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1.
Nat Commun ; 10(1): 1834, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015422

RESUMO

Prevention of inflammatory bowel disease (IBD) relies on tight control of inflammatory, cell death and autophagic mechanisms, but how these pathways are integrated at the molecular level is still unclear. Here we show that the anti-inflammatory protein A20 and the critical autophagic mediator Atg16l1 physically interact and synergize to regulate the stability of the intestinal epithelial barrier. A proteomic screen using the WD40 domain of ATG16L1 (WDD) identified A20 as a WDD-interacting protein. Loss of A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology, as characterized by severe inflammation and increased intestinal epithelial cell death in both small and large intestine. Mechanistically, absence of A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or expression of WDD-deficient Atg16l1 stabilizes A20. Collectively our data show that A20 and Atg16l1 cooperatively control intestinal homeostasis by acting at the intersection of inflammatory, autophagy and cell death pathways.


Assuntos
Proteínas de Transporte/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Repetições WD40/genética , Animais , Autofagia/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Endoscopia , Feminino , Homeostase/imunologia , Humanos , Doenças Inflamatórias Intestinais/diagnóstico por imagem , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/imunologia , Proteômica , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Repetições WD40/imunologia
2.
Lett Appl Microbiol ; 69(1): 11-15, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004518

RESUMO

Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a wide range of hosts, including poultry, and causes acute gastroenteritis in humans that may result in death. Superoxide dismutase (SOD) is an important antioxidant enzyme present in nearly all living cells exposed to oxygen. Recently, we reported the novel roles of SOD in serum resistance and biofilm formation in S. Typhimurium. This study was designed to explore the effect of infection with sodA mutant of S. Typhimurium on the autophagic response of macrophages. Murine macrophage cell line RAW264·7 was infected with wild-type (LSM52), a sodA deletion mutant (LSM52ΔsodA) and complemented strain (LSM52CΔsodA). We found that sodA deletion triggered remarkable autophagic responses in infected cells, shown as higher concentrations of LC3-II or Beclin-1 than those infected with the wild-type or complemented strain during the first hour post-infection in S. Typhimurium. Consistent with these results, the number of viable bacteria in cells infected with the sodA mutant was significantly lower than those infected with wild-type or complemented strains at 1 h, 2 h and 3 h post-infection in S. Typhimurium. All results indicated that infection with sodA mutant of S. Typhimurium leads to up-regulation of autophagy in Raw264·7 macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: Autophagy plays an important role in Salmonella infection although the role of autophagy in Salmonella infection remains unclear. This study was designed to explore the effect of sodA on the autophagic response of macrophage. We found that infection with sodA mutant of Salmonella Typhimurium could lead to up-regulation of autophagy in Raw264·7 macrophages.


Assuntos
Autofagia/imunologia , Proteínas de Bactérias/genética , Macrófagos/imunologia , Macrófagos/microbiologia , Salmonella typhimurium/genética , Superóxido Dismutase/genética , Animais , Proteína Beclina-1/metabolismo , Linhagem Celular , Humanos , Camundongos , Células RAW 264.7 , Infecções por Salmonella , Deleção de Sequência/genética , Regulação para Cima , Virulência
3.
Science ; 363(6434)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30923193

RESUMO

A paradox of tumor immunology is that tumor-infiltrating lymphocytes are dysfunctional in situ, yet are capable of stem cell-like behavior including self-renewal, expansion, and multipotency, resulting in the eradication of large metastatic tumors. We find that the overabundance of potassium in the tumor microenvironment underlies this dichotomy, triggering suppression of T cell effector function while preserving stemness. High levels of extracellular potassium constrain T cell effector programs by limiting nutrient uptake, thereby inducing autophagy and reduction of histone acetylation at effector and exhaustion loci, which in turn produces CD8+ T cells with improved in vivo persistence, multipotency, and tumor clearance. This mechanistic knowledge advances our understanding of T cell dysfunction and may lead to novel approaches that enable the development of enhanced T cell strategies for cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Potássio/metabolismo , Células-Tronco/imunologia , Acetilcoenzima A/metabolismo , Acetilação , Animais , Autofagia/imunologia , Restrição Calórica , Diferenciação Celular/genética , Epigênese Genética , Histonas/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Microambiente Tumoral
4.
Viruses ; 11(2)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717138

