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1.
Fish Shellfish Immunol ; 116: 30-41, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34147615

RESUMO

Beclin-1, the ortholog of yeast autophagy-related gene 6 (Atg6), has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, and lifespan extension. However, understanding of function of fish Beclin-1 is limited now. In this study, the complete Beclin-1 cDNA of large yellow croaker Larimichthys crocea (LcBeclin-1) was cloned, whose open reading frame (ORF) is 1344 bp long and encodes a protein of 447 amino acids (aa). The deduced LcBeclin-1 possesses a typical Bcl-2 homology domain 3(BH3) and an APG6 domain that contains a central coiled-coil domain (CCD, residues 174 to 231) and a C-terminal evolutionarily conserved domain (ECD, residues 241 to 334). LcBeclin-1 shared a high amino acid identity of 81.66-98.66% with reported Beclin-1 molecules from other vertebrate species. LcBeclin-1 gene was constitutively expressed in all tissues tested, with the highest levels in heart. LcBeclin-1 transcripts were also detected in primary head kidney granulocytes (PKGs), primary head kidney macrophages (PKMs), primary head kidney leukocytes (PKLs), and large yellow croaker head kidney cell line (LYCK), and were significantly upregulated by poly (I:C) in PKMs and LYCK cells. Subcellular localization showed that LcBeclin-1 was evenly distributed in the cytoplasm and nucleus of LYCK cells. Overexpression of LcBeclin-1 significantly increased the replication of SVCV, as evidenced by increased severity of the cytopathic effects, enhanced viral titre, and upregulated transcriptional levels of viral genes. Further studies showed that LcBeclin-1 induced the occurrence of autophagy in LYCK cells. Additionally, LcBeclin-1 also decreased the expression levels of large yellow croaker interferons (IFNs; IFNc, IFNd, and IFNh), interferon regulatory factor 3 (IRF3) and IRF7, IFN-stimulated genes (ISGs; Mx, PKR, and Viperin) in LYCK cells. All these data suggest that LcBeclin-1 promoted the viral replication possibly by inducing autophagy or negatively modulating IFN response, which will help us to further understand the function of fish Beclin-1.


Assuntos
Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perciformes/genética , Perciformes/imunologia , Viroses/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Rim Cefálico/citologia , Rim Cefálico/imunologia , Leucócitos/imunologia , Macrófagos/imunologia
2.
FEBS J ; 288(10): 3164-3185, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33830641

RESUMO

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.


Assuntos
Apresentação de Antígeno/genética , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H1N1/genética , Macroautofagia/genética , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Brefeldina A/farmacologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Feminino , Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade Classe II/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Macroautofagia/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Transfecção
3.
Int Immunopharmacol ; 94: 107460, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33621850

RESUMO

Allergic asthma is a common chronic inflammatory disease characterized by airway inflammation, mucus hypersecretion and airway remodeling. Autophagy is a highly conserved intracellular degradation pathway in eukaryotic cells. There is growing evidence suggesting that dysregulation of autophagy is involved in the pathological process of asthma. Luteolin is a typical flavonoid compound with anti-inflammatory, anti-allergic and immune-enhancing functions. Previous studies have shown that luteolin can attenuate airway inflammation and hypersensitivity in asthma. However, whether luteolin can play a role in treating asthma by regulating autophagy remains unclear. The aim of the present study was to evaluate the therapeutic effect of luteolin on ovalbumin (OVA)-induced asthmatic mice, observe its effect on the level of autophagy in lung tissues, and further elucidate its underlying mechanism. The results showed that OVA-induced mice developed airway hyperresponsiveness, mucus over-production and collagen deposition. The number of inflammatory cells, levels of interleukin (IL)-4, IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF) and OVA-specific IgE in serum were significantly increased. Furthermore, the infiltration of inflammatory cells was observed along with the activation of autophagy in lung tissues. Luteolin treatment significantly inhibited the OVA-induced inflammatory responses and the level of autophagy in lung tissues as well. Moreover, luteolin activated the PI3K/Akt/mTOR pathway and inhibited the Beclin-1-PI3KC3 protein complex in lung tissues of asthmatic mice. In conclusion, this study explored the regulatory mechanism of luteolin on autophagy in allergic asthma, providing biologic evidence for its clinical application.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Autofagia/efeitos dos fármacos , Luteolina/uso terapêutico , Alérgenos , Animais , Antiasmáticos/farmacologia , Asma/imunologia , Proteína Beclina-1/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Citocinas/imunologia , Feminino , Luteolina/farmacologia , Camundongos Endogâmicos BALB C , Ovalbumina , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/imunologia
4.
Oncol Rep ; 45(3): 987-996, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33469679

