RESUMO
Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology.
Assuntos
Apresentação de Antígeno , Mitocôndrias/imunologia , Doença de Parkinson/imunologia , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Dendríticas/patologia , Modelos Animais de Doenças , Inflamação/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Doença de Parkinson/patologia , Proteínas Quinases/genética , Vesículas Transportadoras/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Viral proteins are usually processed by the 'classical' major histocompatibility complex (MHC) class I presentation pathway. Here we showed that although macrophages infected with herpes simplex virus type 1 (HSV-1) initially stimulated CD8(+) T cells by this pathway, a second pathway involving a vacuolar compartment was triggered later during infection. Morphological and functional analyses indicated that distinct forms of autophagy facilitated the presentation of HSV-1 antigens on MHC class I molecules. One form of autophagy involved a previously unknown type of autophagosome that originated from the nuclear envelope. Whereas interferon-gamma stimulated classical MHC class I presentation, fever-like hyperthermia and the pyrogenic cytokine interleukin 1beta activated autophagy and the vacuolar processing of viral peptides. Viral peptides in autophagosomes were further processed by the proteasome, which suggests a complex interaction between the vacuolar and MHC class I presentation pathways.
Assuntos
Apresentação de Antígeno/imunologia , Autofagia/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Animais , Antígenos Virais/imunologia , Citocinas/imunologia , Imunofluorescência , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Microscopia Eletrônica de Transmissão , Fagossomos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Vacúolos/imunologiaRESUMO
Macrophages play an important role in innate and adaptive immunity as professional phagocytes capable of internalizing and degrading pathogens to derive antigens for presentation to T cells. They also produce pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that mediate local and systemic responses and direct the development of adaptive immunity. The present work describes the use of label-free quantitative proteomics to profile the dynamic changes of proteins from resting and TNF-α-activated mouse macrophages. These analyses revealed that TNF-α activation of macrophages led to the down-regulation of mitochondrial proteins and the differential regulation of several proteins involved in vesicle trafficking and immune response. Importantly, we found that the down-regulation of mitochondria proteins occurred through mitophagy and was specific to TNF-α, as other cytokines such as IL-1ß and IFN-γ had no effect on mitochondria degradation. Furthermore, using a novel antigen presentation system, we observed that the induction of mitophagy by TNF-α enabled the processing and presentation of mitochondrial antigens at the cell surface by MHC class I molecules. These findings highlight an unsuspected role of TNF-α in mitophagy and expanded our understanding of the mechanisms responsible for MHC presentation of self-antigens.
Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Mitofagia/efeitos dos fármacos , Proteômica/métodos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antígenos/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular , Biologia Computacional , Regulação para Baixo/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteoma/metabolismoRESUMO
Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ≈ 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.
Assuntos
Retículo Endoplasmático/química , Macrófagos/metabolismo , Fagossomos/química , Proteômica/métodos , Animais , Biomarcadores/análise , Western Blotting , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Endossomos/química , Endossomos/metabolismo , Endossomos/ultraestrutura , Imunofluorescência , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Marcação por Isótopo , Lisossomos/química , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Macrófagos/citologia , Macrófagos/ultraestrutura , Espectrometria de Massas , Camundongos , Oligopeptídeos , Fagocitose , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Plasmídeos , Sinais Direcionadores de Proteínas , Proteínas Qa-SNARE , TransfecçãoRESUMO