RESUMO

Autophagy exhibits dual effects during viral infections, promoting the clearance of viral components and activating the immune system to produce antiviral cytokines. However, some viruses impair immune defenses by collaborating with autophagy. Mounting evidence suggests that the interaction between autophagy and innate immunity is critical to understanding the contradictory roles of autophagy. Type I interferon (IFN-I) is a crucial antiviral factor, and studies have indicated that autophagy affects IFN-I responses by regulating IFN-I and its receptors expression. Similarly, IFN-I and interferon-stimulated gene (ISG) products can harness autophagy to regulate antiviral immunity. Crosstalk between autophagy and IFN-I responses could be a vital aspect of the molecular mechanisms involving autophagy in innate antiviral immunity. This review briefly summarizes the approaches by which autophagy regulates antiviral IFN-I responses and highlights the recent advances on the mechanisms by which IFN-I and ISG products employ autophagy against viruses.


Assuntos
Autofagia/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Interferon Tipo I/imunologia , Viroses/imunologia , Regulação da Expressão Gênica , Humanos , Interferon-alfa/imunologia , Interferon beta/imunologia , Transdução de Sinais/imunologia
5.
Mol Cancer ; 18(1): 17, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678689

RESUMO

Autophagy is a genetically well-controlled cellular process that is tightly controlled by a set of core genes, including the family of autophagy-related genes (ATG). Autophagy is a "double-edged sword" in tumors. It can promote or suppress tumor development, which depends on the cell and tissue types and the stages of tumor. At present, tumor immunotherapy is a promising treatment strategy against tumors. Recent studies have shown that autophagy significantly controls immune responses by modulating the functions of immune cells and the production of cytokines. Conversely, some cytokines and immune cells have a great effect on the function of autophagy. Therapies aiming at autophagy to enhance the immune responses and anti-tumor effects of immunotherapy have become the prospective strategy, with enhanced antigen presentation and higher sensitivity to CTLs. However, the induction of autophagy may also benefit tumor cells escape from immune surveillance and result in intrinsic resistance against anti-tumor immunotherapy. Increasing studies have proven the optimal use of either ATG inducers or inhibitors can restrain tumor growth and progression by enhancing anti-tumor immune responses and overcoming the anti-tumor immune resistance in combination with several immunotherapeutic strategies, indicating that induction or inhibition of autophagy might show us a prospective therapeutic strategy when combined with immunotherapy. In this article, the possible mechanisms of autophagy regulating immune system, and the potential applications of autophagy in tumor immunotherapy will be discussed.


Assuntos
Autofagia/imunologia , Sistema Imunitário/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Citocinas/imunologia , Humanos , Imunoterapia/métodos
6.
Methods Mol Biol ; 1880: 455-477, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610715

RESUMO

Macroautophagy is a ubiquitous degradative pathway involved in innate and adaptive immunity. Its molecular machinery has been described to deliver intracellular and extracellular antigens to MHC class II loading compartment by regulating autophagosome and phagosome maturation. We recently found that the respective Atg proteins can contribute to MHC class I-restricted antigen presentation to CD8+ T cells by regulating MHC class I surface levels in mouse dendritic cell. Indeed, we determined that MHC class I molecules are stabilized on the cell surface of murine antigen presenting cells deficient for core components of the macroautophagy machinery such as Atg5 and Atg7. This stabilization seems to result from defective internalization of MHC class I molecules dependent on adaptor protein kinase 1 (AAK1), involved in clathrin-mediated endocytosis. Moreover, macroautophagy-dependent stabilization of MHC class I molecules leads to enhanced CD8+ T cell priming during influenza A virus infection in vivo, resulting in decreased pathology. In this chapter, we describe four experiments to monitor, characterize, and quantify the effect of macroautophagy deficiency on MHC class I molecule trafficking and the subsequent CD8+ T cell priming. First, we will show how to monitor MHC class I internalization in lung CD11c+ cells from mice lacking key components of the macroautophagy machinery. Then, we will propose a method to characterize the interaction between either MHC class I or Atg8/LC3 with AAK1. Finally, we will describe how to evaluate the influenza A-specific CD8+ T cell response in mice conditionally depleted for Atg5 in their DC compartment. This set of experiments allows to characterize MHC class I internalization with the help of the molecular machinery of macroautophagy.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/imunologia , Bioensaio/métodos , Antígenos de Histocompatibilidade Classe I/metabolismo , Animais , Apresentação do Antígeno/imunologia , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Bioensaio/instrumentação , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Separação Celular/instrumentação , Separação Celular/métodos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Endocitose/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vírus da Influenza A/imunologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo
7.
Methods Mol Biol ; 1880: 679-690, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610731