RESUMO

Non­Hodgkin lymphoma (NHL) is a form of lymphoid malignancy, with diffuse large B cell lymphoma (DLBCL) being the most common NHL isoform. Approximately half of patients with DLBCL are successfully cured via first­line Rituximab, Cyclophosphamide, Epirubicin, Vindesine, Prednisolone (R­CHOP) treatment. However, 30­40% of patients with DLBCL ultimately suffer from treatment­refractory or relapsed disease. These patients often suffer from high mortality rates owing to a lack of suitable therapeutic options, and all patients are at a high risk of serious treatment­associated dose­dependent toxicity. As such, it is essential to develop novel treatments for NHL that are less toxic and more efficacious. Oncolytic Vaccinia virus (OVV) has shown promise as a means of treating numerous types of cancer. Gene therapy strategies further enhance OVV­based therapy by improving tumor cell recognition and immune evasion. Beclin1 is an autophagy­associated gene that, when upregulated, induces excess autophagy and cell death. The present study aimed to develop an OVV­Beclin1 therapy capable of inducing autophagic tumor cell death. OVV­Beclin1 was able to efficiently kill NHL cells and to increase the sensitivity of these cells to R­CHOP, thereby decreasing the dose­dependent toxic side effects associated with this chemotherapeutic regimen. The combination of OVV­Beclin1 and R­CHOP also significantly improved tumor growth inhibition and survival in a BALB/c murine model system owing to the synergistic induction of autophagic cell death. Together, these findings suggest that OVV­Beclin1 infection can induce significant autophagic cell death in NHL, highlighting this as a novel means of inducing tumor cell death via a mechanism that is distinct from apoptosis and necrosis.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Proteína Beclina-1/imunologia , Linfoma Difuso de Grandes Células B/terapia , Terapia Viral Oncolítica/métodos , Vaccinia virus/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Morte Celular Autofágica/efeitos dos fármacos , Morte Celular Autofágica/imunologia , Proteína Beclina-1/genética , Biópsia , Linhagem Celular Tumoral , Terapia Combinada/métodos , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Engenharia Genética , Humanos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Vírus Oncolíticos/imunologia , Prednisona/administração & dosagem , Rituximab/administração & dosagem , Evasão Tumoral/efeitos dos fármacos , Vaccinia virus/imunologia , Vincristina/administração & dosagem , Vindesina/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Autophagy ; 17(11): 3408-3423, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33459125

RESUMO

Although macroautophagy/autophagy has been proposed as a critical defense mechanism against HIV-1 by targeting viral components for degradation, its contribution as a catabolic process in providing optimal anti-HIV-1 immunity has never been addressed. The failure to restore proper antiviral CD8A/CD8 T-cell immunity, especially against HIV-1, is still the major limitation of current antiretroviral therapies. Consequently, it is of clinical imperative to provide new strategies to enhance the function of HIV-1-specific CD8A T-cells in patients under antiretroviral treatments (ART). Here, we investigated whether targeting autophagy activity could be an optional solution to make this possible. Our data show that, after both polyclonal and HIV-1-specific activation, CD8A T-cells from ART displayed reduced autophagy-dependent degradation of lysosomal contents when compared to naturally HIV-1 protected elite controllers (EC). We further confirmed in EC, by using specific BECN1 gene silencing and lysosomal inhibitors, the critical role of active autophagy in superior CD8A T-cell protection against HIV-1. More importantly, we found that an IL21 treatment was effective in rescuing the antiviral CD8A T-cell immunity from ART in an autophagy-dependent manner. Finally, we established that IL21-dependent rescue occurred due to the enhanced degradation of endogenous lipids via autophagy, referred to as lipophagy, which fueled the cellular rates of mitochondrial beta-oxidation. In summary, our data show that autophagy/lipophagy can be considered as a therapeutic tool to elicit functional antiviral CD8 T-cell responses. Our results also provide additional insights toward the development of improved T-cell-based prevention and cure strategies against HIV-1.Abbreviations: ART: patients under antiretroviral therapy; BaF: bafilomycin A1; BECN1: beclin 1; CEF: cytomegalo-, Epstein-Barr- and flu-virus peptide pool; Chloro.: chloroquine; EC: elite controllers; FAO: fatty acid beta-oxidation; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBMC: peripheral blood mononuclear cells; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.


