RESUMEN
The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.
Asunto(s)
Endorribonucleasas , Quinasa I-kappa B , Inflamación , Macrófagos , Proteínas Serina-Treonina Quinasas , Receptores Toll-Like , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Citocinas/metabolismo , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/genética , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Receptores Toll-Like/metabolismo , Ubiquitina/metabolismoRESUMEN
The DNA exonuclease three-prime repair exonuclease 1 (TREX1) is critical for preventing autoimmunity in mice and humans by degrading endogenous cytosolic DNA, which otherwise triggers activation of the innate cGAS/STING pathway leading to the production of type I IFNs. As tumor cells are prone to aberrant cytosolic DNA accumulation, we hypothesized that they are critically dependent on TREX1 activity to limit their immunogenicity. Here, we show that in tumor cells, TREX1 restricts spontaneous activation of the cGAS/STING pathway, and the subsequent induction of a type I IFN response. As a result, TREX1 deficiency compromised in vivo tumor growth in mice. This delay in tumor growth depended on a functional immune system, systemic type I IFN signaling, and tumor-intrinsic cGAS expression. Mechanistically, we show that tumor TREX1 loss drove activation of CD8+ T cells and NK cells, prevented CD8+ T-cell exhaustion, and remodeled an immunosuppressive myeloid compartment. Consequently, TREX1 deficiency combined with T-cell-directed immune checkpoint blockade. Collectively, we conclude that TREX1 is essential to limit tumor immunogenicity, and that targeting this innate immune checkpoint remodels the tumor microenvironment and enhances antitumor immunity by itself and in combination with T-cell-targeted therapies. See related article by Toufektchan et al., p. 673.
Asunto(s)
Exodesoxirribonucleasas , Inmunidad Innata , Proteínas de la Membrana , Nucleotidiltransferasas , Fosfoproteínas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Animales , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Ratones , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/genética , Interferón Tipo I/metabolismo , Ratones Noqueados , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Transducción de Señal , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéuticoRESUMEN
Immune cell locomotion is associated with amoeboid migration, a flexible mode of movement, which depends on rapid cycles of actin polymerization and actomyosin contraction1. Many immune cells do not necessarily require integrins, the major family of adhesion receptors in mammals, to move productively through three-dimensional tissue spaces2,3. Instead, they can use alternative strategies to transmit their actin-driven forces to the substrate, explaining their migratory adaptation to changing external environments4-6. However, whether these generalized concepts apply to all immune cells is unclear. Here, we show that the movement of mast cells (immune cells with important roles during allergy and anaphylaxis) differs fundamentally from the widely applied paradigm of interstitial immune cell migration. We identify a crucial role for integrin-dependent adhesion in controlling mast cell movement and localization to anatomical niches rich in KIT ligand, the major mast cell growth and survival factor. Our findings show that substrate-dependent haptokinesis is an important mechanism for the tissue organization of resident immune cells.
Asunto(s)
Actinas , Integrinas , Animales , Integrinas/metabolismo , Actinas/metabolismo , Mastocitos/metabolismo , Movimiento Celular , Leucocitos/metabolismo , Adhesión Celular , Mamíferos/metabolismoRESUMEN
Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.
Asunto(s)
Caspasa 8/metabolismo , Citocinas/inmunología , Inmunidad Innata/inmunología , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Citocinas/antagonistas & inhibidores , Humanos , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.
Asunto(s)
Muerte Celular , Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/metabolismo , Inflamación/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinación , Animales , Caspasa 8/genética , Caspasa 8/metabolismo , Muerte Celular/genética , Enzimas Desubicuitinizantes/genética , Pérdida del Embrión/genética , Endopeptidasas/genética , Inflamación/enzimología , Inflamación/genética , Interferón Tipo I/biosíntesis , Ratones , Ratones Endogámicos C57BL , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ubiquitinación/genética , Pérdida de Peso/genéticaRESUMEN
NKT cells represent a small subset of glycolipid-recognizing T cells that are heavily implicated in human allergic, autoimmune, and malignant diseases. In the thymus, precursor cells recognize self-glycolipids by virtue of their semi-invariant TCR, which triggers NKT cell lineage commitment and maturation. During their development, NKT cells are polarized into the NKT1, NKT2, and NKT17 subsets, defined through their cytokine-secretion patterns and the expression of key transcription factors. However, we have largely ignored how the differentiation into the NKT cell subsets is regulated. In this article, we describe the mRNA-binding Roquin-1 and -2 proteins as central regulators of murine NKT cell fate decisions. In the thymus, T cell-specific ablation of the Roquin paralogs leads to a dramatic expansion of NKT17 cells, whereas peripheral mature NKT cells are essentially absent. Roquin-1/2-deficient NKT17 cells show exaggerated lineage-specific expression of nearly all NKT17-defining proteins tested. We show through mixed bone marrow chimera experiments that NKT17 polarization is mediated through cell-intrinsic mechanisms early during NKT cell development. In contrast, the loss of peripheral NKT cells is due to cell-extrinsic factors. Surprisingly, Roquin paralog-deficient NKT cells are, in striking contrast to conventional T cells, compromised in their ability to secrete cytokines. Altogether, we show that Roquin paralogs regulate the development and function of NKT cell subsets in the thymus and periphery.