RESUMO

Bacteria that escape from membrane-enclosed vacuoles to the cytosol of cells are targeted by autophagy, which recognizes and captures bacteria into autophagosomes wherein their proliferation is restricted. Here we discuss two means by which antibacterial autophagy is assessed: (1) the visualization and enumeration of autophagy protein recruitment to the vicinity of cytosolic bacteria by means of immunofluorescence microscopy and (2) the measurement of autophagy-dependent restriction of bacterial proliferation by means of colony-forming unit assay.


Assuntos
Autofagossomos/imunologia , Autofagia/imunologia , Bioensaio/métodos , Interações Hospedeiro-Patógeno/imunologia , Autofagossomos/microbiologia , Bioensaio/instrumentação , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Contagem de Colônia Microbiana/instrumentação , Contagem de Colônia Microbiana/métodos , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Salmonella typhimurium/isolamento & purificação , Transformação Bacteriana , Vacúolos/imunologia , Vacúolos/microbiologia
8.
Fish Shellfish Immunol ; 86: 1009-1018, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30586633

RESUMO

Autophagy plays a vital role in innate and adaptive immunity against invading microorganisms, such as virus and bacteria. However, the mechanism underlying autophagy in shrimp is still limited. In our study, we challenged white shrimp L. vannamei with rapamycin to induce autophagy and employed Solexa/Illumina high-throughput RNA-seq method to examine the differences of transcriptome from gills of shrimps treated with or without rapamycin. More than 22.64 Gb raw data were produced, which were assembled into 62, 503 unigenes, with 14,126 unigenes over 1 kb in length. We then performed differential expression analysis and identified a total of 3050 differentially expressed genes (DEGs). Among them, 1456 were upregulated and 1594 were downregulated. We further annotated DEGs by matching against non-redundant protein sequence (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups of proteins (COG), euKaryotic Orthologous Groups (KOG), Gene ontology (GO), and Pfam databases. The assembled and annotated DEGs will facilitate our understanding of the molecular mechanism underlying autophagy and promote the studies on the role of autophagy in innate immunity of L. vannamei and other crustaceans.


Assuntos
Autofagia/imunologia , Penaeidae/genética , Penaeidae/imunologia , Transcriptoma , Animais , Autofagia/efeitos dos fármacos , Perfilação da Expressão Gênica , Brânquias/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Imunidade Inata/genética , Sirolimo/farmacologia
9.
PLoS One ; 13(12): e0209444, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30571757

RESUMO

Lung ischemia and reperfusion injury (LIRI) were mediated by several processes including over-production of reactive oxygen species (ROS) and inflammatory activation. ROS generated by nicotinamide adenine dinucletide phosphate (NADPH) oxidase (Nox) may play a pivotal role in pathophysiological changes in a range of disease. However, it was poorly understood in LIRI. Thus, the purpose of our study was to explore whether GKT137831, as a special dual inhibitor of Nox1 and 4, could alleviate LIRI in mice model and explore the minimal dose. According to the protocol, this study was divided into two parts. The first part was to determine the minimal dose of Nox1/4 inhibitor in attenuating LIRI via histopathology and apoptosis analysis. Eighteen C57BL/6J male wild-type mice were randomly divided in to sham, 2.5Nox+sham, 5.0Nox+sham, IR, 2.5Nox+IR and 5.0Nox+IR groups. According to the different group, mice were pretreated with corresponding dose of Nox1/4 inhibitors or normal saline. After LIRI, the results showed 5.0mg/kg Nox1/4 inhibitor could be considered as the minimal dose to alleviate injury by decreasing of lung injury score and the number of TUNEL-positive cells. The second part was to further verify the benefit of 5.0mg/kg Nox1/4 inhibitor in lung protective effects. Thirty-seven C57BL/6J male wild-type mice were divided in to sham, IR and 5.0Nox+IR groups randomly. The results showed that expressions of inflammatory, autophagy cytokines were markedly elevated and PH value was declined after LIRI. However, 5.0 mg/kg Nox1/4 inhibitor significantly attenuated cytokine production as reflected by immunohistochemistry, western blotting and Q-PCR analysis. In conclusion, our findings suggested that 5.0mg/kg Nox1/4 inhibitor contributed to protect lung tissue damage after LIRI via the suppression of inflammatory and autophagy activation.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Pulmão/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/uso terapêutico , Humanos , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 1/antagonistas & inibidores , NADPH Oxidase 1/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/metabolismo , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia
10.
Int J Mol Sci ; 19(12)2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30544507

