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1.
Cell Rep ; 42(2): 112046, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36708514

RESUMO

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.


Assuntos
Fagócitos , Análise de Célula Única , Animais , Camundongos
2.
Nat Immunol ; 23(5): 705-717, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35487985

RESUMO

Caspase-11 detection of intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria mediates noncanonical activation of the NLRP3 inflammasome. While avirulent bacteria do not invade the cytosol, their presence in tissues necessitates clearance and immune system mobilization. Despite sharing LPS, only live avirulent Gram-negative bacteria activate the NLRP3 inflammasome. Here, we found that bacterial mRNA, which signals bacterial viability, was required alongside LPS for noncanonical activation of the NLRP3 inflammasome in macrophages. Concurrent detection of bacterial RNA by NLRP3 and binding of LPS by pro-caspase-11 mediated a pro-caspase-11-NLRP3 interaction before caspase-11 activation and inflammasome assembly. LPS binding to pro-caspase-11 augmented bacterial mRNA-dependent assembly of the NLRP3 inflammasome, while bacterial viability and an assembled NLRP3 inflammasome were necessary for activation of LPS-bound pro-caspase-11. Thus, the pro-caspase-11-NLRP3 interaction nucleated a scaffold for their interdependent activation explaining their functional reciprocal exclusivity. Our findings inform new vaccine adjuvant combinations and sepsis therapy.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Caspase 1/metabolismo , Caspases , Bactérias Gram-Negativas , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RNA Mensageiro
3.
Nature ; 560(7719): E33, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29950723

RESUMO

The microarray data generated and analysed in this Article have been uploaded to the Gene Expression Omnibus (GEO) under accession number GSE113383 . Accordingly, the 'Data availability' section of the Methods of the original Article has been rephrased online.

4.
Nature ; 548(7665): 52-57, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28746310

RESUMO

It has been proposed that the hypothalamus helps to control ageing, but the mechanisms responsible remain unclear. Here we develop several mouse models in which hypothalamic stem/progenitor cells that co-express Sox2 and Bmi1 are ablated, as we observed that ageing in mice started with a substantial loss of these hypothalamic cells. Each mouse model consistently displayed acceleration of ageing-like physiological changes or a shortened lifespan. Conversely, ageing retardation and lifespan extension were achieved in mid-aged mice that were locally implanted with healthy hypothalamic stem/progenitor cells that had been genetically engineered to survive in the ageing-related hypothalamic inflammatory microenvironment. Mechanistically, hypothalamic stem/progenitor cells contributed greatly to exosomal microRNAs (miRNAs) in the cerebrospinal fluid, and these exosomal miRNAs declined during ageing, whereas central treatment with healthy hypothalamic stem/progenitor cell-secreted exosomes led to the slowing of ageing. In conclusion, ageing speed is substantially controlled by hypothalamic stem cells, partially through the release of exosomal miRNAs.


Assuntos
Envelhecimento/genética , Envelhecimento/fisiologia , Exossomos/genética , Hipotálamo/citologia , Hipotálamo/fisiologia , Longevidade/fisiologia , MicroRNAs/genética , Células-Tronco Neurais/fisiologia , Envelhecimento/líquido cefalorraquidiano , Envelhecimento/patologia , Animais , Microambiente Celular , Exossomos/metabolismo , Hipotálamo/patologia , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inflamação , Longevidade/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/líquido cefalorraquidiano , MicroRNAs/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Complexo Repressor Polycomb 1/deficiência , Proteínas Proto-Oncogênicas/deficiência , Fatores de Transcrição SOXB1/deficiência , Fatores de Tempo
5.
J Virol ; 89(4): 2041-51, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25428880

RESUMO

UNLABELLED: Hepatitis B, which caused by hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation are the main driving factors of liver fibrosis and cirrhosis in chronic hepatitis B. Proinflammatory tumor necrosis factor alpha (TNF-α) has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B virus core (HBc) protein made cells susceptible to TNF-α-induced apoptosis. We found by tandem affinity purification and mass spectrometry that receptor of activated protein kinase C 1 (RACK1) interacted with HBc. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase (JNK). In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the proapoptotic activity of HBc. Capsid particle formation was not obligatory for HBc proapoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus the first indication of the pathogenic effects of HBc in liver injury during hepatitis B. IMPORTANCE: Our study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The proapoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with severe hepatitis may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.