Assuntos
Autofagia/imunologia , Autofagia/fisiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , HIV-1/imunologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Autofagia/efeitos dos fármacos , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Estudos de Casos e Controles , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Paciente HIV Positivo não Progressor , Humanos , Técnicas In Vitro , Interleucinas/imunologia , Metabolismo dos Lipídeos/imunologia , Ativação Linfocitária , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Oxirredução
6.
J Exp Med ; 218(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33355624

RESUMO

Autophagy programs the metabolic and functional fitness of regulatory T (T reg) cells to establish immune tolerance, yet the mechanisms governing autophagy initiation in T reg cells remain unclear. Here, we show that the E3 ubiquitin ligase ZFP91 facilitates autophagy activation to sustain T reg cell metabolic programming and functional integrity. T reg cell-specific deletion of Zfp91 caused T reg cell dysfunction and exacerbated colonic inflammation and inflammation-driven colon carcinogenesis. TCR-triggered autophagy induction largely relied on T reg cell-derived ZFP91 to restrict hyperglycolysis, which is required for the maintenance of T reg cell homeostasis. Mechanistically, ZFP91 rapidly translocated from the nucleus to the cytoplasm in response to TCR stimulation and then mediated BECN1 ubiquitination to promote BECN1-PIK3C3 complex formation. Therefore, our results highlight a ZFP91-dependent mechanism promoting TCR-initiated autophagosome maturation to maintain T reg cell homeostasis and function.


Assuntos
Homeostase/imunologia , Linfócitos T Reguladores/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Autofagia/imunologia , Proteína Beclina-1/imunologia , Carcinogênese/imunologia , Colo/imunologia , Modelos Animais de Doenças , Feminino , Tolerância Imunológica/imunologia , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/imunologia , Ubiquitinação/imunologia
7.
Int J Mol Sci ; 21(4)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32098061

RESUMO

The screening of biologically active chemical compound libraries can be an efficient way to reposition Food and Drug Adminstration (FDA)-approved drugs or to discover new therapies for human diseases. Particulate matter with an aerodynamic diameter equal to or less than 2.5 µm (PM2.5) is a form of air pollutant that causes significant lung damage when inhaled. This study illustrates drug repositioning with biapenem (BIPM) for the modulation of PM-induced lung injury. Biapenem was used for the treatment of severe infections. Mice were treated with BIPM via tail-vein injection after the intratracheal instillation of PM2.5. Alterations in the lung wet/dry weight, total protein/total cell count and lymphocyte count, inflammatory cytokines in the bronchoalveolar lavage fluid (BALF), vascular permeability, and histology were monitored in the PM2.5-treated mice. BIPM effectively reduced the pathological lung injury, lung wet/dry weight ratio, and hyperpermeability caused by PM2.5. Enhanced myeloperoxidase (MPO) activity by PM2.5 in the pulmonary tissue was inhibited by BIPM. Moreover, increased levels of inflammatory cytokines and total protein by PM2.5 in the BALF were also decreased by BIPM treatment. In addition, BIPM markedly suppressed PM2.5-induced increases in the number of lymphocytes in the BALF. Additionally, the activity of mammalian target of rapamycin (mTOR) was increased by BIPM. Administration of PM2.5 increased the expression levels of toll-like receptor 4 (TLR4), MyD88, and the autophagy-related proteins LC3 II and Beclin 1, which were suppressed by BIPM. In conclusion, these findings indicate that BIPM has a critical anti-inflammatory effect due to its ability to regulate both the TLR4-MyD88 and mTOR-autophagy pathways, and may thus be a potential therapeutic agent against diesel PM2.5-induced pulmonary injury.


