RESUMEN
Dendritic cells (DCs) are key players in the immune system. Much of their biology has been elucidated via culture systems in which hematopoietic precursors differentiate into DCs under the aegis of cytokines. A widely used protocol involves the culture of murine bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate BM-derived DCs (BMDCs). BMDCs express CD11c and MHC class II (MHCII) molecules and share with DCs isolated from tissues the ability to present exogenous antigens to T cells and to respond to microbial stimuli by undergoing maturation. We demonstrate that CD11c(+)MHCII(+) BMDCs are in fact a heterogeneous group of cells that comprises conventional DCs and monocyte-derived macrophages. DCs and macrophages in GM-CSF cultures both undergo maturation upon stimulation with lipopolysaccharide but respond differentially to the stimulus and remain separable entities. These results have important implications for the interpretation of a vast array of data obtained with DC culture systems.
Asunto(s)
Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Animales , Presentación de Antígeno , Antígeno CD11c/metabolismo , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunofenotipificación , Lipopolisacáridos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , TranscriptomaRESUMEN
DNGR-1 is a C-type lectin receptor that binds F-actin exposed by dying cells and facilitates cross-presentation of dead cell-associated antigens by dendritic cells. Here we present the structure of DNGR-1 bound to F-actin at 7.7 Å resolution. Unusually for F-actin binding proteins, the DNGR-1 ligand binding domain contacts three actin subunits helically arranged in the actin filament, bridging over two protofilaments, as well as two neighboring actin subunits along one protofilament. Mutation of residues predicted to mediate ligand binding led to loss of DNGR-1-dependent cross-presentation of dead cell-associated antigens, formally demonstrating that the latter depends on F-actin recognition. Notably, DNGR-1 has relatively modest affinity for F-actin but multivalent interactions allow a marked increase in binding strength. Our findings shed light on modes of actin binding by cellular proteins and reveal how extracellular detection of cytoskeletal components by dedicated receptors allows immune monitoring of loss of cellular integrity.
Asunto(s)
Actinas/química , Reactividad Cruzada , Células Dendríticas/inmunología , Lectinas Tipo C/química , Modelos Moleculares , Receptores Inmunológicos/química , Actinas/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Mutación , Unión ProteicaRESUMEN
Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf-/-), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf-/- mouse are much greater than in those of Cdnf+/+ mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf-/- mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf-/- male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf-/- mouse brain is altered. The deficiencies of Cdnf-/- mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.
Asunto(s)
Encéfalo/patología , Encéfalo/fisiología , Dopamina/metabolismo , Sistema Nervioso Entérico/patología , Sistema Nervioso Entérico/fisiopatología , Factores de Crecimiento Nervioso/fisiología , Neuronas/patología , Neuronas/fisiología , Animales , Apoptosis , Autofagia , Femenino , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso/genéticaRESUMEN
Phosphoinositide-3-kinases have been shown to be involved in influenza virus pathogenesis. They are targeted directly by virus proteins and are essential for efficient viral replication in infected lung epithelial cells. However, to date the role of PI3K signaling in influenza infection in vivo has not been thoroughly addressed. Here we show that one of the PI3K subunits, p110γ, is in fact critically required for mediating the host's antiviral response. PI3Kγ deficient animals exhibit a delayed viral clearance and increased morbidity during respiratory infection with influenza virus. We demonstrate that p110γ is required for the generation and maintenance of potent antiviral CD8+ T cell responses through the developmental regulation of pulmonary cross-presenting CD103+ dendritic cells under homeostatic and inflammatory conditions. The defect in lung dendritic cells leads to deficient CD8+ T cell priming, which is associated with higher viral titers and more severe disease course during the infection. We thus identify PI3Kγ as a novel key host protective factor in influenza virus infection and shed light on an unappreciated layer of complexity concerning the role of PI3K signaling in this context.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/citología , Pulmón/virología , Infecciones por Orthomyxoviridae/virología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Células Epiteliales/virología , Pulmón/inmunología , Activación de Linfocitos/inmunología , Ratones , Replicación Viral/fisiologíaRESUMEN
Being deeply connected to signalling, cell dynamics, growth, regulation, and defence, endocytic processes are linked to almost all aspects of cell life and disease. In this review, we focus on endosomes in the classical endocytic pathway, and on the programme of changes that lead to the formation and maturation of late endosomes/multivesicular bodies. The maturation programme entails a dramatic transformation of these dynamic organelles disconnecting them functionally and spatially from early endosomes and preparing them for their unidirectional role as a feeder pathway to lysosomes.
Asunto(s)
Endocitosis/fisiología , Endosomas/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Autofagia/fisiología , Proteína Coat de Complejo I/metabolismo , Endosomas/ultraestructura , Exosomas/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Cullin-3 (Cul3) functions as a scaffolding protein in the Bric-a-brac, Tramtrack, Broad-complex (BTB)-Cul3-Rbx1 ubiquitin E3 ligase complex. Here, we report a previously undescribed role for Cul3 complexes in late endosome (LE) maturation. RNAi-mediated depletion of Cul3 results in a trafficking defect of two cargoes of the endolysosomal pathway, influenza A virus (IAV) and epidermal growth factor receptor (EGFR). IAV is able to reach an acidic endosomal compartment, coinciding with LE/lysosome (LY) markers. However, it remains trapped or the capsid is unable to uncoat after penetration into the cytosol. Similarly, activation and subsequent ubiquitination of EGFR appear normal, whereas downstream EGFR degradation is delayed and its ligand EGF accumulates in LE/LYs. Indeed, Cul3-depleted cells display severe morphological defects in LEs that could account for these trafficking defects; they accumulate acidic LE/LYs, and some cells become highly vacuolated, with enlarged Rab7-positive endosomes. Together, these results suggest a crucial role of Cul3 in regulating late steps in the endolysosomal trafficking pathway.
Asunto(s)
Proteínas Cullin/metabolismo , Endosomas/metabolismo , Compartimento Celular , Línea Celular Tumoral , Endosomas/virología , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Humanos , Virus de la Influenza A/fisiología , Lisosomas/metabolismo , Proteolisis , ARN Interferente Pequeño/metabolismo , Coloración y Etiquetado , Ubiquitinación , Internalización del VirusRESUMEN
Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Cullin/metabolismo , Endocitosis , Endosomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transporte Biológico , Línea Celular , Humanos , Virus de la Influenza A/fisiología , Internalización del VirusRESUMEN
Many enveloped and non-enveloped animal viruses delay the penetration into the cytosol of host cells until they have arrived to endocytic vacuoles deep in the cytoplasm. The late timing is generally determined by a low pH-threshold for the acid-activated penetration process (pH 6.2-4.9), but there can be a combination of other reasons for a delay. Since late-penetrating viruses (L-PVs) must be sorted into the degradative pathway, they are particularly sensitive to perturbations that interfere with molecular sorting and proper maturation of endosomes, including switching of Rabs, formation of intraluminal vesicles, and microtubule-mediated transport. In this short review, we focus on L-PVs from several virus families, and their interactions with the endocytic machinery.