Assuntos
Apoptose , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Fator de Necrose Tumoral alfa/imunologia , Animais , Células Cultivadas , Proteínas de Ligação ao GTP/metabolismo , Hepatócitos/fisiologia , Humanos , MAP Quinase Quinase 7/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de Quinase C Ativada , Receptores de Superfície Celular/metabolismo
6.
J Virol ; 89(1): 415-27, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25320320

RESUMO

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus with latent and lytic reactivation cycles. The mechanism by which KSHV evades the innate immune system to establish latency has not yet been precisely elucidated. Toll-like receptors (TLRs) are the first line of defense against viral infections. Myeloid differentiation factor 88 (MyD88) is a key adaptor that interacts with all TLRs except TLR3 to produce inflammatory factors and type I interferons (IFNs), which are central components of innate immunity against microbial infection. Here, we found that KSHV replication and transcription activator (RTA), which is an immediate-early master switch protein of viral cycles, downregulates MyD88 expression at the protein level by degrading MyD88 through the ubiquitin (Ub)-proteasome pathway. We identified the interaction between RTA and MyD88 in vitro and in vivo and demonstrated that RTA functions as an E3 ligase to ubiquitinate MyD88. MyD88 also was repressed at the early stage of de novo infection as well as in lytic reactivation. We also found that RTA inhibited lipopolysaccharide (LPS)-triggered activation of the TLR4 pathway by reducing IFN production and NF-κB activity. Finally, we showed that MyD88 promoted the production of IFNs and inhibited KSHV LANA-1 gene transcription. Taken together, our results suggest that KSHV RTA facilitates the virus to evade innate immunity through the degradation of MyD88, which might be critical for viral latency control. IMPORTANCE: MyD88 is an adaptor for all TLRs other than TLR3, and it mediates inflammatory factors and IFN production. Our study demonstrated that the KSHV RTA protein functions as an E3 ligase to degrade MyD88 through the ubiquitin-proteasome pathway and block the transmission of TLRs signals. Moreover, we found that KSHV inhibited MyD88 expression during the early stage of de novo infection as well as in lytic reactivation. These results provide a potential mechanism for the virus to evade innate immunity.


Assuntos
Herpesvirus Humano 8/imunologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Fator 88 de Diferenciação Mieloide/metabolismo , Transativadores/metabolismo , Linhagem Celular , Humanos , Mapeamento de Interação de Proteínas , Proteólise , Ubiquitina/metabolismo
7.
J Virol ; 88(5): 3004-15, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24371056

RESUMO

UNLABELLED: We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection. IMPORTANCE: Adenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.


Assuntos
Adenoviridae/genética , Epitopos/genética , Vetores Genéticos/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Adenoviridae/imunologia , Administração Intravenosa , Animais , Linhagem Celular , Modelos Animais de Doenças , Epitopos/imunologia , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/terapia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Imunização , Imunoterapia , Fígado/imunologia , Fígado/metabolismo , Fígado/virologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Subpopulações de Linfócitos T/imunologia , Transdução Genética , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Vírion/fisiologia , Montagem de Vírus , Replicação Viral
8.
J Biol Chem ; 288(3): 1785-94, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23212909

RESUMO

Arkadia is a RING-based ubiquitin ligase that positively regulates TGF-ß signaling by targeting several pathway components for ubiquitination and degradation. However, little is known about the mechanisms controlling Arkadia activity. Here we show that the LIM-only protein FHL2 binds and synergistically cooperates with Arkadia to activate Smad3/Smad4-dependent transcription. Knockdown of FHL2 by RNA interference decreases Arkadia level and restricts the amplitude of Arkadia-induced TGF-ß target gene responses. We found that Arkadia is ubiquitinated via K63- and K27-linked polyubiquitination. A single mutation at the RING domain that abolishes the E3 activity diminishes Arkadia ubiquitination, indicating that this modification partly involves autocatalytic process. Mutation of seven lysines at the C-terminal region of Arkadia severely impairs ubiquitination through the K27 but not the K63 linkage and slows down the turnover of Arkadia, suggesting that K27-linked polyubiquitination might promote proteolysis-dependent regulation of Arkadia. We show that FHL2 increases the half-life of Arkadia through inhibition of ubiquitin chain assembly on the protein, which provides a molecular basis for functional cooperation between Arkadia and FHL2 in enhancing TGF-ß signaling. Our study uncovers a novel regulatory mechanism of Arkadia by ubiquitination and identifies FHL2 as important regulator of Arkadia ubiquitination and TGF-ß signal transduction.