Assuntos
Reposicionamento de Medicamentos , Lesão Pulmonar , Pulmão , Material Particulado/toxicidade , Tienamicinas/farmacologia , Animais , Proteína Beclina-1/imunologia , Lavagem Broncoalveolar , Citocinas/imunologia , Pulmão/imunologia , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/imunologia , Lesão Pulmonar/patologia , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Serina-Treonina Quinases TOR/imunologia , Receptor 4 Toll-Like/imunologia
8.
Nat Microbiol ; 5(2): 272-281, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959973

RESUMO

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens1,2. However, if left uncontrolled, these responses may lead to detrimental inflammation3. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism4. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation5-12. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200-but not of other essential autophagy components ATG5, ATG16L1 or ATG7-in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection.


Assuntos
Autofagia/genética , Listeria monocytogenes/patogenicidade , Macrófagos Peritoneais/imunologia , Animais , Autofagia/imunologia , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proliferação de Células , Suscetibilidade a Doenças/imunologia , Feminino , Predisposição Genética para Doença , Interferon gama/imunologia , Listeria monocytogenes/imunologia , Listeriose/etiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Front Immunol ; 10: 2203, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31620128

RESUMO

Cereblon (CRBN) as a multifunctional protein has been extensively studied. Here, we show that CRBN is a negative regulator of bactericidal activity and autophagy activation. Mitochondrial localization of CRBN was significantly increased in response to Toll-like receptor 4 (TLR4) stimulation. CRBN interrupted the association of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT)-TNF-receptor associated factor 6 (TRAF6) complex, thereby inhibiting the ubiquitination of ECSIT, which plays a pivotal role for the production of mitochondrial reactive oxygen species (mROS). Subsequently, mROS levels were markedly elevated in CRBN-knockdown (CRBNKD) THP-1 cells, and that led to resistance against S. typhimurium infection, indicating CRBN is a negative regulator of bactericidal activity through the regulation of mROS. Additionally, CRBN inhibited TRAF6-induced ubiquitination of BECN1 (Beclin 1), and that induced autophagy activation in CRBNKD THP-1, CRBN-knockout (CRBNKO) H1299, and CRBNKO MCF7 cancer cells in response to TLR4 stimulation. Notably, we found that the ability of cancer migration and invasion was significantly enhanced in CRBNKO H1299 and CRBNKO MCF7 cancer cells, as compared with those of control cancer cells. Collectively, these results suggest that CRBN is a negative regulator of bactericidal activity and autophagy activation through inhibiting the TRAF6-induced ubiquitination of ECSIT and BECN1, respectively.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Estresse Oxidativo/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Proteína Beclina-1/imunologia , Humanos , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/fisiologia
10.
Biol Cell ; 111(12): 308-318, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31628772