Asunto(s)
Diferenciación Celular/inmunología , Células T Asesinas Naturales/inmunología , Subgrupos de Linfocitos T/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Citometría de Flujo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken ß actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology.
Asunto(s)
Adenoviridae/genética , Marcación de Gen , Genes Reporteros , Vectores Genéticos/genética , Recombinación Homóloga , Integrasas/metabolismo , Transducción Genética , Animales , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Expresión Génica , Marcación de Gen/métodos , Humanos , Integrasas/genética , Leucocitos/inmunología , Leucocitos/metabolismo , Ratones , Ratones Transgénicos , Células Mieloides/citología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Especificidad de ÓrganosRESUMEN
Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR.
Asunto(s)
Autoinmunidad/inmunología , Factores de Transcripción Forkhead/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Reguladores/inmunología , Animales , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Metilación de ADN/inmunología , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/genética , Inflamación/inmunología , Factores Reguladores del Interferón/biosíntesis , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Timocitos/citologíaRESUMEN
The immunoglobulin E (IgE)-mediated mast cell (MC) response is central to the pathogenesis of type I allergy and asthma. IκB kinase 2 (IKK2) was reported to couple IgE-induced signals to MC degranulation by phosphorylating the SNARE protein SNAP23. We investigated MC responses in mice with MC-specific inactivation of IKK2 or NF-κB essential modulator (NEMO), or animals with MC-specific expression of a mutant, constitutively active IKK2. We show that the IgE-induced late-phase cytokine response is reduced in mice lacking IKK2 or NEMO in MCs. However, anaphylactic in vivo responses of these animals are not different from those of control mice, and in vitro IKK2-deficient MCs readily phosphorylate SNAP23 and degranulate similarly to control cells in response to allergen or calcium ionophore. Constitutive overactivation of the NF-κB pathway has only slight effects on allergen-triggered MC responses. Thus, IKK2 is dispensable for MC degranulation, and the important question how IgE-induced signals trigger MC vesicle fusion remains open.
Asunto(s)
Degranulación de la Célula , Citocinas/metabolismo , Quinasa I-kappa B/metabolismo , Inmunoglobulina E/inmunología , Mastocitos/metabolismo , Alérgenos/inmunología , Anafilaxia/metabolismo , Animales , Citocinas/genética , Quinasa I-kappa B/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mastocitos/inmunología , Mastocitos/fisiología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismoRESUMEN
Mast cells are implicated in the pathogenesis of inflammatory and autoimmune diseases. However, this notion based on studies in mast cell-deficient mice is controversial. We therefore established an in vivo model for hyperactive mast cells by specifically ablating the NF-κB negative feedback regulator A20. While A20 deficiency did not affect mast cell degranulation, it resulted in amplified pro-inflammatory responses downstream of IgE/FcεRI, TLRs, IL-1R, and IL-33R. As a consequence house dust mite- and IL-33-driven lung inflammation, late phase cutaneous anaphylaxis, and collagen-induced arthritis were aggravated, in contrast to experimental autoimmune encephalomyelitis and immediate anaphylaxis. Our results provide in vivo evidence that hyperactive mast cells can exacerbate inflammatory disorders and define diseases that might benefit from therapeutic intervention with mast cell function.