RESUMO

Innate immunity is the first line of defense against invading pathogens and plays an essential role in defending the brain against infection, injury, and disease. It is currently well recognized that central nervous system (CNS) infections can result in long-lasting neurological sequelae and that innate immune and inflammatory reactions are highly implicated in the pathogenesis of neurodegeneration. Due to the conservation of the mechanisms that govern neural development and innate immune activation from flies to mammals, the lack of a classical adaptive immune system and the availability of numerous genetic and genomic tools, the fruit fly Drosophila melanogaster presents opportunities to investigate the cellular and molecular mechanisms associated with immune function in brain tissue and how they relate to infection, injury and neurodegenerative diseases. Here, we present an overview of currently identified innate immune mechanisms specific to the adult Drosophila brain.


Assuntos
Encéfalo/imunologia , Imunidade Inata/imunologia , Animais , Autofagia/imunologia , Autofagia/fisiologia , Encéfalo/metabolismo , Drosophila , Imunidade Inata/fisiologia , Modelos Animais , Fagocitose/imunologia , Fagocitose/fisiologia
11.
Immunol Res ; 66(6): 655-662, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30574665

RESUMO

Microparticles (MPs) are small membrane vesicles released by many cell types under physiological and pathological conditions. In the last years, these particles were considered as inert cell debris, but recently many studies have demonstrated they could have a role in intercellular communication. Increased levels of MPs have been reported in various pathological conditions including infections, malignancies, and autoimmune diseases, such as rheumatoid arthritis (RA). RA is an autoimmune systemic inflammatory disease characterized by chronic synovial inflammation, resulting in cartilage and bone damage with accelerated atherosclerosis increasing mortality. According to the literature data, also MPs could have a role in endothelial dysfunction, contributing to atherosclerosis in RA patients. Moreover many researchers have shown that a dysregulated autophagy seems to be involved in endothelial dysfunction. Autophagy is a reparative process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. It has been shown in many works that basal autophagy is essential to proper vascular function. Taking into account these considerations, we hypothesized that in RA patients MPs could contribute to atherosclerosis process by dysregulation of endothelial autophagy process.


Assuntos
Artrite Reumatoide/imunologia , Aterosclerose/imunologia , Autofagia/imunologia , Micropartículas Derivadas de Células/imunologia , Animais , Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Humanos , Inflamação/imunologia
12.
Nihon Saikingaku Zasshi ; 73(3): 193-199, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-30158393

RESUMO

Autophagy acts as an intracellular host defense system against invading pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we revealed Rab GTPase network that regulate autophagosome formation against GAS. A unique set of Rab GTPases coordinates autophagy to enable to form huge autophagosomes surrounding GAS by linking recycling endosomes and trans Golgi-network. We also found that NLRP4, one of intracellular pathogen recognition receptor, directs Rho signaling to facilitate autophagosome formation. In this article, we would like to show our findings on how host autophagy regulators coordinate autophagy during GAS infection.


Assuntos
Autofagia/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes , Autofagossomos/imunologia , Endossomos/imunologia , Humanos , Membranas Intracelulares/imunologia , Membranas Intracelulares/metabolismo , Transporte Proteico , Proteínas Repressoras/imunologia , Transdução de Sinais/imunologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/patogenicidade , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/imunologia , Rede trans-Golgi/imunologia
13.
PLoS One ; 13(5): e0196504, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29718959

RESUMO

The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.


Assuntos
Imunidade Adaptativa/genética , Autofagossomos/imunologia , Autofagia/imunologia , Linfócitos T CD4-Positivos/imunologia , GTP Fosfo-Hidrolases/fisiologia , Animais , Linhagem Celular , GTP Fosfo-Hidrolases/genética , Células HEK293 , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/fisiologia , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Sequestossoma-1/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia
14.
Cell Host Microbe ; 23(5): 644-652.e5, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29746835

RESUMO

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.