RESUMO

BACKGROUND INFORMATION: Autophagy is induced during HIV-1 entry into CD4 T cells by the fusion of the membranes triggered by the gp41 envelope glycoprotein. This anti-HIV-1 mechanism is inhibited by the viral infectivity factor (Vif) neosynthesized after HIV-1 integration to allow viral replication. However, autophagy is very rapidly controlled after HIV-1 entry by a still unknown mechanism. As HIV-1 viral protein R (Vpr) is the only auxiliary protein found within the virion in substantial amount, we studied its capability to control the early steps of HIV-1 envelope-mediated autophagy. RESULTS: We demonstrated that ectopic Vpr inhibits autophagy in both the Jurkat CD4 T cell line and HEK.293T cells. Interestingly, Vpr coming from the virus also blocks autophagy in CD4 T cells, the main cell target of HIV-1. Furthermore, Vpr decreases the expression level of two essential autophagy proteins (ATG), LC3B and Beclin-1, and an important autophagy-related protein, BNIP3 as well as the level of their mRNA. We also demonstrated in HEK.293T cells that Vpr degrades the FOXO3a transcription factor through the ubiquitin proteasome system. CONCLUSION: Vpr, the only well-expressed HIV-1 auxiliary protein incorporated into viruses, is able to negatively control autophagy induced during HIV-1 entry into CD4 T cells. SIGNIFICANCE: We provide insights of how HIV-1 controls autophagy very early after its entry into CD4 T cells and discovered a new function of Vpr. These results open the route to a better understanding of the roles of Vpr during HIV-1 infection through FOXO3a degradation and could be important to consider additional therapies that counteract the role of Vpr on autophagy.


Assuntos
Autofagia/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1 , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/imunologia , Proteína Beclina-1/imunologia , Linfócitos T CD4-Positivos/citologia , Células HEK293 , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Células Jurkat , Proteínas de Membrana/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Proteínas Supressoras de Tumor/imunologia , Replicação Viral
11.
Fish Shellfish Immunol ; 94: 336-345, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31521781

RESUMO

Beclin-1 is an essential autophagic regulator that plays diverse roles in physiology and disease. However, reports about the function of fish Beclin-1 during pathogen infection are still very limited. In this study, a Beclin-1 homolog (EcBeclin-1) from orange-spotted grouper (Epinephelus coioides) was identified and its roles in viral infection were investigated. EcBeclin-1 encoded 447amino acids protein with a BH3 domain, a CCD domain and an ECD domain, which shared high identities (97%-82%) with reported Beclin-1 proteins from mammal to fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBeclin-1 was predominantly expressed in brain and muscle of healthy grouper. Using fluorescence microscopy, we found that EcBeclin-1 was co-localized with endoplasmic reticulum (ER) in grouper spleen cells (EAGS). After red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro, EcBeclin-1 transcript was significantly up-regulated, implying that EcBeclin-1 might be involved in viral infection. Furthermore, the in vitro studies of EcBeclin-1 overexpression promoted RGNNV induced autophagy, as well as the expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcBeclin-1 suppressed the expressions of interferon pathway-related factors, inflammatory-related factors and activities of NF-κB and ISRE. Additionally, EcBeclin-1 could interact with EcBcl-xL in vitro. These data suggest that EcBeclin-1 affect viral replication through modulating IFN and inflammatory responses, as well as virus-induced cell death, which will help us to further explore the immune response of fish during viral infection.


Assuntos
Imunidade Adaptativa/genética , Bass/genética , Bass/imunologia , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Beclina-1/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/imunologia , Filogenia , Alinhamento de Sequência/veterinária
12.
Eur J Histochem ; 63(2)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31189296

RESUMO

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Cóclea/embriologia , Cóclea/metabolismo , Animais , Anticorpos/imunologia , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proteína Beclina-1/metabolismo , Caspase 3/genética , Caspase 3/imunologia , Caspase 3/metabolismo , Cóclea/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/imunologia , Proteína Sequestossoma-1/metabolismo , Fatores de Tempo
13.
Fish Shellfish Immunol ; 89: 207-216, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30936045

