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1.
Proc Natl Acad Sci U S A ; 120(34): e2211281120, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37579175

RESUMO

Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release.


Assuntos
Capsídeo , Infecções por Vírus Epstein-Barr , Humanos , Capsídeo/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Envelope Viral/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo
2.
Methods Mol Biol ; 2692: 311-336, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37365477

RESUMO

Canonical autophagy and the non-canonical autophagy pathway LC3-associated phagocytosis (LAP) play crucial roles in the immune system by processing antigens for major histocompatibility complex (MHC) class II restricted presentation to CD4+ T cells. Recent studies offer insight into the relationship between LAP, autophagy, and antigen processing in macrophages and dendritic cells; however their involvement during antigen processing in B cells is less well understood.In this chapter, we describe how to monitor, manipulate, and understand the role of LAP and classical autophagy during MHC-restricted antigen presentation by human monocyte-derived macrophages as well as in B cell lymphoblastoid cell lines (LCLs). It includes an explanation on how to generate LCLs and monocyte-derived macrophages from primary human cells. Then we describe two different approaches to manipulate the autophagy pathways: silencing of the atg4b gene using CRISPR/Cas9 technology and a lentivirus delivery system for specific ATG4B overexpression. We also propose a method for triggering LAP and measuring different ATG proteins using Western blot and immunofluorescence. Finally, we show an approach to investigate MHC class II antigen presentation by an in vitro co-culture assay that uses the measurement of secreted cytokines, released by activated CD4+ T cells, as readout.


Assuntos
Apresentação de Antígeno , Fagocitose , Humanos , Macrófagos/metabolismo , Autofagia/genética , Antígenos de Histocompatibilidade Classe II/metabolismo
3.
Blood ; 137(23): 3225-3236, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33827115

RESUMO

Primary immunodeficiencies in the costimulatory molecule CD27 and its ligand, CD70, predispose for pathologies of uncontrolled Epstein-Barr virus (EBV) infection in nearly all affected patients. We demonstrate that both depletion of CD27+ cells and antibody blocking of CD27 interaction with CD70 cause uncontrolled EBV infection in mice with reconstituted human immune system components. While overall CD8+ T-cell expansion and composition are unaltered after antibody blocking of CD27, only some EBV-specific CD8+ T-cell responses, exemplified by early lytic EBV antigen BMLF1-specific CD8+ T cells, are inhibited in their proliferation and killing of EBV-transformed B cells. This suggests that CD27 is not required for all CD8+ T-cell expansions and cytotoxicity but is required for a subset of CD8+ T-cell responses that protect us from EBV pathology.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunidade Celular , Fosfoproteínas/imunologia , Transativadores/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Linfócitos B/imunologia , Transformação Celular Viral/genética , Transformação Celular Viral/imunologia , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fosfoproteínas/genética , Transativadores/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética
4.
Nat Commun ; 12(1): 1508, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686057

RESUMO

LC3-associated phagocytosis (LAP) contributes to a wide range of cellular processes and notably to immunity. The stabilization of phagosomes by the macroautophagy machinery in human macrophages can maintain antigen presentation on MHC class II molecules. However, the molecular mechanisms involved in the formation and maturation of the resulting LAPosomes are not completely understood. Here, we show that reactive oxygen species (ROS) produced by NADPH oxidase 2 (NOX2) stabilize LAPosomes by inhibiting LC3 deconjugation from the LAPosome cytosolic surface. NOX2 residing in the LAPosome membrane generates ROS to cause oxidative inactivation of the protease ATG4B, which otherwise releases LC3B from LAPosomes. An oxidation-insensitive ATG4B mutant compromises LAP and thereby impedes sustained MHC class II presentation of exogenous Candida albicans antigens. Redox regulation of ATG4B is thereby an important mechanism for maintaining LC3 decoration of LAPosomes to support antigen processing for MHC class II presentation.


Assuntos
Apresentação de Antígeno/fisiologia , Autofagia/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Fagossomos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos de Fungos , Proteínas Relacionadas à Autofagia , Candida albicans , Classe III de Fosfatidilinositol 3-Quinases , Cisteína Endopeptidases/metabolismo , Células HEK293 , Humanos , Macroautofagia , Macrófagos/metabolismo , NADPH Oxidase 2/metabolismo , Oxirredução , Fagocitose/fisiologia , Espécies Reativas de Oxigênio/metabolismo
5.
Autophagy ; 17(5): 1244-1258, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32401602

