , Humanos , Quinasas Janus/metabolismo , Legionelosis/microbiología , Macrófagos/citología , Masculino , Ratones Endogámicos C57BL , Neumonía/microbiología , Neumonía/patología , Receptor de Interferón alfa y beta/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Vacuolas/metabolismoRESUMEN
Stimulator of interferon genes (STING) was initially described as a sensor of intracellular bacterial and viral DNA and a promising adjuvant target in innate immune cells; more recently STING has also been shown to detect endogenous DNA and play a role in tumor immunity and autoimmune disease development. Thus far STING has been studied in macrophages and dendritic cells. In this study, to our knowledge we provide the first evidence of STING activation in T cells, in which STING agonists not only provoke type I IFN production and IFN-stimulated gene expression, mirroring the response of innate cells, but are also capable of activating cell stress and death pathways. Our results suggest a re-evaluation of STING agonist-based therapies may be necessary to identify the possible effects on the T cell compartment. Conversely, the effects of STING on T cells could potentially be harnessed for therapeutic applications.
Asunto(s)
Muerte Celular , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Proteínas de la Membrana/metabolismo , Linfocitos T/inmunología , Animales , Estrés del Retículo Endoplásmico , Inmunidad Innata , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Análisis de Secuencia de ARN , Linfocitos T/metabolismo , Linfocitos T/fisiologíaRESUMEN
cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP-deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3' UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.
Asunto(s)
Empalme Alternativo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Receptor fas/metabolismo , Empalme Alternativo/genética , Animales , Anticuerpos , Apoptosis , Emparejamiento Base/genética , Secuencia de Bases , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Susceptibilidad a Enfermedades , Proteína Ligando Fas/metabolismo , Ligamiento Genético , Sitios Genéticos , Genoma , Hígado/metabolismo , Hígado/patología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Carácter Cuantitativo Heredable , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
With the stimulator of IFN genes (STING) C terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. In this article, we identify novel mutations in NTD of Sting of the MOLF strain in response to HSV and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFN-ß caused by failure of MOLF STING to translocate from the endoplasmic reticulum. These data provide evidence that the NTD of STING affects DNA responses via control of trafficking. They also show that the genetic diversity of wild-derived mice resembles the diversity observed in humans. Several human alleles of STING confer attenuated IFN-I production similar to what we observe with the MOLF Sting allele, a crucial functional difference not apparent in classical inbred mice. Thus, understanding the functional significance of polymorphisms in MOLF STING can provide basic mechanistic insights relevant to humans.