RESUMO

Beclin-1, the mammalian ortholog of yeast Atg6, plays essential roles in the regulation of various processes, including autophagy, apoptosis, embryonic development and immune responses in vertebrates. However, the information about Beclin-1 in invertebrates especially in crustaceans is still very limited. In the present study, a novel Beclin-1 (designated as EsBeclin-1) was identified from Chinese mitten crab Eriocheir sinensis. The open reading frame of EsBeclin-1 cDNA was of 1,275 bp, encoding a typical APG6 domain. The deduced amino acid sequence of EsBeclin-1 shared high similarity ranging from 42.9% to 63.6% with the previously identified Beclins. In the phylogenetic tree, EsBeclin-1 was firstly clustered with Drosophila melanogaster Atg6 and then assigned into the branch of invertebrate Beclin-1. The mRNA transcripts of EsBeclin-1 were highly expressed in hepatopancreas, hemocytes and gill. After lipopolysaccharide (LPS) and Aeromonas hydrophila stimulations, the relative mRNA expression of EsBeclin-1 in hemocytes was significantly increased from 3 to 24 h with the peak level of 4.70-fold (p < 0.01) and 2.91-fold (p < 0.01) at 6 h, respectively. EsBeclin-1 protein was diffusely distributed in the cytoplasm of crab hemocytes under normal conditions, whereas it displayed predominantly punctuate distribution after LPS stimulation. After EsBeclin-1 was interfered with specific EsBeclin-1-dsRNA, the mRNA transcripts of some antimicrobial peptides, including EsALF2, EsLYZ, EsCrus and EsCrus2 in crab hemocytes were significantly decreased at 6 h post LPS stimulation. These results implicated that EsBeclin-1 played a role in regulating the antimicrobial peptides expressions in the immune responses of E. sinensis.


Assuntos
Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Proteína Beclina-1/química , Perfilação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Filogenia , Alinhamento de Sequência
14.
Virulence ; 10(1): 151-165, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30829115

RESUMO

Autophagy is a catabolic pathway required for cellular and organism homeostasis. Autophagy participates in the innate and adaptive immune responses at different levels. Xenophagy is a class of selective autophagy that involves the elimination of intracellular pathogens. Trypanosoma cruzi is the causative agent of Chagas, a disease that affects 8 million individuals worldwide. Previously, our group has demonstrated that autophagy participates in the invasion of T. cruzi in non-phagocytic cells. In this work we have studied the involvement of autophagy in the development of T. cruzi infection in mice. Beclin-1 is a protein essential for autophagy, required for autophagosome biogenesis and maturation. We have performed an acute model of infection on the autophagic deficient Beclin-1 heterozygous knock-out mice (Bcln±) and compared to control Bcln+/+ animals. In addition, we have analyzed the infection process in both peritoneal cells and RAW macrophages. Our results have shown that the infection was more aggressive in the autophagy-deficient mice, which displayed higher numbers of parasitemia, heart´s parasitic nests and mortality rates. We have also found that peritoneal cells derived from Bcln± animals and RAW macrophages treated with autophagy inhibitors displayed higher levels of infection compared to controls. Interestingly, free cytosolic parasites recruited LC3 protein and other markers of xenophagy in control compared to autophagy-deficient cells. Taken together, these data suggest that autophagy plays a protective role against T. cruzi infection in mice, xenophagy being one of the processes activated as part of the repertoire of immune responses generated by the host.


Assuntos
Autofagia , Doença de Chagas/imunologia , Interações Hospedeiro-Patógeno , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Doença de Chagas/mortalidade , Modelos Animais de Doenças , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , Trypanosoma cruzi
15.
Biochem Biophys Res Commun ; 509(1): 194-200, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30579601

RESUMO

Hepatocellular carcinoma (HCC) is associated with poor prognosis due to many unknowns about its inflammatory microenvironment. As a pivotal proinflammatory cytokine, IL-17A exerts a protective effect on the survival and function of HCC cells. It is widely accepted that IL-17A plays an important role in regulating autophagy. Bcl2, a key molecule promoting the survival of HCC cells, also plays an indispensable role as an autophagy regulator. The aim of this study was to investigate the role of Bcl2 in IL-17A-regulated autophagy of HCC cells. The results showed that IL-17A not only inhibited autophagic activity, but also increased Bcl2 levels in HCC cells under starvation. Besides, IL-17A could prevent the dissociation of autophagy protein Beclin1 from Bcl2-Beclin1 complex upon starvation. Overexpression of Beclin1 rescued the autophagy deficiency of HCC cells in presence of IL-17A. Moreover, RNAi-induced Bcl2 silencing impaired the function of IL-17A in inhibiting the activation of autophagy, subsequently reducing the viability and migration of HCC cells, while the inhibition of Beclin1 by spautin-1 could reduce autophagic activity to a certain degree, thus restoring the viability and migration of HCC cells. In summary, it was suggested that the inhibition of Bcl2 degradation may be an important mechanism by which IL-17A inhibits autophagy response, subsequently maintaining the survival in HCC cells.