Assuntos
Autofagia/efeitos dos fármacos , Autofagia/imunologia , Bactérias/imunologia , Proteínas de Transporte/imunologia , Complemento C3/imunologia , Complemento C3/farmacologia , Interações Hospedeiro-Patógeno/imunologia , Animais , Bactérias/patogenicidade , Proteínas da Membrana Bacteriana Externa/imunologia , Disenteria Bacilar/imunologia , Disenteria Bacilar/microbiologia , Células Epiteliais , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Shigella flexneri/imunologia , Shigella flexneri/patogenicidade , Células THP-1
15.
Biochem Biophys Res Commun ; 496(4): 1148-1154, 2018 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-29402409

RESUMO

Age-associated dysfunction of retinal pigment epithelial cells (RPEs) is considered to be the initial trigger of retinal diseases such as age-related macular degeneration. Although autophagy is upregulated in RPEs during the course of aging, little is known about how autophagy is regulated and its functional role in RPEs. In this study, we found that expression of Sirtuin 6 (SIRT6) and autophagic markers are upregulated in RPEs of aged mice where subretinal deposition of amyloid-ß is accumulated and in amyloid-ß stimulated RPEs. In addition, gain and loss-of-function studies confirmed the positive role of SIRT6 in regulating autophagy. Interesting, inhibition of autophagy attenuates amyloid-ß stimulated inflammatory response in RPEs. Collectively, our findings uncover the autophagy modulated by SIRT6 may be a proinflammatory mechanism for amyloid-ß induced RPE dysfunction.


Assuntos
Peptídeos beta-Amiloides/imunologia , Autofagia/imunologia , Células Epiteliais/imunologia , Mediadores da Inflamação/imunologia , Epitélio Pigmentado da Retina/imunologia , Retinite/imunologia , Sirtuínas/imunologia , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Retinite/induzido quimicamente , Retinite/patologia
16.
PLoS Pathog ; 14(1): e1006877, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29381763

RESUMO

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-ß and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN's protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7's antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.


Assuntos
Antivirais/farmacologia , Autofagia , Interferons/farmacologia , Paramyxovirinae/fisiologia , Ribonucleoproteínas/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Autofagia/genética , Autofagia/imunologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , Ribonucleoproteínas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Replicação Viral/genética
17.
Cell Host Microbe ; 23(2): 191-202.e4, 2018 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-29358084

RESUMO

Genome-wide association studies have linked polymorphisms in the autophagy gene ATG16L1 with susceptibility to inflammatory bowel disease (IBD). However, the cell-type-specific effects of autophagy on the regulation of chronic intestinal inflammation have not been investigated. Here, we assessed the effect of myeloid-specific or intestinal epithelial cell (IEC)-specific deletion of Atg16l1 on chronic colitis triggered by the intestinal opportunistic pathogen Helicobacter hepaticus in mice. Although Atg16l1 deficiency in myeloid cells had little effect on disease, mice selectively lacking Atg16l1 in IECs (Atg16l1VC) developed severely exacerbated pathology, accompanied by elevated pro-inflammatory cytokine secretion and increased IEC apoptosis. Using ex vivo IEC organoids, we demonstrate that autophagy intrinsically controls TNF-induced apoptosis and in vivo blockade of TNF attenuated the exacerbated pathology in Atg16l1VC mice. These findings suggest that the IBD susceptibility gene ATG16L1 and the process of autophagy within the epithelium control inflammation-induced apoptosis and barrier integrity to limit chronic intestinal inflammation.


Assuntos
Apoptose/imunologia , Autofagia/imunologia , Proteínas de Transporte/genética , Colite/imunologia , Células Caliciformes/imunologia , Celulas de Paneth/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/genética , Autofagia/genética , Linhagem Celular , Citrobacter rodentium/patogenicidade , Células HEK293 , Helicobacter hepaticus/patogenicidade , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia
18.
Cell Host Microbe ; 23(2): 177-190.e4, 2018 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-29358083