The invariant chain (Ii) has pleiotropic functions and is a key factor in antigen presentation. Ii associates with major histocompatibility complex class II molecules in the endoplasmic reticulum (ER) and targets the complex in the endocytic pathway to allow antigenic peptide loading. The human Iip35 isoform includes a cytoplasmic extension containing a di-arginine motif causing ER retention. This minor isoform does not exist in mice and its function in humans has not been thoroughly investigated. We have recently generated transgenic mice expressing Iip35 and these were crossed with Ii-deficient mice to generate animals (Tgp35/mIiKO) expressing exclusively the human isoform. In these mice, we show that Iip35 is expressed in antigen presenting cells and is inducible by interferon gamma (IFN-γ). Despite the low constitutive expression of the protein and some minor differences in the Vß repertoire of Tgp35/mIiKO mice, Iip35 restored thymic selection of CD4(+) T cells and of invariant natural killer T cells. In vitro functional assays using purified primary macrophages treated with IFN-γ showed that Iip35 allows presentation of an Ii-dependent ovalbumin T-cell epitope. Altogether, our results suggest that Iip35 is functional and does not require co-expression of other isoforms for antigen presentation.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Timo/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Ovalbumina/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Timócitos/imunologia , Timócitos/metabolismo , Timo/citologia , Timo/metabolismoRESUMO
The classical view that endogenous antigens are processed by the proteasome and loaded on MHC class I molecules in the endoplasmic reticulum, while exogenous antigens taken up by endocytosis or phagocytosis are degraded and loaded on MHC class II in lysosome-derived organelles, has evolved along with the improvement of our understanding of the cell biology of antigen-presenting cells. In recent years, evidence for alternative presentation pathways has emerged. Exogenous antigens can be processed by the proteasome and loaded on MHC class I through a pathway called cross-presentation. Moreover, endogenous antigens can be targeted to lytic organelles for presentation on MHC class II through autophagy, a highly conserved cellular process of self-eating. Recent evidence indicates that the vacuolar degradation of endogenous antigens is also beneficial for presentation on MHC class I molecules. This review focuses on how various forms of autophagy participate to presentation of these antigens on MHC class I.
Assuntos
Apresentação de Antígeno/imunologia , Autofagia/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Animais , Apresentação Cruzada/imunologia , Lisossomos/imunologia , Camundongos , Peptídeos/imunologia , Fagocitose/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Vacúolos/imunologia , Viroses/imunologiaRESUMO
Most soluble lysosomal proteins carry Man6P (mannose 6-phosphate), a specific carbohydrate marker that enables their binding to cellular MPRs (Man6P receptors) and their subsequent targeting towards the lysosome. This characteristic was exploited to identify novel soluble lysosomal proteins by proteomic analysis of Man6P proteins purified from a human cell line. Among the proteins identified during the course of the latter study [Journet, Chapel, Kieffer, Roux and Garin (2002) Proteomics, 2, 1026-1040], some had not been previously described as lysosomal proteins. We focused on a protein detected at 76 kDa by SDS/PAGE. We named this protein 'p76' and it appeared later in the NCBI protein database as the 'hypothetical protein LOC196463'. In the present paper, we describe the identification of p76 by MS and we analyse several of its biochemical characteristics. The presence of Man6P sugars was confirmed by an MPR overlay experiment, which showed the direct and Man6P-dependent interaction between p76 and the MPR. The presence of six N-glycosylation sites was validated by progressive peptide-N-glycosidase F deglycosylation. Experiments using N- and C-termini directed anti-p76 antibodies provided insights into p76 maturation. Most importantly, we were able to demonstrate the lysosomal localization of this protein, which was initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates.
Assuntos
Glicoproteínas/metabolismo , Lisossomos/metabolismo , Animais , Biomarcadores , Linhagem Celular , Glicosilação , Humanos , Fígado/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional , RatosAssuntos
Antígenos Virais/fisiologia , Autofagia/fisiologia , Ativação Linfocitária/fisiologia , Animais , Antígenos Virais/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Ativação Linfocitária/imunologia , Proteínas Virais/imunologia , Proteínas Virais/fisiologiaRESUMO
Complex membrane trafficking events are involved in the regulation of antigen processing and presentation of both endogenous and exogenous antigens. While these processes were believed to involve mainly organelles along the endo/phagocytic and the biosynthetic pathways, recent studies have shown that autophagy also participates actively in both innate and adaptive immunity. We have shown recently that, in macrophages infected with the Herpes simplex type 1 virus, autophagy plays a key role in the targeting of viral proteins to hydrolytic compartments, and their processing for presentation on MHC class I molecules. This pathway involves a novel type of autophagosomes formed by coiling of the nuclear membrane where viral proteins are highly enriched. The ability to enhance the contribution of autophagy to antigen presentation in various conditions suggests that this pathway could be used to boost the immune response against viral infection and develop new vaccines.