Assuntos
Autofagia , Carcinoma Hepatocelular/imunologia , Interleucina-17/imunologia , Neoplasias Hepáticas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteína Beclina-1/análise , Proteína Beclina-1/imunologia , Carcinoma Hepatocelular/patologia , Sobrevivência Celular , Células Hep G2 , Humanos , Interleucina-17/análise , Neoplasias Hepáticas/patologia , Lisossomos/imunologia , Lisossomos/patologia , Proteólise , Proteínas Proto-Oncogênicas c-bcl-2/análise
16.
Front Immunol ; 9: 2096, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30258449

RESUMO

Autophagy has been implicated in innate immune responses against various intracellular pathogens. Recent studies have reported that autophagy can be triggered by pathogen recognizing sensors, including Toll-like receptors and cyclic guanosine monophosphate-adenosine monophosphate synthase, to participate in innate immunity. In the present study, we examined whether the RIG-I signaling pathway, which detects viral infections by recognizing viral RNA, triggers the autophagic process. The introduction of polyI:C into the cytoplasm, or Sendai virus infection, significantly induced autophagy in normal cells but not in RIG-I-deficient cells. PolyI:C transfection or Sendai virus infection induced autophagy in the cells lacking type-I interferon signaling. This demonstrated that the effect was not due to interferon signaling. RIG-I-mediated autophagy diminished by the deficiency of mitochondrial antiviral signaling protein (MAVS) or tumor necrosis factor receptor-associated factor (TRAF)6, showing that the RIG-I-MAVS-TRAF6 signaling axis was critical for RIG-I-mediated autophagy. We also found that Beclin-1 was translocated to the mitochondria, and it interacted with TRAF6 upon RIG-I activation. Furthermore, Beclin-1 underwent K63-polyubiquitination upon RIG-I activation, and the ubiquitination decreased in TRAF6-deficient cells. This suggests that the RIG-I-MAVS-TRAF6 axis induced K63-linked polyubiquitination of Beclin-1, which has been implicated in triggering autophagy. As deficient autophagy increases the type-I interferon response, the induction of autophagy by the RIG-I pathway might also contribute to preventing an excessive interferon response as a negative-feedback mechanism.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Autofagia/imunologia , Proteína Beclina-1/imunologia , Proteína DEAD-box 58/imunologia , Transdução de Sinais/imunologia , Fator 6 Associado a Receptor de TNF/imunologia , Viroses/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Receptores Imunológicos
17.
Fish Shellfish Immunol ; 77: 8-12, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29551665

RESUMO

In mammals, beclin-1 is a key player that regulates autophagic activity. In fish, the immune function of beclin-1 is essentially unknown. In this study, we analyzed the involvement of tongue sole (Cynoglossus semilaevis) beclin-1 (named CsBECN1) in antibacterial immunity. CsBECN1 is composed of 451 amino acid residues and shares 84.5-95.1% overall sequence identities with other teleost beclin-1. CsBECN1 possesses a typical Bcl-2 homology domain 3 and an Atg6 domain. Expression of CsBECN1 occurred in multiple tissues and was upregulated during bacterial infection. Knockdown of CsBECN1 significantly enhanced bacterial dissemination in the tissues of tongue sole, whereas overexpression of CsBECN1 significantly reduced bacterial dissemination. Taken together, these results indicate that CsBECN1 is required for the antibacterial immunity of tongue sole.