RESUMO

The protozoan parasite Toxoplasma gondii triggers severe small intestinal immunopathology characterized by IFN-γ- and intestinal microbiota-mediated inflammation, Paneth cell loss, and bacterial dysbiosis. Paneth cells are a prominent secretory epithelial cell type that resides at the base of intestinal crypts and releases antimicrobial peptides. We demonstrate that the microbiota triggers basal Paneth cell-specific autophagy via induction of IFN-γ, a known trigger of autophagy, to maintain intestinal homeostasis. Deletion of the autophagy protein Atg5 specifically in Paneth cells results in exaggerated intestinal inflammation characterized by complete destruction of the intestinal crypts resembling that seen in pan-epithelial Atg5-deficient mice. Additionally, lack of functional autophagy in Paneth cells within intestinal organoids and T. gondii-infected mice causes increased sensitivity to the proinflammatory cytokine TNF along with increased intestinal permeability, leading to exaggerated microbiota- and IFN-γ-dependent intestinal immunopathology. Thus, Atg5 expression in Paneth cells is essential for tissue protection against cytokine-mediated immunopathology during acute gastrointestinal infection.


Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia/imunologia , Interferon gama/imunologia , Celulas de Paneth/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/patologia , Animais , Proteína 5 Relacionada à Autofagia/genética , Linfócitos T CD4-Positivos/imunologia , Disbiose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/parasitologia , Fator de Necrose Tumoral alfa/imunologia
19.
DNA Cell Biol ; 37(4): 287-290, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29350547

RESUMO

Macroautophagy, hereafter autophagy, is a catabolic process that is important for maintaining cellular homeostasis. It can also be used by cells to remove intracellular microbial pathogens. However, the studies on hepatitis C virus (HCV) in recent years indicated that this virus could regulate this cellular pathway and use it to enhance its replication. HCV could temporally control the autophagic flux and use the autophagic membranes for the assembly of its RNA replication complex. In this report, we will discuss the biogenesis of autophagosomes induced by HCV and how HCV uses this autophagic pathway for its RNA replication.


Assuntos
Autofagia/fisiologia , Hepacivirus/metabolismo , RNA Viral/fisiologia , Autofagossomos , Autofagia/imunologia , Replicação do DNA , Hepacivirus/patogenicidade , Humanos , Fagossomos/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral/genética , Replicação Viral/fisiologia
20.
Biomed Pharmacother ; 97: 1053-1060, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29136784

RESUMO

Naturally regulatory T cells (Tregs) play a critical role in the regulation of T cell-mediated immune responses in atherosclerosis. However, the regulatory mechanism underlying Tregs upon long-term development of atherosclerosis remains unknown. Therefore, in this study, atherosclerotic model was induced in ApoE-/- mice by feeding fat-diet for 10 weeks. Quantification of atherosclerotic lesions was done by calculating the lesion size in the aortic sinus every 2 weeks. The lipid levels and inflammatory mediators were detected in serum sample. The populations of CD4+CD25+Foxp3+ Tregs were compared between ApoE-/- mice (ApoE-/-) and wild type C57BL/6 littermates (WT). The expression levels of autophagy and apoptosis signaling related regulators were determined by flow cytomery, RT-qPCR, and western blot assays in the CD4+CD25+Foxp3+ Tregs isolated from ApoE-/- and WT. We found that the sizes of plaque lesions in atherosclerotic ApoE-/- mice were larger than those in WT group during 10 weeks' detection (all P<0.05); Whereas, flow cytometry assay showed that the populations of CD4+CD25+Foxp3+ Tregs were significantly reduced in atherosclerotic ApoE-/- mice compared with those in corresponding WT group from the 4th weeks' detection (all P<0.05). The lipid accumulation and increased pro-inflammatory mediators were correlated with the developmental progression of atherosclerosis. Furthermore, compared to WT group, the functional properties of CD4+CD25+Foxp3+ Tregs from ApoE-/- mice showed a gradually decreased autophagic activity with aberrant expressions of LC3, Beclin1, ATG5, ATG7, p62 (all P<0.05), and a gradually increased apoptotic activity with abnormal expressions of cleaved caspase 3, Bim, Bcl-2 (all P<0.05) during the 10 weeks' detection period. Taken together, our data demonstrated that the population of CD4+CD25+Foxp3+ Tregs was reversely correlated with plaque forming in atherosclerotic ApoE-/- mice during atherosclerosis development. And the autophagy/apoptosis-dependent Tregs might play a crucial role for the maintenance of CD4 9+CD25+Foxp3+ Tregs survival during atherosclerosis progression.


Assuntos
Apoptose/imunologia , Aterosclerose/imunologia , Autofagia/imunologia , Linfócitos T Reguladores/imunologia , Animais , Apolipoproteínas E/genética , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Progressão da Doença , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Mediadores da Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lipídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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