Assuntos
Proteínas com Homeodomínio LIM/genética , Proteínas Musculares/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Meia-Vida , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Luciferases , Camundongos , Proteínas Musculares/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
Antivir Chem Chemother ; 22(3): 119-30, 2011 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-22095520

RESUMO

BACKGROUND: Because of high mutation rates, new drug-resistant viruses are rapidly evolving, thus making the necessary control of influenza virus infection difficult. METHODS: We screened a constrained cysteine-rich peptide library mimicking µ-conotoxins from Conus geographus and a proline-rich peptide library mimicking lebocin 1 and 2 from Bombyx mori by using influenza virus RNA polymerase (PB1, PB2 and PA) and nucleoprotein (NP) as baits. RESULTS: Among the 22 peptides selected from the libraries, we found that the NP-binding proline-rich peptide, PPWCCCSPMKRASPPPAQSDLPATPKCPP, inhibited influenza replicon activity to mean±sd 40.7%±15.8% when expressed as a GFP fusion peptide in replicon cells. Moreover, when the GFP fusion peptide was transduced into cells by an HIV-TAT protein transduction domain sequence, the replication of influenza virus A/WSN/33 (WSN) at a multiplicity of infection of 0.01 was inhibited to 20% and 69% at 12 and 24 h post-infection, respectively. In addition, the TAT-GFP fusion peptide was able to slightly protect Balb/c mice from WSN infection when administrated prior to the infection. CONCLUSIONS: These results suggest the potential of this peptide as the seed of an anti-influenza drug and reveal the usefulness of the constrained peptide strategy for generating inhibitors of influenza infection. The results also suggest that influenza NP, which is conserved among the influenza A viruses, is a good target for influenza inhibition, despite being the most abundant protein in infected cells.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Peptídeos/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas do Core Viral/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Cães , Feminino , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/metabolismo , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo , Biblioteca de Peptídeos , Proteínas de Ligação a RNA/metabolismo , Células Vero , Proteínas do Core Viral/metabolismo
10.
Anesthesiology ; 108(3): 426-33, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18292680

RESUMO

BACKGROUND: Volatile anesthetics (VAs) have been found to induce a delayed protective response called preconditioning to subsequent hypoxic/ischemic injury. VA preconditioning has been primarily studied in canine and rodent heart. A more genetically tractable model of VA preconditioning would be extremely useful. Here, the authors report the development of the nematode Caenorhabditis elegans as a model of VA preconditioning. METHODS: Wild-type and mutant C. elegans were exposed to isoflurane, halothane, or air under otherwise identical conditions. After varying recovery periods, the animals were challenged with hypoxic, azide, or hyperthermic incubations. After recovery from these incubations, mortality was scored. RESULTS: Isoflurane- and halothane-preconditioned animals had significantly reduced mortality to all three types of injuries compared with air controls. Concentrations as low as 1 vol% isoflurane (0.64 mm) and halothane (0.71 mm) induced significant protection. The onset and duration of protection after anesthetic were 6 and 9 h, respectively. A mutation that blocks inhibition of neurotransmitter release by isoflurane did not attenuate the preconditioning effect. A loss-of-function mutation of the Apaf-1 homolog CED-4 blocked the preconditioning effect of isoflurane, but mutation of the downstream caspase CED-3 did not. CONCLUSIONS: Volatile anesthetic preconditioning extends beyond the vertebrate subphylum. This markedly broadens the scope of VA preconditioning and suggests that its mechanisms are widespread across species and is a fundamental and evolutionarily conserved cellular response. C. elegans offers a means to dissect genetically the mechanism for VA preconditioning as illustrated by the novel finding of the requirement for the Apaf-1 homolog CED-4.


Assuntos
Anestésicos Inalatórios/administração & dosagem , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Precondicionamento Isquêmico/métodos , Animais , Hipóxia/fisiopatologia , Hipóxia/terapia , Volatilização
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