Assuntos
Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Doenças dos Peixes/imunologia , Linguados , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Animais , Proteína Beclina-1/química , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Distribuição Aleatória , Análise de Sequência de DNA , Vibrio/fisiologia , Vibrioses
18.
IUBMB Life ; 70(3): 207-214, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29369472

RESUMO

Hepatitis E virus (HEV) infection remains a serious threat to life and productivity in developing world. Vaccine seems to be an effective, safe, and affordable approach to address HEV disease burden. The HEV genome consists of three open reading frames (ORFs). Of these, ORF2 encodes a single structural protein (pORF2) for the HEV capsid which has been studied extensively as vaccine candidates. Recently, it has been recognized that autophagy plays an important role in innate and adaptive immunity defense against intracellular pathogens. This mechanism could therefore promote a protective immune response by inducing CD4+ and CD8+ T cells. In this study, HEV 239 and Beclin1 proteins were expressed in prokaryotic host cell [Escherichia coli (BL21)]. HEV 239 protein with different formulations (+Alum, +Beclin1, and +Alum-Beclin1) were used as candidate vaccines and administrated subcutaneously in BALB/c mice on 0, 14, and 28 days. Finally, elicited cellular and humoral immunity were evaluated. Taken together, although our results indicated that mice immunized with HEV 239 protein formulated with Alum, Beclin1, and Alum + Beclin1 displayed humoral and cellular response that was not significant in comparison with each other (P > 0.05); whereas they were significant while compared with control groups (P < 0.05). A comprehensive understanding of the intricate interplay between autophagy and immune response remains to be unraveled. Further study will clear the detailed impact of autophagy manipulation to enhance vaccine efficacy and boost the immune responses against the disease. © 2018 IUBMB Life, 70(3):207-214, 2018.


Assuntos
Vírus da Hepatite E/imunologia , Hepatite E/imunologia , Imunidade Humoral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas Virais/administração & dosagem , Imunidade Adaptativa/imunologia , Animais , Autofagia/imunologia , Proteína Beclina-1/administração & dosagem , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Modelos Animais de Doenças , Escherichia coli/genética , Genoma Viral/imunologia , Hepatite E/prevenção & controle , Hepatite E/virologia , Vírus da Hepatite E/patogenicidade , Humanos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia
19.
Nat Commun ; 8: 15676, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28607490

RESUMO

In response to virus infection, RIG-I-like receptors (RLRs) sense virus RNA and induce MAVS to form prion-like aggregates to further propagate antiviral signalling. Although monomeric MAVS recombinant protein can assemble into prion-like filaments spontaneously in vitro, endogenous MAVS in cells is prevented from aggregation until viral infection. The mechanism preventing cellular MAVS from spontaneous aggregation is unclear. Here we show that multiple N-terminal truncated isoforms of MAVS are essential in preventing full-length MAVS from spontaneous aggregation through transmembrane domain-mediated homotypic interaction. Without these shorter isoforms, full-length MAVS is prone to spontaneous aggregation and Nix-mediated mitophagic degradation. In the absence of N-terminally truncated forms, blocking Nix-mediated mitophagy stabilizes full-length MAVS, which aggregates spontaneously and induces the subsequent expression of type I interferon and other proinflammatory cytokines. Our data thus uncover an important mechanism preventing spontaneous aggregation of endogenous MAVS to avoid accidental activation of antiviral innate immune signalling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Imunidade Inata , Infecções por Vírus de RNA/imunologia , Autofagia , Proteína 5 Relacionada à Autofagia/imunologia , Proteína Beclina-1/imunologia , Citometria de Fluxo , Deleção de Genes , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Inflamação , Mitofagia , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
20.
Sci Rep ; 6: 34440, 2016 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-27694929

RESUMO

The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C- macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.


Assuntos
Autofagia/imunologia , Macrófagos/imunologia , Microdomínios da Membrana/imunologia , Pinocitose/imunologia , Receptores Depuradores Classe B/imunologia , Animais , Autofagia/genética , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Complexo de Golgi/genética , Complexo de Golgi/imunologia , Listeria monocytogenes/imunologia , Listeriose/genética , Listeriose/imunologia , Listeriose/patologia , Hepatopatias/genética , Hepatopatias/imunologia , Hepatopatias/patologia , Macrófagos/patologia , Microdomínios da Membrana/genética , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Pinocitose/genética , Receptores Depuradores Classe B/genética , Esplenopatias/genética , Esplenopatias/imunologia , Esplenopatias/patologia
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