RESUMO

Whereas central nervous system (CNS) homeostasis is highly dependent on tissue surveillance by immune cells, dysregulated entry of leukocytes during autoimmune neuroinflammation causes severe immunopathology and neurological deficits. To invade the CNS parenchyma, encephalitogenic T helper (TH) cells must encounter their cognate antigen(s) presented by local major histocompatibility complex (MHC) class II-expressing antigen-presenting cells (APCs). The precise mechanisms by which CNS-associated APCs facilitate autoimmune T cell reactivation remain largely unknown. We previously showed that mice with conditional deletion of the gene encoding the essential autophagy protein ATG5 in dendritic cells (DCs) are resistant to EAE development. Here, we report that the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2, also known as CYBB/NOX2, in conventional DCs (cDCs) regulates endocytosed MOG (myelin oligodendrocyte protein) antigen processing and supports MOG-antigen presentation to CD4+ T cells through LC3-associated phagocytosis (LAP). Genetic ablation of Cybb in cDCs is sufficient to restrain encephalitogenic TH cell recruitment into the CNS and to ameliorate clinical disease development upon the adoptive transfer of MOG-specific CD4+ T cells. These data indicate that CYBB-regulated MOG-antigen processing and LAP in cDCs licenses encephalitogenic TH cells to initiate and sustain autoimmune neuroinflammation.Abbreviations: Ag: antigen; APC: antigen-presenting cell; AT: adoptive transfer; ATG/Atg: autophagy-related; BAMs: border-associated macrophages; BMDC: bone marrow-derived DC; CD: cluster of differentiation; CNS: central nervous system; CSF2/GM-CSF: colony stimulating factor 2 (granulocyte-macrophage); CYBB/NOX2/gp91phox: cytochrome b-245, beta polypeptide; DC: dendritic cell; EAE: experimental autoimmune encephalomyelitis; fl: floxed; FOXP3: forkhead box P3; GFP: green fluorescent protein; H2-Ab: histocompatibility 2, class II antigen A, beta 1; IFN: interferon; IL: interleukin; ITGAX/CD11c: integrin subunit alpha X; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: median fluorescence intensity; MG: microglia; MHCII: major histocompatibility complex class II; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; NADPH: nicotinamide adenine dinucleotide phosphate; ODC: oligodendroglial cell; OVA: ovalbumin; pDC: plasmacytoid DC; Ptd-L-Ser: phosphatidylserine; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; SLE: systemic lupus erythematosus; TH cells: T helper cells; TLR: toll-like receptor; ZBTB46: zinc finger and BTB domain containing 46.


Assuntos
Autofagia/fisiologia , Células Dendríticas/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Inflamação/metabolismo , NADPH Oxidase 2/metabolismo , Linfócitos T/metabolismo , Animais , Encefalomielite Autoimune Experimental/imunologia , Ativação Linfocitária/imunologia , Macrófagos/metabolismo , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/metabolismo , Fagocitose/imunologia
6.
Curr Protoc Immunol ; 123(1): e60, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30253073

RESUMO

LC3-associated phagocytosis (LAP) is an unconventional form of autophagy that relies on parts of the canonical autophagy machinery for its function. LAP is triggered upon receptor-mediated phagocytosis and is characterized by the formation of a single-membrane vesicle decorated with the autophagy protein LC3. In professional phagocytic cells, such as macrophages, the role of LAP in immune processes has been characterized, although how LAP functions at the molecular level remains poorly defined. It is important to point out that as for all autophagic pathways, the study of LAP is still challenging for the scientific community because it is a dynamic and complex process, requiring interactions among several proteins. Here, we describe the most common methods used to monitor and quantify the formation of LC3-coated single-membrane endosomes, or so-called LAPosomes, and to validate the involvement of LAP in immunological processes of human macrophages. © 2018 by John Wiley & Sons, Inc.


Assuntos
Apresentação de Antígeno , Endossomos/imunologia , Macrófagos/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Fagocitose , Animais , Humanos
7.
Cell Rep ; 15(5): 1076-1087, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-27117419

RESUMO

The macroautophagy machinery has been implicated in MHC class II restricted antigen presentation. Here, we report that this machinery assists in the internalization of MHC class I molecules. In the absence of the autophagy factors Atg5 and Atg7, MHC class I surface levels are elevated due to decreased endocytosis and degradation. Internalization of MHC class I molecules occurs less efficiently if AAK1 cannot be recruited via Atg8/LC3B. In the absence of Atg-dependent MHC class I internalization, dendritic cells stimulate CD8(+) T cell responses more efficiently in vitro and in vivo. During viral infections, lack of Atg5 results in enhanced influenza- and LCMV-specific CD8(+) T cell responses in vivo. Elevated influenza-specific CD8(+) T cell responses are associated with better immune control of this infection. Thus, the macroautophagy machinery orchestrates T cell immunity by supporting MHC class II but compromises MHC class I restricted antigen presentation.


Assuntos
Proteína 5 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Apresentação de Antígeno/imunologia , Células Cultivadas , Endocitose/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Autophagy ; 10(9): 1588-602, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25046114

RESUMO

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking autophagosome acidification. In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy. Here, we address the question of the membrane traffic during internalization of Yersinia investigating the role of vesicle- associated membrane proteins (VAMPs). First, we show that as in epithelial cells, Yersinia pseudotuberculosis replicates mainly in nonacidic LC3-positive vacuoles. Second, in these cells, we unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single membranes using correlative light-electron microscopy. Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3. Fourth, we show that VAMP7 knockdown alters LC3's association with single-and multimembrane-YCVs. Finally, in uninfected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher number of double-membrane, LC3-positive vesicles. Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion proteins involved in Yersinia pseudotuberculosis' intravesicular life cycle.


Assuntos
Autofagia/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas R-SNARE/metabolismo , Transdução de Sinais , Vacúolos/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Yersinia pseudotuberculosis/metabolismo , Linhagem Celular , Humanos , Macrófagos/citologia , Microscopia Eletrônica , Fagossomos/ultraestrutura
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