Asunto(s)
Anafilaxia/inmunología , Artritis Experimental/inmunología , Proteínas de Unión al ADN/deficiencia , Encefalomielitis Autoinmune Experimental/inmunología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Mastocitos/inmunología , Ubiquitina-Proteína Ligasas/deficiencia , Anafilaxia/inducido químicamente , Anafilaxia/metabolismo , Anafilaxia/patología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/metabolismo , Artritis Experimental/patología , Colágeno Tipo II/administración & dosificación , Cisteína Endopeptidasas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Dinitrofenoles/administración & dosificación , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Expresión Génica , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Interleucinas/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Masculino , Mastocitos/metabolismo , Mastocitos/patología , Ratones , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , FN-kappa B/genética , FN-kappa B/inmunología , Fragmentos de Péptidos/administración & dosificación , Neumonía/inducido químicamente , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/patología , Pyroglyphidae/inmunología , Receptores de IgE/genética , Receptores de IgE/inmunología , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Albúmina Sérica Bovina/administración & dosificación , Receptores Toll-Like/genética , Receptores Toll-Like/inmunologíaRESUMEN
Mast cells are abundantly situated at contact sites between the body and its environment, such as the skin and, especially during certain immune responses, at mucosal surfaces. They mediate allergic reactions and degrade toxins as well as venoms. However, their roles during innate and adaptive immune responses remain controversial and it is likely that major functions remain to be discovered. Recent developments in mast cell-specific conditional gene targeting in the mouse promise to enhance our understanding of these fascinating cells. To complete the genetic toolbox to study mast cell development, homeostasis and function, it is imperative to inducibly manipulate their gene expression. Here, we report the generation of a novel knock-in mouse line expressing a tamoxifen-inducible version of the Cre recombinase from within the endogenous c-Kit locus. We demonstrate highly efficient and specific inducible expression of a fluorescent reporter protein in mast cells both in vivo and in vitro. Furthermore, induction of diphtheria toxin A expression allowed selective and efficient ablation of mast cells at various anatomical locations, while other hematopoietic cells remain unaffected. This novel mouse strain will hence be very valuable to study mast cell homeostasis and how specific genes influence their functions in physiology and pathology.
Asunto(s)
Toxina Diftérica/metabolismo , Marcación de Gen/métodos , Integrasas/metabolismo , Mastocitos/inmunología , Ratones Transgénicos/inmunología , Fragmentos de Péptidos/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Toxina Diftérica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Sustitución del Gen , Sitios Genéticos/genética , Integrasas/genética , Mastocitos/efectos de los fármacos , Mastocitos/patología , Ratones , Especificidad de Órganos , Fragmentos de Péptidos/genética , Proteínas Proto-Oncogénicas c-kit/genética , Tamoxifeno/administración & dosificación , Transgenes/genéticaRESUMEN
Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.
Asunto(s)
Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Animales , Linaje de la Célula , Citocinas/metabolismo , Técnicas de Sustitución del Gen , Homeostasis , Inmunidad Innata/inmunología , Inflamación/inmunología , Inflamación/patología , Recuento de Linfocitos , Ratones , Ratones Transgénicos , Fenotipo , Transducción de Señal/inmunología , Factores de TiempoRESUMEN
The Roquin-1 protein binds to messenger RNAs (mRNAs) and regulates gene expression posttranscriptionally. A single point mutation in Roquin-1, but not gene ablation, increases follicular helper T (Tfh) cell numbers and causes lupus-like autoimmune disease in mice. In T cells, we did not identify a unique role for the much lower expressed paralog Roquin-2. However, combined ablation of both genes induced accumulation of T cells with an effector and follicular helper phenotype. We showed that Roquin-1 and Roquin-2 proteins redundantly repressed the mRNA of inducible costimulator (Icos) and identified the Ox40 costimulatory receptor as another shared mRNA target. Combined acute deletion increased Ox40 signaling, as well as Irf4 expression, and imposed Tfh differentiation on CD4(+) T cells. These data imply that both proteins maintain tolerance by preventing inappropriate T cell activation and Tfh cell differentiation, and that Roquin-2 compensates in the absence of Roquin-1, but not in the presence of its mutated form.
Asunto(s)
Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , ARN Mensajero/metabolismo , Receptores OX40/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antígenos CD4/metabolismo , Diferenciación Celular/genética , Células HEK293 , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Unión Proteica , Receptores OX40/genética , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Lipid droplets (LDs) are cellular storage organelles for neutral lipids that vary in size and abundance according to cellular needs. Physiological conditions that promote lipid storage rapidly and markedly increase LD volume and surface. How the need for surface phospholipids is sensed and balanced during this process is unknown. Here, we show that phosphatidylcholine (PC) acts as a surfactant to prevent LD coalescence, which otherwise yields large, lipolysis-resistant LDs and triglyceride (TG) accumulation. The need for additional PC to coat the enlarging surface during LD expansion is provided by the Kennedy pathway, which is activated by reversible targeting of the rate-limiting enzyme, CTP:phosphocholine cytidylyltransferase (CCT), to growing LD surfaces. The requirement, targeting, and activation of CCT to growing LDs were similar in cells of Drosophila and mice. Our results reveal a mechanism to maintain PC homeostasis at the expanding LD monolayer through targeted activation of a key PC synthesis enzyme.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/metabolismo , Metabolismo de los Lípidos/fisiología , Fosfatidilcolinas/fisiología , Animales , Citidililtransferasa de Colina-Fosfato/antagonistas & inhibidores , Citidililtransferasa de Colina-Fosfato/genética , Drosophila , Lipólisis , Ratones , Ácido Oléico/metabolismo , Fosfatidilcolinas/biosíntesis , Interferencia de ARN , Triglicéridos/metabolismoRESUMEN
The ubiquitin-editing enzyme A20/TNFAIP3 is essential for controlling signals inducing the activation of nuclear factor-κB transcription factors. Polymorphisms and mutations in the TNFAIP3 gene are linked to various human autoimmune conditions, and inactivation of A20 is a frequent event in human B-cell lymphomas characterized by constitutive nuclear factor-κB activity. Through B cell-specific ablation in the mouse, we show here that A20 is required for the normal differentiation of the marginal zone B and B1 cell subsets. However, loss of A20 in B cells lowers their activation threshold and enhances proliferation and survival in a gene-dose-dependent fashion. Through the expression of proinflammatory cytokines, most notably interleukin-6, A20-deficient B cells trigger a progressive inflammatory reaction in naive mice characterized by the expansion of myeloid cells, effector-type T cells, and regulatory T cells. This culminates in old mice in an autoimmune syndrome characterized by splenomegaly, plasma cell hyperplasia, and the presence of class-switched, tissue-specific autoantibodies.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/patología , Cisteína Endopeptidasas/deficiencia , Cisteína Endopeptidasas/inmunología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/inmunología , Envejecimiento/inmunología , Envejecimiento/patología , Animales , Autoinmunidad , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Diferenciación Celular , Cisteína Endopeptidasas/genética , Dosificación de Gen , Humanos , Técnicas In Vitro , Inflamación/etiología , Inflamación/inmunología , Inflamación/patología , Interleucina-6/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/patología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Intact antibodies and antigen binding fragments (Fab) have been previously shown to form an alternatively folded state (AFS) at low pH. This state consists primarily of secondary structure interactions, with reduced tertiary structure content. The AFS can be distinguished from the molten globule state by the formation of nonnative structure and, in particular, its high stability. In this study, the isolated domains of the MAK33 (murine monoclonal antibody of the subtype kappa/IgG1) Fab fragment were investigated under conditions that have been reported to induce the AFS. Surprising differences in the ability of individual domains to form the AFS were observed, despite the similarities in their native structures. All Fab domains were able to adopt the AFS, but only for V(H) (variable domain of the heavy chain) could a significant amount of tertiary structure be detected and different conditions were needed to induce the AFS. V(H), the least stable of the domains under physiological conditions, was the most stable in the AFS, yet all domains showed significant stability against thermal and chemical unfolding in their AFS. Formation of the AFS was found to generally proceed via the unfolded state, with similar rates for most of the domains. Taken together, our data reveal striking differences in the biophysical properties of the AFS of individual antibody domains that reflect the variation possible for domains of highly homologous native structures. Furthermore, they allow individual domain contributions to be dissected from specific oligomer effects in the AFS of the antibody Fab fragment.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Animales , Fragmentos Fab de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/metabolismo , Ratones , Microscopía de Fuerza Atómica , Modelos Moleculares , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Interferon-producing killer dendritic cells (IKDCs) have been described as possessing the lytic potential of NK cells and the antigen-presenting capacity of dendritic cells (DCs). In this study, we examine the lytic function and antigen-presenting capacity of mouse spleen IKDCs, including those found in DC preparations. IKDCs efficiently killed NK cell targets, without requiring additional activation stimuli. However, in our hands, when exposed to protein antigen or to MHC class II peptide, IKDCs induced little or no T cell proliferation relative to conventional DCs or plasmacytoid DCs, either before or after activation with CpG, or in several disease models. Certain developmental features indicated that IKDCs resembled NK cells more than DCs. IKDCs, like NK cells, did not express the transcription factor PU.1 and were absent from recombinase activating gene-2-null, common gamma-chain-null (Rag2(-/-)Il2rg(-/-)) mice. When cultured with IL-15 and -18, IKDCs proliferated extensively, like NK cells. Under these conditions, a proportion of expanded IKDCs and NK cells expressed high levels of surface MHC class II. However, even such MHC class II(+) IKDCs and NK cells induced poor T cell proliferative responses compared with DCs. Thus, IKDCs resemble NK cells functionally, and neither cell type could be induced to be effective antigen-presenting cells.